Maria Christina F. de Mello

Regulation of acetylcholine synthesis and storage

Acetylcholine is one of the major modulators of brain functions and it is the main neurotransmitter at the peripheral nervous system. Modulation of acetylcholine release is crucial for nervous system function. Moreover, dysfunction of cholinergic transmission has been linked to a number of pathological conditions. In this manuscript, we review the cellular mechanisms involved with regulation of acetylcholine synthesis and storage. We focus on how phosphorylation of key cholinergic proteins can participate in the physiological regulation of cholinergic nerve-endings.

Direct inhibition of the N-methyl-D-aspartate receptor channel by dopamine and (+)-SKF38393

Dopamine is known to modulate glutamatergic synaptic transmission in the retina and in several brain regions by activating specific G‐protein‐coupled receptors. We have examined the possibility of a different type of mechanism for this modulation, one involving direct interaction of dopamine with ionotropic glutamate receptors. Ionic currents induced by fast application of N‐methyl‐D‐aspartate (NMDA) were recorded under whole‐cell patch‐clamp in cultured striatal, thalamic and hippocampal neurons of the rat and in retinal neurons of the chick. Dopamine at concentrations above 100 μM inhibited the NMDA response in all four neuron types, exhibiting an IC50 of 1.2 mM in hippocampal neurons. The time course of this inhibition was fast, developing in less than 100 ms. The D1 receptor agonist (+)‐SKF38393 mimicked the effect of dopamine, with an IC50 of 58.9 μM on the NMDA response, while the enantiomer (−)‐SKF38393 was ineffective at 50 μM. However, the D1 antagonist R(+)‐SCH23390 did not prevent the inhibitory effect of (+)‐SKF38393. The degree of inhibition by dopamine and (+)‐SKF38393 depended on transmembrane voltage, increasing 2.7 times with a hyperpolarization of about 80 mV. The voltage‐dependent block by dopamine was also observed in the presence of MgCl2 1 mM. Single‐channel recordings showed that the open times of NMDA‐gated channels were shortened by (+)‐SKF38393. These data suggested that the site to which the drugs bound to produce the inhibitory effect was distinct from the classical D1‐type dopamine receptor sites, possibly being located inside the NMDA channel pore. It is concluded that dopamine and (+)‐SKF38393 are NMDA channel ligands.

L-DOPA supply to the neuro retina activates dopaminergic communication at the early stages of embryonic development

DOPA decarboxylase (DDC; aromatic‐l‐amino acid decarboxylase; EC 4.1.1.28) is absent in retinas from 6‐day‐old chicken embryos (E6) but is expressed in retina of E8 embryos, in the presumptive outer plexiform layer. Thereafter, DDC appears in cell bodies of presumptive amacrine cells. The dopamine (DA) content of E9/10 and E15/16 retinas, pre‐incubated with l‐DOPA for 1 h, increased 250‐ and 600‐fold, respectively, showing that DDC is active since early in development. Intercellular communication, measured by endogenous cyclic AMP accumulation, was observed when retinas from E9/10 to E15/16 were pre‐incubated for 1 h with 1 mm l‐DOPA, washed and followed by incubation in the presence of 0.5 mm 3‐isobutyl‐1‐methylxanthine, a phosphodiesterase inhibitor. Cyclic AMP accumulation was prevented when pre‐incubation with l‐DOPA was carried out in the presence of carbidopa. Moreover, the accumulation of cyclic AMP was inhibited by SCH 23390 (2 µm). The incubation of retinas in medium previously conditioned by retina‐pigmented epithelium (RPE) also increased its cyclic AMP content with the characteristics described for l‐DOPA. Our results show that dopaminergic communication takes place in the embryonic retina, before tyrosine hydroxylase expression, provided l‐DOPA is supplied to the tissue. It also shows that RPE is a potential source of l‐DOPA early in development.

Expression of functional dopaminergic phenotype in purified cultured Müller cells from vertebrate retina

Purified retina glial Müller cells can express the machinery for dopamine synthesis and release when maintained in culture. Dopamine is detected in cell extracts of cultures exposed to its precursor, L-DOPA. A large portion of synthesized dopamine is recovered in the superfusing medium showing the tendency of the accumulated dopamine to be released. Müller cells purified from developing chick and mouse retinas express L-DOPA decarboxylase (DDC; aromatic-L-amino-acid decarboxylase; EC 4.1.1.28) and the dopamine transporter DAT. The synthesis of dopamine from L-DOPA supplied to Müller cultures is inhibited by m-hydroxybenzylhydrazine, a DDC inhibitor. Dopamine release occurs via a transporter-mediated process and can activate dopaminergic D(1) receptors expressed by the glia population. The synthesis and release of dopamine were also observed in Müller cell cultures from mouse retina. Finally, cultured avian Müller cells display increased expression of tyrosine hydroxylase, under the influence of agents that increase cAMP levels, which results in higher levels of dopamine synthesized from tyrosine. A large proportion of glial cells in culture do express Nurr1 transcription factor, consistent with the dopaminergic characteristics displayed by these cells in culture. The results show that Müller cells, deprived of neuron influence, differentiate dopaminergic properties thought to be exclusive to neurons.

Atypical effect of dopamine in modulating the functional inhibition of NMDA receptors of cultured retina cells.

Cultured retina cells released accumulated [3H]GABA (gamma-aminobutyric acid) when stimulated by L-glutamate, N-methyl-D-aspartate (NMDA) and kainate. In the absence of Mg2+, dopamine at 200 microM (IC50 60 microM), inhibited in more than 50% the release of [3H]GABA induced by L-glutamate and NMDA, but not by kainate. This effect was not blocked by the D1-like dopamine receptor antagonist, R-(+)-7-chloro-8-hydroxy-3-methyl- -phenyl-2,3,4,5-tetrahydro- H-3-benzazepine hydrochloride (SCH 23390), neither by haloperidol nor spiroperidol (dopamine D2-like receptor antagonists). The dopamine D1-like receptor agonist R(+)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,diol hydrochloride (SKF 38393) at 50 microM, but not its enantiomer, also inhibited the release of [3H]GABA induced by NMDA, but not by kainate; an effect that was not prevented by the antagonists mentioned above. (+/-)-6-Chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepin e hydrobromide (SKF 812497) had no effect. Neither 8BrcAMP (5 mM) nor forskolin (10 microM) inhibited the release of [3H]GABA. Our results suggest that dopamine and (+)-SKF 38393 inhibit the glutamate and NMDA-evoked [3H]GABA release through mechanisms that seem not to involve known dopaminergic receptor systems.

Norepinephrine acts as D1-dopaminergic agonist in the embryonic avian retina: Late expression of β1-adrenergic receptor shifts norepinephrine specificity in the adult tissue

Dopamine is the main catecholamine found in the chick retina whereas norepinephrine is only found in trace amounts. We compared the effectiveness of dopamine and norepinephrine in promoting cyclic AMP accumulation in retinas at embryonic day 13 (E13) and from post-hatched chicken (P15). Dopamine (EC(50)=10microM) and norepinephrine (EC(50)=30microM), but not the beta(1)-adrenergic agonist isoproterenol, stimulated over seven-fold the production of cyclic AMP in E13 retina. The cyclic AMP accumulation induced by both catecholamines in embryonic tissue was entirely blocked by 2microM SCH23390, a D(1) receptor antagonist, but not by alprenolol (beta-adrenoceptor antagonist). In P15 retinas, 100microM isoproterenol stimulated five-fold the accumulation of cAMP. This effect was blocked by propanolol (10microM), but not by 2microM SCH23390. Embryonic and adult retina display beta(1) adrenergic receptor mRNA as detected by RT-PCR, but the beta(1) adrenergic receptor protein was detected only in post-hatched tissue. We conclude that norepinephrine cross-reacts with D(1) dopaminergic receptor with affinity similar to that of dopamine in the embryonic retina. In the mature retina, however, D(1) receptors become restricted to activation by dopamine. Moreover, as opposed to the embryonic tissue, norepinephrine seems to stimulate cAMP accumulation via beta(1)-like adrenergic receptors in the mature tissue.

Local Differences in GABA Release Induced by Excitatory Amino Acids During Retina Development: Selective Activation of NMDA Receptors by Aspartate in the Inner Retina

Glutamate and GABA are the major excitatory and inhibitory neurotransmitters in the CNS. In the retina, it has been shown that glutamate and aspartate and their agonists kainate and NMDA promote the release of GABA. In the chick retina, at embryonic day 14 (E14), glutamate and kainate were able to induce the release of GABA from amacrine and horizontal cells as detected by GABA-immunoreactivity. NMDA also induced GABA release restricted to amacrine cell population and its projections to the inner plexiform layer (E14 and E18). Although aspartate reduced GABA immunoreactivity, specifically in amacrine cells of E18 retinas, it was not efficient to promote GABA release from retinas at E14. As observed in differentiated retinas, dopamine inhibited the GABA release promoted by NMDA and aspartate but not by kainate. Our data show that different retinal sites respond to distinct EAAs via different receptor systems.

Transient coupling of NMDA receptor with ip3 production in cultured cells of the avian retina

The mobilization of inositol triphosphate ip3 by N-methyl D-aspartate (NMDA) and kainate, two excitatory amino acid EAA receptor agonists, was studied in cultured chick retina cells as a function of culture differentiation. Kainate (EC50 = 30 microM) stimulated from 6 to 9-fold the production of [3H]ip3 between E8C3 (embryonic day 8 plus 3 days in vitro) and E8C13. The kainate response was blocked by CNQX (100 microM) by more than 80% until stage E8C9. MK-801, however, was totally ineffective in preventing the kainate induced ip3 generation. [3H]ip3 production evoked by NMDA was increased 4-fold above basal levels at E8C3. As cultures differentiated, [3H]ip3 production promoted by NMDA decreased to 2.5-fold at E8C6 to 1.6-fold the basal levels in cultures at later stages of differentiation. The removal of Mg2+ from the incubating medium at E8C3 increased the NMDA mediated [3H]ip3 production by 80%. However, at more differentiated stages of the cultures, when cells were not responsive to NMDA, removal of Mg2+ plus the addition of 1 mM glycine did not change the pattern of the response. Although NMDA mediated ip3 production is almost absent in more differentiated cultures, NMDA is able to induce [3H]GABA release in E8C3 and E8C13 cultures with characteristics that reflect typical NMDA receptor activation: it is highly potentiated by the absence of Mg2+ and by the presence of glycine. The NMDA induced production of [3H]ip3 at E8C3 was entirely blocked by MK-801 (100 microM) and APV (100 microM) but not by CNQX.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of choline acetyltransferase by excitatory amino acids as a possible mechanism for cholinergic dysfunction in the central nervous system

Choline acetyltransferase (ChAT) activity was reduced by more than 85% in cultured retina cells after 16 h treatment with 150 µm kainate (T1/2 : 3.5 h). Glutamate, AMPA and quisqualate also inhibited the enzyme in equivalent proportion. Cell lesion measured by lactate dehydrogenase (LDH) release, 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide ‐ thiazolyl blue (MTT) reduction and microscopic observation was not detected even after 48 h with kainate. Other retina neurochemical markers were not affected by kainate and full recovery of the enzyme was achieved 9 days after kainate removal. Moreover, hemicolinium‐3 sensitive choline uptake and hemicolinium‐3 binding sites were maintained intact after kainate treatment. The immunoblot and immunohistochemical analysis of the enzyme revealed that ChAT molecules were maintained in cholinergic neurons. The use of antagonists showed that ionotropic and group 1 metabotropic receptors mediated the effect of glutamate on ChAT inhibition, in a calcium dependent manner. The quisqualate mediated ChAT inhibition and part of the kainate effect (30%) was prevented by 5 mmNG‐nitro‐l‐arginine methyl ester (l‐NAME). Veratridine (3μM) also reduced ChAT by a Ca2+ dependent, but glutamate independent mechanism and was prevented by 1μM tetrodotoxin.

GABA uptake by purified avian Müller glia cells in culture.

GABA is the main inhibitory aminoacid transmitter present in neurons and glial cells. Its uptake is carried out by specific high-affinity Na+/Cl− dependent transporters (GATs). It has been reported in the past that, in the avian retina, [3H]GABA appears to be exclusively accumulated by horizontal and amacrine cells in the inner nuclear layer, and also by ganglion cells. Purified chick Müller glia cultures were able to take up [3H]GABA in a Na+ and Cl− dependent way. Increasing GABA concentration increases GABA uptake by these cells, reaching half-maximal transport efficiency (EC50) around 0.3 mM. [3H]GABA uptake by Müller glia neuronal-free cultures was not mediated by neuronal transporters since it was not blocked by NNC-711, but was inhibited by beta-alanine, a specific glial transporter inhibitor. Chick Müller glia in culture express both GAT-1 and GAT-3 GABA transporters. Although mixed neuron-glial dense cultures released GABA upon glutamate, high K+ or veratridine stimulation, Müller glial cells did not release [3H]GABA upon treatment with these agents, suggesting that different from neurons, transporter mediated GABA release is not a common mechanism operating in these cells. The data also suggest that Müller cells take up GABA unidirectionally, which may constitute an important mechanism of inactivating GABA activity mediated by neurons.