Tachykinins protect cholinergic neurons from quinolinic acid excitotoxicity in striatal cultures

The neuroprotective effect of tachykinins against excitotoxic death of cholinergic neurons was studied in rat striatal cell cultures. Quinolinic acid (QUIN) and kainic acid (KA) produced a dose dependent decrease in choline acetyttransferase activity, but KA was more potent. Our results show that substance P (SP) totally reversed the toxicity induced by 125 μM QUIN but not by 40 μM KA. This effect was also observed using protease inhibitors or a SP-analog resistant to degradation, [Sar⁹]-Substance P. The survival of neuron specific enolase- and acetylcholinesterase (AChE)-positive cells after treatment with QUIN alone or in the presence of SP was also examined. We observed that, while a decrease in total cell number produced by QUIN was not prevented by SP treatment, AChE-positive cells were rescued from the toxic damage. To characterize the SP protective effect we used more selective agonists of the three classes of neurokinin (NK) receptors. [Sar⁹, Met(O₂)¹¹]-Substance P (NK₁ receptor agonist), [Nle¹⁰]-Neurokinin A (NK₂ receptor agonist) or [Me-Phe⁷]-Neurokinin B (NK₃ receptor agonist) were all able to block the toxic effect of QUIN on cholinergic activity. These results show that tachykinins provide an important protective support for striatal neurons, suggesting a possible therapeutical benefit in neurodegenerative disorders affecting cholinergic neurons.     

Pharmacological characterization of grooming induced by a selective NK-1 tachykinin receptor agonist

Bilateral intranigral administration of the selective NK-1 tachykinin receptor agonist [AcArg⁶, Sar⁹, Met(O₂)¹¹]SP_6–11 (0–11 nmol total bilateral dose) selectively induced grooming in rats. This response was blocked by concurrent intranigral administration of the NK-1 tachykinin receptor antagonist RP 67580 (2 nmol), but not by NK-2 (L-659, 877) or NK-3 ([Trp⁷, β-Ala⁸]NKA_4–10) antagonists. Pretreatment with systemic opioid (naloxone 1.5 mg/kg) and D₁ dopamine (SCH 23390 100 μg/kg) receptor antagonists also attenuated tachykinin-induced grooming, which was unaffected by D₂ dopamine (sulpiride 30 mg/kg) or 5-HT_2A+C (ritanserin 2 mg/kg) antagonists. Grooming induced by intranigral [AcArg⁶, Sar⁹, Met(O₂)¹¹]SP_6–11 was also attenuated by bilateral 6-hydroxydopamine lesions of te substantia nigra. These findings indicate that grooming induced by intranigral tachykinins reflects activation of NK-1 receptors and is dependent upon endogenous dopamine and consequent selective stimulation of D₁ dopamine receptors.     

Development of dopamine receptors in the forebrain of the domestic chick in relation to auditory imprinting. An autoradiographic study

The rostro-medial neostriatum/hyperstriatum ventrale (MNH) and the neostriatum dorsocaudale (Ndc) are forebrain regions which play a role in auditory filial imprinting. Both regions receive a distinct dopaminergic input from the mesencephalon and we were interested to investigate if the dopaminergic system, which is known to play a role in associative learning processes and neuronal plasticity is involved in auditory imprinting. Using ligand autoradiography we studied the distribution and density of dopamine receptors (D₁ and D₂ type) in the forebrain of socially isolated chicks during the first postnatal week and compared these data with the values of age-matched imprinted chicks. D₁- and D₂-receptors were present in the chick forebrain on the day of hatching and they showed in general, the same distribution until postnatal day 7. Between days 0 and 2 the D₂-receptor density increased significantly in the lobus parolfactorius and paleostriatum augmentatum while for D₁-receptor density no significant changes were detectable. The receptor densities in the investigated forebrain regions did not differ significantly between imprinted and control chicks. These results suggest that auditory imprinting does not induce alterations of dopamine receptor density, however, more subtle changes can not be excluded. The presented detailed data about the developmental profile of dopamine receptors within distinct brain regions is a further step towards a more specific interpretation of behavioral effects of dopamine receptor agonists or antagonists at different postnatal ages.     

Rat/mouse hemokinin-1, a mammalian tachykinin peptide, markedly potentiates the antinociceptive effects of morphine administered at the peripheral and supraspinal level

Rat/mouse hemokinin 1 (r/m HK-1) is a mammalian tachykinin peptide whose biological functions are not fully understood. Our recent report showed that i.c.v. administration of r/m HK-1 could produce dose- and time-related antinociceptive effect at nanomole concentration, and naloxone significantly antagonized this effect. Thus, we provide indirect evidence favoring a role of NK₁ supraspinal receptors in the inhibitory control of descending pain pathways, a role that seems to partially involve the activation of the endogenous opioid systems. Based on this report, the present study was conducted to further investigate the direct functional interaction between supraspinal tachykinin (r/m HK-1) and opioid systems. The results demonstrate that i.c.v. administration of r/m HK-1 (5 nmol/kg) could significantly potentiate the antinociceptive effects of morphine which was injected at peripheral and supraspinal level. These antinociceptive effects were blocked by prior treatment with the classical opioid receptors antagonist naloxone, indicating that the potentiated analgesic response is mediated by opioid-responsive neurons. Consistent with previous biochemical data, a likely mechanism underlying the peptide-mediated enhancement of opioid analgesia may center on the ability of r/m HK-1 to release endogenous opioid peptides. We suggest that there may be a cascade amplification mechanism in pain modulation when the two agents were co-administrated. The synergistic analgesic relationship of morphine and r/m HK-1 established here supports the hypothesis that supraspinal tachykinin and peripheral and central opioid systems have a direct functional interaction in the modulation of local nociceptive responses.     

Differential effects of the adenosine A(1) receptor allosteric enhancer PD 81,723 on agonist binding to brain and adipocyte membranes.

The benzoylthiophene analog, PD 81,723, has been shown to allosterically enhance agonist binding and functional activation of the mammalian adenosine (ADO) A₁ receptor subtype by putatively maintaining the receptor in a high affinity state. The present studies were conducted to evaluate the ability of PD 81,723 to enhance the binding of [³H]cyclohexyladenosine ([³H]CHA) to A₁ receptors of neural (cerebral cortex) and non-neural (adipocyte) origin in three different species; rat, guinea pig and dog. PD 81,723 (0.3–100 μM) produced a concentration-dependent enhancement of [³H]CHA binding to rat brain A₁ receptors. These effects were also species-dependent with larger enhancements (150–200% of control) observed in guinea pig and dog brain membranes as compared to the rat (120% of control). In contrast, PD 81,723 did not produce any enhancement of [³H]CHA binding to A₁ receptors in adipocyte membranes from any of the species examined. Additional binding studies were conducted using pharmacological manipulations that have previously been shown to enhance the allosteric effects of PD 81,723. In the presence of 1 mM GTP, the allosteric effects of PD 81,723 (15 μM) were increased in rat, guinea pig and dog brain membranes, however, in adipocyte membranes from each species, no significant alteration in agonist binding was observed. Similarly, the A₁ receptor selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (added to effectively reduce the intrinsic antagonist properties of PD 81,723) was found to enhance the allosteric effects of PD 81,723 (15 μM) in brain, but produce no alteration of agonist binding in adipocyte membranes from each species. Examination of the dissociation kinetics of [³H]CHA binding from rat brain and adipocyte membranes revealed that PD 81,723 (15 μM) differentially slowed agonist dissociation from brain, but not adipocyte, membranes. Taken together, the present data support the hypothesis that in tissues that are sensitive to PD 81,723, this benzyolthiophene functions to maintain the A₁ receptor in a high-affinity state and that the relative proportions of high-affinity A₁ receptors present in specific tissues may contribute, at least in part, to the apparent differential effects of PD 81,723 on agonist binding. The tissue specific modulation of A₁ receptor function by PD 81,723 also illustrates the possibility that the locus of allosteric modulation by PD 81,723 may be manifest via a specific, but indirect and tissue-dependent, interaction with the A₁ receptor.     

Rats bred for enhanced apomorphine susceptibility have elevated tyrosine hydroxylase mRNA and dopamine D2-receptor binding sites in nigrostriatal and tuberoinfundibular dopamine systems

From a Wistar population two rat lines were generated using as criterion the behavioral response to the dopamine agonist apomorphine. Rats of the apomorphine-susceptible (apo-sus) line revealed a vigorous gnawing response to apomorphine administration while the other rat line, the apomorphine-unsusceptible (apo-unsus) line, was selected for lack of response to the drug. In the present study using the 12th and 13th generation of these genetically selected lines, we have investigated whether this difference in apomorphine responsiveness was correlated with changes in dopamine neurochemistry. Therefore, we measured tyrosine hydroxylase (TH), the rate limiting enzyme in dopamine synthesis, as well as dopamine D₁ and D₂ receptor mRNA levels in discrete brain regions by in situ hybridization. Dopamine (D₂/D₃) receptor binding was assessed with [¹²⁵I]iodosulpride in a membrane binding assay and by quantitative autoradiography on tissue sections. [³H]SCH 23390 was used to analyze D₁ receptor binding. Apo-sus rats displayed significantly higher TH mRNA levels in the A₉ cell group of the substantia nigra pars compacta and in the A₁₂ cell group of the arcuate nucleus. No difference was found in the A₁₀ cell group of the VTA and the A₆ cell group of the locus coeruleus. The density ofD_2/3 binding sites as well as D₁ receptor mRNA levels in the striatal projection area of the A₉ substantia nigra neurons, were significantly elevated in apo-sus rats. Dopamine D₂ receptor mRNA and D₁ receptor binding levels in caudate putamen and nucleus accumbens, however, were similar in rats of both lines. In conclusion, high apomorphine susceptibility is related to a potentially enhanced dopamine responsiveness selective for the nigrostriatal and tuberoinfundibular pathways.     

μ-Opioid receptor-induced Ca mobilization and astroglial development: morphine inhibits DNA synthesis and stimulates cellular hypertrophy through a Ca-dependent mechanism

Morphine, a preferential μ-opioid receptor agonist, alters astroglial development by inhibiting cell proliferation and by promoting cellular differentiation. Although morphine affects cellular differentiation through a Ca²⁺-dependent mechanism, few studies have examined whether Ca²⁺ mediates the effect of opioids on cell proliferation, or whether a particular Ca²⁺ signal transduction pathway mediates opioid actions. Moreover, it is uncertain whether one or more opioid receptor types mediates the developmental effects of opioids. To address these questions, the present study examined the role of μ-opioid receptors and Ca²⁺ mobilization in morphine-induced astrocyte development. Morphine (1 gmM) and non-morphine exposed cultures enriched in murine astrocytes were incubated in Ca²⁺-free media supplemented with < 0.005, 0.3, 1.0, or 3.0 mM Ca²⁺ ([Ca²⁺]_o), or in unmodified media containing Ca²⁺ ionophore (A23187), nifedipine (1 μM), dantrolene (10 μM), thapsigargin (100 nM), or l-glutamate (100 μM) for 0-72 h. μ-Opioid receptor expression was examined immunocytochemically using specific (MOR1) antibodies. Intracellular Ca²⁺ ([Ca²⁺]ⁱ) was measured by microfluorometric analysis using fura-2. Astrocyte morphology and bromodeoxyuridine (BrdU) incorporation (DNA synthesis) were assessed in glial fibrillary acidic protein (GFAP) immunoreactive astrocytes. The results showed that morphine inhibited astroglial growth by activating μ-opioid receptors. Astrocytes expressed MOR1 immunoreactivity and morphine's actions were mimicked by the selective μ, agonist PL017. In addition, morphine inhibited DNA synthesis by mobilizing [Ca²⁺]_i in developing astroglia. At normal [Ca²⁺]_o, morphine attenuated DNA synthesis by increasing [Ca²⁺]_i; low [Ca²⁺]_o (0.3 mM) blocked this effect, while treatment with Ca²⁺ ionophore or glutamate mimicked morphine's actions. At extremely low [Ca²⁺]_o (< 0.005 mM), morphine paradoxically increased BrdU incorporation. Although opioids can increase [Ca²⁺]_i in astrocytes through several pathways, not all affect DNA synthesis or cellular morphology. Nifedipine (which blocks L-type Ca²⁺ channels) did not prevent morphine-induced reductions in BrdU incorporation or cellular differentiation, while thapsigargin (which depletes IP₃-sensitive Ca²⁺ stores) severely affected inhibited DNA synthesis and cellular differentiation-irrespective of morphine treatment. However, dantrolene (an inhibitor of Ca²⁺-dependent Ca²⁺ release) selectively blocked the effects of morphine. Collectively, the findings suggest that opioids suppress astroglial DNA synthesis and promote cellular hypertrophy by inhibiting Ca²⁺-dependent Ca²⁺ release from dantrolene-sensitive intracellular stores. This implies a fundamental mechanism by which opioids affect central nervous system maturation.     

Interrelationship between secretion, protein phosphorylation and intracellular Ca2+ concentration in platelets stimulated by thrombin or thromboxane A2 analogue

The interrelationship between ATP-secretion, protein phosphorylation and intracellular Ca²⁺ concentration ([Ca²⁺]i) was studied in both ³²P and quin 2 loaded human platelets stimulated by thrombin or thromboxane A₂ analogue (STA₂). In platelets stimulated by thrombin, the degree of 47,000 dalton polypeptides (P47) phosphorylation was observed in completely dose-related manner, regardless of the amount of [Ca²⁺]i. In the same condition, the degree of myosin light chain (P20) phosphorylation, however, was well correlated with ATP secretion and [Ca²⁺]i, when platelets were stimulated by lower dose of thrombin. The similar results were obtained in platelets stimulated by STA₂. These findings suggested that P20, but not P47, phosphorylation in activated platelets is mediated by a rise of [Ca²⁺]i and is well correlated with the secretory reaction. It was unlikely that P47 phosphorylation plays any role in promoting platelet activation.     

Patterns of tachykinin expression and localization in developing feline neostriatum

The development of tachykinins in the neostriatum was determined qualitatively in order to characterize the ontogeny of an early-forming neostriatal peptidergic system. Tachykinins were detected by immunohistochemistry in fetal, postnatal, and adult cats. Neostriatal cells and neurites expressed tachykinins as early as fetal age 30 and increased in frequency progressively with age. Initial tachykinin expression occurred in neostriatal neurons during their postmitotic migration. In the head of the caudate nucleus, clusters of tachykinin-containing cells and fibers formed between fetal days 35 and 45, when the distribution of labeled neurons changed from a dispersed to an aggregated pattern. Between fetal days 45 and 50, tachykinin-rich neuronal clusters increased in frequency and were distributed throughout the rostral caudate nucleus. In contrast to neurons in clusters, neurons in the complementary neuropil expressed tachykinins largely postnatally. Postnatal morphological maturation of tachykinin-containing neurons paralleled the morphogenesis of medium spiny neostriatal cells. In addition, the caudate nucleus and putamen followed different spatiotemporal gradients of tachykinin expression. These results indicate that tachykinins are expressed in neostriatal neurons during the early ontogeny of the neostriatum and may function as trophic factors before synaptogenesis.     

Cholinergic Responses in Developing Outer Hair Cells of the Rat Cochlea

Acetylcholine-evoked currents were investigated using the conventional whole-cell patch-clamp recording technique in developing outer hair cells (OHCs). The cells were isolated from the rat cochlea at different stages of postnatal development ranging from day 4 (P4) to P30. Acetylcholine-evoked currents could be recorded at P6 and P8. At this developmental stage, the majority of OHCs displayed inward nicotinic-like currents near the resting membrane potential. These cholinergic currents zeroed near 0 mV, as expected for a non-selective cation current, and could be reversibly blocked by d-tubocurarine. At P12 and adult stage, the cholinergic response of OHCs switched to an outward current reversing near E _K and displaying a bell shape peaking between -40 and -30 mV. This change in polarity of the acetylcholine response during postnatal development might be explained by progressive functional coupling between acetylcholine ionotropic receptors permeable to Ca²⁺ and nearby Ca²⁺-activated K⁺ channels at the synaptic pole of OHCs.     

Production and characterization of polyclonal and monoclonal antibodies to the zeta (ξ) opioid receptor

The opioid growth factor, [Met⁵]-enkephalin, is an inhibitory agent of cell proliferation and maturation that interacts with the zeta (ξ) opioid receptor to modulate growth. In order to learn more about this receptor, polyclonal and monoclonal antibodies were raised against binding subunits identified on two-dimensional gels by ligand blotting. Using Western blotting, the polyclonal antibodies and some of the monoclonal antibodies recognized all 4 binding polypeptides (32, 30, 17, and 16 kDA) in developing rat cerebellum; no reaction was recorded in adult cerebellum. In addition, other monoclonals were able to distinguish only certain subunits (e.g. 17 kDa). The monoclonal antibodies and their F(ab′)₂ fragments, as well as the polyclonal antibodies, blocked the binding of [³H][Met⁵]-enkephalin to preparations of developing cerebellum. Both the polyclonal and monoclonal antibodies immunoprecipitated ξ opioid binding polypeptides from 6-day-old cerebellar homogenates solubilized by the zwitterionic detergent, CHAPS. Immunocytochemistry performed with polyclonal antibodies showed immunoreactivity associated with proliferating and differentiating cerebellar cells, but no specific staining was detected in the adult cerebellum. These results have identified and characterized antibodies to the ξ opioid receptor, and the antibodies were used to localize this receptor; these antibodies will be valuable to further cellular and molecular studies.     

Downregulation of muscarinic receptors in the rat caudate-putamen after lesioning of the ipsilateral nigrostriatal dopamine pathway with 6-hydroxydopamine (6-OHDA): normalization by fetal mesencephalic transplants

The autoradiographic distribution and density of muscarinic receptors was studied in the neostriatum of rats with long-term unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal dopaminergic pathway and in lesioned rats who had additionally received embryonic substantia nigra grafts in the dopamine denervated striatum. Muscarinic receptors were labeled with [³H]quinuclidinyl benzilate (QNB), M₁ receptors were directly labeled with [³H]pirenzepine (PZ) and non-M₁ receptors were labeled by the competition of 100 nM PZ with [³H]QNB. The density and distribution of muscarinic receptors were directly compared to the sodium-dependent, high-affinity, choline uptake sites as labeled with [³H]hemicholinium-3 (HC-3). In the 6-OHDA-lesioned animals, there was a 25% reduction in muscarinic receptors labeled with [³H]QNB. Subtype analysis showed that there was a reduction of both M₁ (−26%) and non-M₁ (−33%) receptors. A normal density of both muscarinic receptor populations was found in animals with successful transplants. Saturation analysis demonstrated that the changes, in muscarinic receptor density, were due to a change in receptor number (B_max) and not affinity (K_d). There was no significant change in [³H]HC-3 binding in the 6-OHDA-lesioned or transplanted animals, indicating that alterations in muscarinic receptors were not due to transynaptic degeneration of striatal cholinergic interneurons. The findings of downregulation of muscarinic receptors following long-term dopamine denervation and the subsequent normalization of muscarinic receptor density after fetal mesencephalic transplantation suggests that transplanted substantia nigra cells are able to restore inhibitory control on striatal cholinergic interneurons.     

Lateralization of opioid receptors in turtle visual cortex

The opioid μ-agonist morphine, the δ-agonist [ -Ala², -Leu⁵]enkephalin (DADL) and the κ-agonist bremazocine locally applied to the surface of turtle visual cortex inhibited the orthodromic evoked potential (EP; fast negative component N₁). After application of the σ-agonist SKF 10.047 the inhibition was followed by facilitation of EP. The lack of cross-desensitization to the inhibitory action of opioids upon EP indicates that the drugs exert their effects via different opioid receptors. Bremazocine and morphine predominantly inhibited the left cortical EP, whereas DADL was a potent inhibitor of the right cortical EP. Thus, the opioid receptors which modulate evoked electrical activity of the left visual cortex (LVC) apparently belong mostly to the κ- and μ-type, while σ-receptors were predominantly responsible for the modulation of electrical activity in the right visual cortex (RVC). Besides, we have studied in vivo binding of the κ-agonist [³H]ethylketocyclazocine ([³H]EKC) and the σ-agonist [³H]DADL to LVC and RVC. The binding was specific and could be accounted for by the interaction with membrane opioid receptors and/or specific uptake of these drugs by cortex cells. [³H]EKC and [³H]DADL exhibited a more effective binding to LVC and RVC membranes, respectively. The results obtained in vitro apparently indicate that LVC and RVC differ in the number of κ- and σ-binding sites. Thus, turtle brain may have a side-specific mechanism for selective neurochemical regulation of neuron activity in LVC and RVC realized predominantly via κ- and σ-receptor, respectively.     

Ontogeny of mu-, delta- and kappa-opioid receptors mediating inhibition of neurotransmitter release and adenylate cyclase activity in rat brain

The ontogeny was examined of functional opioid receptors mediating presynaptic inhibition of neurotransmitter release and inhibition of dopamine (DA)-sensitive adenylate cyclase in the rat brain, using highly selective agonists for mu-, delta- and kappa-receptors. On gestational day 17 (E17) strong inhibitory effects of the selective mu-agonist DAGO on the electrically evoked release of [3H]noradrenaline from cortical slices and of the selective kappa-agonist U-50,488 on the electrically evoked release of [3H]DA from striatal slices were found. Electrically evoked release of [3H]acetylcholine from striatal slices was not detectable before postnatal day 7 (P7), but on that day it was already strongly inhibited by the selective delta-agonist DPDPE. Although mu- and delta-opioid receptors coupled to DA-sensitive adenylate cyclase in the striatum are likely to be physically associated in an opioid receptor complex in the adult, they were found to develop asynchronously. Whereas selective activation of mu-receptors with DAGO resulted in an inhibition of D1 dopamine receptor-stimulated adenylate cyclase activity on E17, activation of delta-receptors with DPDPE was not effective until P14. This study confirms the early appearance of mu- and kappa-opioid receptors and the relatively late development of delta-opioid receptors in the rat brain. Most importantly, it shows that in an early stage of development opioids are already able to mediate modulation of noradrenergic (via activation of mu-receptors) and dopaminergic (via activation of mu- and kappa-receptors) neurotransmission processes. Therefore, these opioid receptor types could play a role in brain development and/or developmental disturbances.     

Selective alterations in glutamate receptor subtypes after unilateral orbital enucleation

Glutamate is the major excitatory neurotransmitter in the rat visual system. Using quantitative autoradiography the effect of unilateral orbital enucleation on [³H]kainate, [³H]-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid ([³H]AMPA) and [³H]glutamate binding to kainate, quisqualate and NMDA receptors respectively has been examined within anatomical components of the visual pathway at 4 time points up to 20 days post-lesion. The time course for the degeneration of retinal projection fibres was assessed in a separate group of animals by quantifying [³H]cyclohexyladenosine ([³H]CHA) binding to presynaptic adenosine A₁ receptors. Over the first 5 days after orbital enucleation, there were no significant alterations in glutamate or adenosine A₁ receptor binding in visual structures of the visually deprived hemisphere. However, at 10 days post-lesion [³H]AMPA binding was significantly reduced (30%) in the visually deprived superior colliculus but unaltered in other visual structures. At this time point there was also a significant reduction (50%) in [³H]CHA binding in the visually deprived superior colliculus but not in other retino-recipient nuclei. There were similar changes in [³H]AMPA and [³H]CHA binding at 20 days post-enucleation. [³H]Kainate binding was significantly increased in the visually deprived superior colliculus only at 20 days post-enucleation. Saturation analysis of [³H]kainate and [³H]AMPA binding at this time point indicated a selective increase in the b_max value for the high affinity [³H]kainate binding site and a concomitant decrease in the b_max value for the high affinity [³H]AMPA binding site in the visually deprived superior colliculus. There were, however, no significant alterations in [³H]AMPA or [³H]kainate binding in other primary projection areas or in secondary visual areas (e.g. visual cortex) at any time point. NMDA sensitive [³H]glutamate binding was unaltered in the visually deprived hemisphere up to 20 days post-enucleation. These results suggest an upregulation of kainate receptors in the visually deprived superior colliculus after orbital enucleation and a loss of presynaptic quisqualate receptors on degenerating retinal fibres. The plastic alterations in kainate receptors in the superior colliculus are supportive of electrophysiological data suggesting a physiological role for these sites in mediating excitatory postsynaptic potentials in tectal neurons.     

Characterization of the depression of rat cerebral cortical neurons by selective adenosine agonists

The identity of the receptors involved in mediating the depressant actions of adenosine and its analogs on the spontaneous firing of rat cerebral cortical neurons was elucidated by evaluating the effects of selective A₁ and A₂ receptor agonists and antagonists. The A₁ agonist N⁶-cyclopentyladenosine (CPA) and the A₂ agonist CGS 21680 both depressed cortical neuronal firing. At low doses (0.001–0.01 mg/kg) the A₁ antagonist DPCPX blocked the effects of CPA, but not those of CGS 21680. At higher doses, it antagonized both agonists. The A₂ antagonist CGS 15943 (0.01–0.1 mg/kg) selectively blocked the actions of CGS 21680. At 1 mg/kg it also antagonized the responses of some neurons to CPA. These results suggest that the depressant actions of adenosinergic agonists on the firing of rat cerebral cortical neurons involve both A₁ and A₂ receptors.     

Adenosine A1 receptor-mediated inhibition of evoked glutamate release is coupled to calcium influx decrease in goldfish brain synaptosomes

Binding of [³H]cyclohexyladenosine (CHA) to the cellular fractions and P₂ subfractions of the goldfish brain was studied. The A₁ receptor density was predominantly in synaptosomal membranes. In goldfish brain synaptosomes (P₂), 30 mM K⁺ stimulated glutamate, taurine and GABA release in a Ca²⁺-dependent fashion, whereas the aspartate release was Ca²⁺-independent. Adenosine, R-phenylisopropyladenosine (R-PIA) and CHA (100 μM) inhibited K⁺-stimulated glutamate release (31%, 34% and 45%, respectively). All of these effects were reversed by the selective adenosine A₁ receptor antagonist, 8-cyclopentyltheophylline (CPT). In the same synaptosomal preparation, K⁺ (30 mM) stimulated Ca²⁺ influx (46.8±6.8%) and this increase was completely abolished by pretreatment with 100 nM ω-conotoxin. Pretreatment with 100 μM R-PIA or 100 μM CHA, reduced the evoked increase of intra-synaptosomal Ca²⁺ concentration, respectively by 37.7±4.3% and 39.7±9.0%. A possible correlation between presynaptic A₁ receptor inhibition of glutamate release and inhibition of calcium influx is discussed.     

Opposite presynaptic regulations by glutamate through NMDA receptors of dopamine synthesis and release in rat striatal synaptosomes

Purified striatal synaptosomes were superfused continuously with L-[3,5-³H]tyrosine to measure simultaneously the synthesis ([³H]water formed during the conversion of [³H]tyrosine into [³H]DOPA) and the release of [³H]dopamine ([³H]DA). Glutamate (10⁻³ M) and NMDA (10⁻³ M, in the absence of Mg²⁺) stimulated the release of [³H]DA, but they reduced the efflux of [³H]water. This reduction of [³H]DA synthesis was blocked by 2-amino-5-phosphonovalerate indicating the involvement of NMDA receptors. Although D,L--amino-3-hydroxy-5-methyl-4-isoxazole-4-propionate (AMPA) and kainate stimulated the release of [³H]DA, they did not affect its synthesis. The glutamate-evoked inhibition of [³H]DA synthesis was prevented when synaptosomes were superfused continuously with adenosine adenosine deaminase plus quinpirole, a treatment which markedly reduces the phosphorylation of tyrosine hydroxylase by cAMP dependent protein kinase. The opposite effects of glutamate on [³H]DA synthesis and release were mimicked by ionomycin (10⁻⁶ M). It is proposed that both an activation of a cyclic nucleotide phosphodiesterase and a dephosphorylation of tyrosine hydroxylase linked to the influx of calcium through NMDA receptors is responsible for the inhibition of dopamine synthesis by glutamate and that calcineurin could play a critical role in these processes.     

Dopaminergic effects after chronic treatment with nandrolone visualized in rat brain by positron emission tomography

Anabolic–androgenic steroids (AAS) have recently been shown to induce neurochemical alterations in areas of the male rat CNS related to behavioural changes that have been observed among AAS misusers. In the present study, positron emission tomography (PET) is suggested as a suitable in vivo method in order to visualize the density of the dopamine transporter ([¹¹C]-FE-β-CIT) as well as the dopamine D₁-like ([¹¹C]-(+)-SCH23390) and the D₂-like receptors ([¹¹C]-raclopride) in the male rat brain. Chronic treatment with the AAS nandrolone decanoate (15 mg/kg/day for 14 days) caused an up-regulation of the binding potential of the dopamine transporter in the striatum.     

Angiotensin receptor binding in human hypothalamus: autoradiographic localization

Binding of ¹²⁵I-[Sar¹,Ile⁸]angiotensin II in the human hypothalamus was mapped by in vitro autoradiography carried out on frozen sections of hypothalamus from two human brains. Regions showing the greatest specific binding of this radioligand were the organum vasculosum of the lamina terminalis, median preoptic nucleus, subfornical organ, median eminence, arcuate nucleus and paraventricular nucleus. These regions may be sites of angiotensin II receptors involved in the regulation of blood pressure, fluid balance and pituitary hormone secretion.