Journal of Neurochemistry

Abstract: Dopamine synthesis rate and cyclic AMP concentration were measured in synaptosomes prepared from rat striatum. Dopamine synthesis rate was decreased by the addition of either adenosine deaminase or 8‐phenyltheophylline, an adenosine receptor blocker, and was increased by the addition of 2‐chloroadenosine. The addition of L‐glutamate in the absence of adenosine deaminase decreased both dopamine synthesis rate and cyclic AMP concentration; in the presence of adenosine deaminase, glutamate had no effect on basal dopamine synthesis, but enhanced K⁺‐stimulated synthesis. Both these effects of glutamate were abolished in Ca²⁺‐free medium or in the presence of 2‐amino‐5‐phosphonovalerate, an N′‐methyl‐D‐aspartate (NMDA) receptor blocker. In Mg²⁺‐free medium with adenosine deaminase, glutamate enhanced both basal and K⁺‐stimulated synthesis. These results suggest that dopaminergic terminals have A₂. adenosine receptors, whose activation can stimulate dopamine synthesis by a cyclic AMP‐dependent mechanism, and NMDA receptors, which modulate dopamine synthesis by a Ca²⁺‐dependent mechanism.

To determine the role of brain‐derived neurotrophic factor (BDNF) in the enhancement of hippocampal neurogenesis resulting from dietary restriction (DR), heterozygous BDNF knockout (BDNF +/–) mice and wild‐type mice were maintained for 3 months on DR or ad libitum (AL) diets. Mice were then injected with bromodeoxyuridine (BrdU) and killed either 1 day or 4 weeks later. Levels of BDNF protein in neurons throughout the hippocampus were decreased in BDNF +/– mice, but were increased by DR in wild‐type mice and to a lesser amount in BDNF +/– mice. One day after BrdU injection the number of BrdU‐labeled cells in the dentate gyrus of the hippocampus was significantly decreased in BDNF +/– mice maintained on the AL diet, suggesting that BDNF signaling is important for proliferation of neural stem cells. DR had no effect on the proliferation of neural stem cells in wild‐type or BDNF +/– mice. Four weeks after BrdU injection, numbers of surviving labeled cells were decreased in BDNF +/– mice maintained on either AL or DR diets. DR significantly improved survival of newly generated cells in wild‐type mice, and also improved their survival in BDNF +/– mice, albeit to a lesser extent. The majority of BrdU‐labeled cells in the dentate gyrus exhibited a neuronal phenotype at the 4‐week time point. The reduced neurogenesis in BDNF +/– mice was associated with a significant reduction in the volume of the dentate gyrus. These findings suggest that BDNF plays an important role in the regulation of the basal level of neurogenesis in dentate gyrus of adult mice, and that by promoting the survival of newly generated neurons BDNF contributes to the enhancement of neurogenesis induced by DR.

The existence on glutamatergic nerve endings of nicotinic acetylcholine receptors (nAChRs) mediating enhancement of glutamate release has often been suggested but not demonstrated directly. Here, we study the effects of nAChR agonists on [³H]‐d‐aspartate ([³H]‐d‐ASP) release from synaptosomes superfused in conditions known to prevent indirect effects. Nicotinic receptor agonists, while unable to modify the basal [³H]‐d‐ASP release from human neocortex or rat striatal synaptosomes, enhanced the Ca²⁺‐dependent exocytotic release evoked by K⁺ (12 mm) depolarization. Their rank order of potency were anatoxin‐a > epibatidine > nicotine > ACh (+ atropine). The anatoxin‐a effect, both in human and rat synaptosomes, was antagonized by mecamylamine, α‐bungarotoxin or methyllycaconitine. The basal release of [³H]ACh from human cortical synaptosomes was increased by (–)‐nicotine (EC₅₀ = 1.16 ± 0.33 µm) or by ACh plus atropine (EC₅₀ = 2.0 ± 0.04 µm). The effect of ACh plus atropine was␣insensitive to α‐bungarotoxin, methyllycaconitine or α‐conotoxin MII, whereas it was totally antagonized by mecamylamine or dihydro‐β‐erythroidine. To conclude, glutamatergic axon terminals in human neocortex and in rat striatum possess α7* nicotinic heteroreceptors mediating enhancement of glutamate release. Release‐enhancing cholinergic autoreceptors in human neocortex are nAChRs with a pharmacological profile compatible with the α4β2 subunit combination.

Alterations of striatal synaptic transmission have been associated with several motor disorders involving the basal ganglia, such as Parkinson’s disease. For this reason, we investigated the role of group‐III metabotropic glutamate (mGlu) receptors in regulating synaptic transmission in the striatum by electrophysiological recordings and by using our novel orthosteric agonist (3S)‐3‐[(3‐amino‐3‐carboxypropyl(hydroxy)phosphinyl)‐hydroxymethyl]‐5‐nitrothiophene (LSP1‐3081) and l‐2‐amino‐4‐phosphonobutanoate (L‐AP4). Here, we show that both drugs dose‐dependently reduced glutamate‐ and GABA‐mediated post‐synaptic potentials, and increased the paired‐pulse ratio. Moreover, they decreased the frequency, but not the amplitude, of glutamate and GABA spontaneous and miniature post‐synaptic currents. Their inhibitory effect was abolished by (RS)‐α‐cyclopropyl‐4‐phosphonophenylglycine and was lost in slices from mGlu4 knock‐out mice. Furthermore, (S)‐3,4‐dicarboxyphenylglycine did not affect glutamate and GABA transmission. Finally, intrastriatal LSP1‐3081 or L‐AP4 injection improved akinesia measured by the cylinder test. These results demonstrate that mGlu4 receptor selectively modulates striatal glutamate and GABA synaptic transmission, suggesting that it could represent an interesting target for selective pharmacological intervention in movement disorders involving basal ganglia circuitry.

Neuronal nicotinic acetylcholine receptors (nAChR) can regulate several neuronal processes through Ca²⁺‐dependent mechanisms. The versatility of nAChR‐mediated responses presumably reflects the spatial and temporal characteristics of local changes in intracellular Ca²⁺ arising from a variety of sources. The aim of this study was to analyse the components of nicotine‐evoked Ca²⁺ signals in SH‐SY5Y cells, by monitoring fluorescence changes in cells loaded with fluo‐3 AM. Nicotine (30 µm) generated a rapid elevation in cytoplasmic Ca²⁺ that was partially and additively inhibited (40%) by α7 and α3β2* nAChR subtype selective antagonists; α3β4* nAChR probably account for the remaining response (60%). A substantial blockade (80%) by CdCl₂ (100 µm) indicates that voltage‐operated Ca²⁺ channels (VOCC) mediate most of the nicotine‐evoked response, although the α7 selective antagonist α‐bungarotoxin (40 nm) further decreased the CdCl₂‐ resistant component. The elevation of intracellular Ca²⁺ levels provoked by nicotine was sustained for at least 10 min and required the persistent activation of nAChR throughout the response. Intracellular Ca²⁺ stores were implicated in both the initial and sustained nicotine‐evoked Ca²⁺ responses, by the blockade observed after ryanodine (30 µm) and the inositoltriphosphate (IP₃)‐receptor antagonist, xestospongin‐c (10 µm). Thus, nAChR subtypes are differentially coupled to specific sources of Ca²⁺: activation of nAChR induces a sustained elevation of intracellular Ca²⁺ levels which is highly dependent on the activation of VOCC, and also involves Ca²⁺ release from ryanodine and IP₃‐dependent intracellular stores. Moreover, the α7, but not α3β2* nAChR, are responsible for a fraction of the VOCC‐independent nicotine‐evoked Ca²⁺ increase that appears to be functionally coupled to ryanodine sensitive Ca²⁺ stores.

Abstract: We have recently reported evidence that a very high affinity interaction between the β‐amyloid peptide Aβ_1‐42 and the α7 nicotinic acetylcholine receptor (α7nAChR) may be a precipitating event in the formation of amyloid plaques in Alzheimer's disease. In the present study, the kinetics for the binding of Aβ_1‐42 to α7nAChR and α4β2nAChR were determined using the subtype‐selective nicotinic receptor ligands [³H]methyllycaconitine and [³H]cytisine. Synaptic membranes prepared from rat and guinea pig cerebral cortex and hippocampus were used as the source of receptors. Aβ_1‐42 bound to the α7nAChR with exceptionally high affinity, as indicated by K_i values of 4.1 and 5.0 pM for rat and guinea pig receptors, respectively. When compared with the α7nAChR, the affinity of Aβ_1‐42 for the α4β2nAChR was ∼5,000‐fold lower, as indicated by corresponding K_i values of 30 and 23nM. The results of this study support the concept that an exceptionally high affinity interaction between Aβ_1‐42 and α7nAChR could serve as a precipitating factor in the formation of amyloid plaques and thereby contribute to the selective degeneration of cholinergic neurons that originate in the basal forebrain and project to the cortex and hippocampus.

Abstract: When primary cultures of cerebellar granule neurons are grown in a physiological concentration of KCl (5 mM) they undergo apoptosis, which can be prevented by growing the cells in the presence of N‐methyl‐d‐aspartate (NMDA). We now show that ethanol inhibits this trophic effect of NMDA, i.e., promotes apoptosis, and also inhibits the NMDA‐induced increase in intracellular Ca²⁺ concentration in cells grown in 5 mM KCl. Both effects of ethanol show a similar concentration dependence and are reversed by a high concentration of glycine, the co‐agonist at the NMDA receptor. The data suggest that the effect of ethanol on apoptosis is mediated, at least in part, by inhibition of NMDA receptor function. This effect of ethanol to increase apoptosis could contribute to the previously described in vivo sensitivity of the developing cerebellum to ethanol‐induced damage.

Abstract: Neural cell adhesion molecules (NCAMs) play critical roles during development of the nervous system. The aim of this study is to investigate the possible effect of ethanol exposure on the pattern of expression and sialylation of NCAM isoforms during postnatal rat brain development because alterations in NCAM content and distribution have been associated with defects in cell migration, synapse formation, and memory consolidation, and deficits in these processes have been observed after in utero alcohol exposure. The expression of NCAM isoforms in the developing cerebral cortex of pups from control and alcohol‐fed mothers was assessed by western blotting, ribonuclease protection assay, and immunocytochemistry. The highly sialylated form of NCAM [polysialic acid (PSA)‐NCAM] is mainly expressed during the neonatal period and then is down‐regulated in parallel with the appearance of NCAM 180 and NCAM 140. Ethanol exposure increases PSA‐NCAM levels during the neonatal period, delays the loss of PSA‐NCAM, decreases the amount of NCAM 180 and NCAM 140 isoforms, and reduces sialyltransferase activity during postnatal brain development. Neuraminidase treatment of ethanol‐exposed neonatal brains leads to more intense band degradation products, suggesting a higher content of NCAM polypeptides carrying PSA in these samples. However, NCAM mRNA levels are not changed by ethanol. Immunocytochemical analysis demonstrates that ethanol triggers an increase in PSA‐NCAM immunolabeling in the cytoplasm of astroglial cells, accompanied by a decrease in immunogold particles over the plasma membrane. These findings indicate that ethanol exposure during brain development alters the pattern of NCAM expression and suggest that modification of NCAM could affect neuronal‐glial interactions that might contribute to the brain defects observed after in utero alcohol exposure.

Abstract: The expression and activity of factors influencing early neuronal development are altered by ethanol. Such factors include growth factors, for example, platelet‐derived growth factor and basic fibroblast growth factor (for cell proliferation), and cell adhesion molecules (for neuronal migration). One agent, transforming growth factor β1 (TGFβ1), may affect both events. We tested the hypothesis that ethanol alters myriad TGFβ1‐mediated activities [i.e., cell proliferation and neural cell adhesion molecule (N‐CAM) expression] using B104 neuroblastoma cells. TGFβ1 inhibited the proliferation of B104 cells as evidenced by decreases in cell number and [³H]thymidine ([³H]dT) incorporation. TGFβ1 induced sustained activation of extracellular signal‐regulated kinases (ERKs), which are part of the family of mitogen‐activated protein kinases (MAPKs). Treatment with PD98059 (a MAPK kinase blocker) abolished TGFβ1‐regulated inhibition of [³H]dT incorporation. TGFβ1‐mediated growth inhibition was potentiated by ethanol exposure. Ethanol also produced prolonged activation of ERK, an effect that was partially eliminated by treatment with PD98059. On the other hand, TGFβ1 up‐regulated N‐CAM expression, and this up‐regulation was not affected by treatment with PD98059. Ethanol inhibited the TGFβ1‐induced up‐regulation of N‐CAM expression in a concentration‐dependent manner. Thus, TGFβ1 affects ERK‐dependent cell proliferation and ERK‐independent N‐CAM expression in B104 cells. Both activities are sensitive to ethanol and may underlie the ethanol‐induced alterations in the proliferation and migration of CNS neurons.

Abstract: Exposure to ethanol during fetal development reduces the astroglial‐specific marker glial fibrillary acidic protein (GFAP) and its mRNA levels in brains of fetal rats and in radial glia in primary culture, affecting the proliferation and differentiation of astrocytes. The objectives of this study were to evaluate the possible effect of ethanol on GFAP mRNA levels in astrocytes and to investigate the molecular mechanism(s) involved in ethanol‐induced changes in GFAP expression by analyzing the GFAP transcription rate, GFAP mRNA stability, and GFAP DNA methylation. We show here that prenatal exposure to ethanol reduces significantly GFAP immunoreactivity and its mRNA levels in both astrocytes in primary culture and brains of pups from alcohol‐fed mothers. Runoff experiments from nuclei of astrocytes indicate that ethanol exposure decreases GFAP transcription rate significantly and reduces GFAP mRNA stability slightly. DNA methylation analysis indicates that prenatal ethanol exposure induces a hypermethylated state of the GFAP DNA in fetal brains. Methylation‐mediated repression of GFAP transcription could be a mechanism involved in ethanol‐induced reduction of GFAP expression. Ethanol‐induced alterations in GFAP expression and astroglial development may underlie the CNS dysfunctions observed after prenatal alcohol exposure.