Specificity and timing of neocortical transcriptome changes in response to BDNF gene ablation during embryogenesis or adulthood.

Brain-derived neurotrophic factor (BDNF) has been reported to be critical for the development of cortical inhibitory neurons. However, the effect of BDNF on the expression of transcripts whose protein products are involved in gamma amino butric acid (GABA) neurotransmission has not been assessed. In this study, gene expression profiling using oligonucleotide microarrays was performed in prefrontal cortical tissue from mice with inducible deletions of BDNF. Both embryonic and adulthood ablation of BDNF gave rise to many shared transcriptome changes. BDNF appeared to be required to maintain gene expression in the SST-NPY-TAC1 subclass of GABA neurons, although the absence of BDNF did not alter their general phenotype as inhibitory neurons. Furthermore, we observed expression alterations in genes encoding early-immediate genes (ARC, EGR1, EGR2, FOS, DUSP1, DUSP6) and critical cellular signaling systems (CDKN1c, CCND2, CAMK1g, RGS4). These BDNF-dependent gene expression changes may illuminate the biological basis for transcriptome changes observed in certain human brain disorders.

Molecular and functional analysis of hyperpolarization-activated pacemaker channels in the hippocampus after entorhinal cortex lesion.

Differential display of hippocampal tissue after entorhinal cortex lesion (ECL) revealed decreases in mRNA encoding the neuronal hyperpolarization-activated, cyclic nucleotide-gated channel HCN1. In situ hybridization confirmed that hippocampal transcripts of HCN1, but not HCN2/3/4, are down-regulated after ECL. Expression recovered at approximately 21 days after lesion (dal). Immunohistochemistry demonstrated a corresponding regulation of HCN1 protein expression in CA1-CA3 dendrites, hilar mossy cells and interneurons, and granule cells. Patch-clamp recordings in the early phase after lesion from mossy cells and hilar interneurons revealed an increase in the fast time constant of current activation and a profound negative shift in voltage activation of Ih. Whereas current activation recovered at 30 dal, the voltage activation remained hyperpolarized in mossy cells and hilar interneurons. Granule cells, however, were devoid of any detectable somatic Ih currents. Hence, denervation of the hippocampus decreases HCN1 and concomitantly the Ih activity in hilar neurons, and the recovery of h-current activation kinetics occurs parallel to postlesion sprouting.

Cloning and localization of the hyperpolarization-activated cyclic nucleotide-gated channel family in rat brain.

Rhythmic firing in brain and heart is mediated by pacemaker channels that are activated by hyperpolarization and regulated directly by cyclic nucleotides. Recent work has identified a new gene family that encodes such channels, which are termed hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels. In this study, we report the molecular cloning and localization by in situ hybridization of HCN1-4 in adult rat brain. The rat HCN1-4 clones show high homology to the deduced amino acid sequence of the mouse channels (>97% identity). The mRNA expression of the four channels in adult brain was evaluated in a systematic manner from the olfactory bulb to lower brain stem nuclei. Each mRNA demonstrated a unique pattern of distribution. HCN1 expression is highly enriched in cerebral cortex, hippocampus, cerebellum, and facial motor nucleus; HCN2 is highly abundant in mamillary bodies, pontine nucleus, ventral cochlear nucleus, and nucleus of the trapezoid body; HCN3 expression is most pronounced in supraoptic nucleus of hypothalamus; and HCN4 expression is most abundant in medial habenula and anterior and principal relay nuclei of the thalamus. These variations in regional specificity of HCN channels could generate important differences in neuronal pacemaker activity across brain systems.

Pharmacology of human sulphonylurea receptor SUR1 and inward rectifier K(+) channel Kir6.2 combination expressed in HEK-293 cells.

1. The pharmacological properties of K(ATP) channels generated by stable co-expression of the sulphonylurea receptor SUR1 and the inwardly rectifying K(+) channel Kir6.2 were characterized in HEK-293 cells. 2. [(3)H]-Glyburide (glibenclamide) bound to transfected cells with a B(max) value of 18.5 pmol mg(-1) protein and with a K(D) value of 0.7 nM. Specific binding was displaced by a series of sulphonylurea analogues with rank order potencies consistent with those observed in pancreatic RINm5F insulinoma and in the brain. 3. Functional activity of K(ATP) channels was assessed by whole cell patch clamp, cation efflux and membrane potential measurements. Whole cell currents were detected in transfected cells upon depletion of internal ATP or by exposure to 500 microM diazoxide. The currents showed weak inward rectification and were sensitive to inhibition by glyburide (IC(50)=0.92 nM). 4. Metabolic inhibition by 2-deoxyglucose and oligomycin treatment triggered (86)Rb(+) efflux from transfected cells that was sensitive to inhibition by glyburide (IC(50)=3.6 nM). 5. Diazoxide, but not levcromakalim, evoked concentration-dependen decreases in DiBAC(4)(3) fluorescence responses with an EC(50) value of 14.1 microM which were attenuated by the addition of glyburide. Diazoxide-evoked responses were inhibited by various sulphonylurea analogues with rank order potencies that correlated well with their binding affinities. 6. In summary, results from ligand binding and functional assays demonstrate that the pharmacological properties of SUR1 and Kir6.2 channels co-expressed in HEK-293 cells resemble those typical of native K(ATP) channels described in pancreatic and neuronal tissues.

Withdrawal from repeated amphetamine administration reduces NMDAR1 expression in the rat substantia nigra, nucleus accumbens and medial prefrontal cortex.

Glutamate plays a critical role in neuroadaptations induced by drugs of abuse. This study determined whether expression of the NMDAR1 subunit of the NMDA receptor is altered by repeated amphetamine administration. We quantified NMDAR1 mRNA (using in situ hybridization with 35S-labelled oligonucleotide probes) and immunolabelling (using immunocytochemistry with 35S-labelled secondary antibodies) in rat ventral midbrain, nucleus accumbens and prefrontal cortex after 3 or 14 days of withdrawal from five daily injections of saline or amphetamine sulphate (5 mg/kg/day). No changes in NMDAR1 expression were observed after 3 days of withdrawal, whereas significant decreases were observed in all regions after 14 days. NMDAR1 mRNA levels in midbrain were too low for reliable quantification, but immunolabelling was decreased significantly in intermediate and caudal portions of the substantia nigra. This may indicate a reduction in excitatory drive to substantia nigra dopaminergic neurons. In the nucleus accumbens, there were significant decreases in NMDAR1 mRNA levels (74.8 +/- 7. 7% of control, P < 0.05) and immunolabelling (76.7 +/- 4.4%, P < 0. 05). This may account for previously-reported decreases in the electrophysiological responsiveness of nucleus accumbens neurons to NMDA after chronic amphetamine treatment, and contribute to dysregulation of goal-directed behaviour. In prefrontal cortex, there was a significant decrease in NMDAR1 mRNA levels (76.1 +/- 7. 1%, P < 0.05) and a trend towards decreased immunolabelling (89.5 +/- 7.0%). This may indicate decreased neuronal excitability within prefrontal cortex. A resultant decrease in activity of excitatory prefrontal cortical projections to nucleus accumbens or midbrain could synergize with local decreases in NMDAR1 to further reduce neuronal excitability in these latter regions.

Pharmacological and molecular characterization of ATP-sensitive K+ channels in the TE671 human medulloblastoma cell line.

ATP-sensitive K+ (K(ATP)) channels in the human medulloblastoma TE671 cell line were characterized by membrane potential assays utilizing a potentiometric fluorescent probe, bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC4(3)), and by mRNA analysis. Membrane potential assays showed concentration-dependent and glyburide-sensitive changes in fluorescence upon addition of (-)-cromakalim, pinacidil, diazoxide and P1075. The rank order of potency for these openers was P1075 > (-)-cromakalim approximately = pinacidil > diazoxide. Additionally, glyburide and glipizide inhibited P1075-evoked responses in TE671 cells with half-maximal inhibitory concentrations of 0.22 and 14 microM, respectively. The rank order potencies of both openers and inhibitors were similar to those observed in the rat smooth muscle A-10 cell line. In contrast, in the rat pancreatic insulinoma RIN-m5F cell line, only diazoxide was effective as an opener. Reverse transcription-polymerase chain reaction (RT-PCR) studies detected sulfonylurea receptors SUR2B and SUR1 mRNA in TE671 cells whereas only SUR2B and SUR1 mRNA were, respectively, detected in A-10 and RIN-m5F cells. The inward rectifier Kir6.2 mRNA was detected in all three cell types whereas Kir6.1 was detected only in A-10 cells. Collectively, the molecular and pharmacologic studies suggest that K(ATP) channels endogenously expressed in TE671 medulloblastoma resemble those present in the smooth muscle.

Gain of function mutation of the alpha7 nicotinic receptor: distinct pharmacology of the human alpha7V274T variant.

In the human alpha7 nicotinic receptor, valine-274 in the pore-lining transmembrane-2 region was mutated to threonine to produce the variant human alpha7V274T, which was evaluated electrophysiologically following expression in Xenopus laevis oocytes. Inward current rectification was strong in human alpha7V274T as in the human alpha7 wild type nicotinic receptor. However, human alpha7V274T was 100-fold more sensitive to the nicotinic receptor agonists acetylcholine, (-)-nicotine and 1,1-dimethyl-4-phenylpiperazinium. Choline also activated human alpha7V274T (EC50 = 12 microM) and was 82-fold more potent than at human alpha7 wild type nicotinic receptor. (-)-Cotinine, (2,4)-dimethoxybenzylidene anabaseine (GTS-21) and 2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine (ABT-089), weak partial agonists at human alpha7 wild type, were much stronger agonists at human alpha7V274T with EC50 values of 70 microM, 4 microM and 28 microM and fractional activation values of 93%, 96% and 40%, respectively. However, (-)-lobeline, a human alpha7 wild type nicotinic receptor antagonist, and dihydro-beta-erythroidine, which activates chick mutagenized alpha7 nicotinic receptors, had only weak agonist-like activity at human alpha7V274T (< or = 4% of the maximal acetylcholine response). Methyllycaconitine, mecamylamine, d-tubocurarine and dihydro-beta-erythroidine retained antagonist activity and, indeed, appeared to be at least as potent at human alpha7V274T as at human alpha7 wild type. These results support and extend the concept that human nicotinic receptor pharmacology can be profoundly altered by single amino acid changes in the pore-lining segment.

Activation of the recombinant human alpha 7 nicotinic acetylcholine receptor significantly raises intracellular free calcium.

The alpha 7 nicotinic acetylcholine receptor (nAChR) subtype, unlike other neuronal nicotinic receptors, exhibits a relatively high permeability to Ca++ ions. Although Ca++ entry through this receptor subtype has been implicated in various Ca(++)-dependent processes in the central nervous system, little is known about how this receptor modulates mammalian intracellular Ca++ dynamics. Intracellular Ca++ responses evoked by activation of the human alpha 7 nAChRs stably expressed in HEK-293 (human embryonic kidney) cells were studied. Inward current and intracellular Ca++ transients were recorded simultaneously in response to a fast drug application system. Current recordings under whole-cell voltage-clamp and fast ratiometric intracellular Ca++ imaging acquisition were synchronized to drug pulses. The mean peak [Ca++]i observed with 100 microM (-)-nicotine was 356 +/- 48 nM (n = 8). The magnitude of the intracellular Ca++ elevation corresponds to a 20% fractional current carried by Ca++ ions. The EC50 of the intracellular Ca++ responses for (-)-nicotine, (+/-)-epibatidine, 1,1 dimethyl-4-phenyl-piperazinium and acetylcholine were 51, 3.5, 75 and 108 microM, respectively. These EC50 values strongly correlate with those recorded for the cationic inward current through alpha 7 nAChR. alpha-Bungarotoxin, methyllcaconitine or extracellular Ca++ chelation ablated (-)-nicotine-evoked increase in intracellular Ca++ concentration. This study provides evidence that cation influx through the human alpha 7 nAChR is sufficient to mediate a significant, transient, rise in intracellular Ca++ concentration.

Stable expression, pharmacologic properties and regulation of the human neuronal nicotinic acetylcholine alpha 4 beta 2 receptor.

(-)-Nicotine, the prototypical agonist for neuronal nicotinic acetylcholine receptors (nAChR) has been shown to bind with high affinity to the rodent and avian alpha 4 beta 2 nAChR subtype. This subtype may represent a primary molecular target for some of the beneficial central nervous system effects i.e., cognitive enhancement, anxiolysis, analgesia, neuroprotection, of (-)-nicotine and related ligands. However, a detailed study of the human alpha 4 beta 2 subunit combination has not yet been reported. In this study, we stably coexpressed the human neuronal alpha 4 and beta 2 nAChR subunits in human embryonic kidney (HEK) 293 cells and studied its pharmacological and regulatory properties. [3H]Cytisine bound to stably transfected cells with high affinity (KD value, 0.2 +/- 0.04 nM) and with a Bmax value of 1359 +/- 91 fmol/mg protein. A good correlation (r = 0.98) was observed between binding affinities in transfected cells and in native neuronal preparations for a series of nAChR ligands. 86Rb+ efflux studies showed that stably transfected cells express functional ion channels that are sensitive to blockade by dihydro-beta-erythroidine. (+/-)-Epibatidine, (-)-nicotine, 1,1-dimethyl-4-phenylpiperazinium, (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole (ABT-418), acetylcholine and (-)-cytisine stimulated 86Rb+ efflux with EC50 values of 0.02, 3.9, 2.5, 10, 44 and 38 microM, respectively. Treatment of transfected cells with (-)-nicotine for 7 days led to a significant increase in the density of [3H](-)-cytisine binding sites (EC50 = 0.56 microM) and a significant enhancement in the sensitivity of ACh. Specific binding or (-)-nicotine-evoked cation efflux was not detected in untransfected cells. Analysis of total cellular RNA from transfected, but not untransfected cells, showed the expected fragment sizes corresponding to the human alpha 4 and beta 2 subunit mRNA. These results demonstrate that stable expression of the human alpha 4 beta 2 nAChR subunit combination can give rise to functional ion channels that bind [3H](-)-cytisine with high affinity, exhibit homologous regulation and evoke agonist-induced cation flux with pharmacological properties consistent with native neuronal alpha 4 beta 2 nAChR.

Cloning and transient expression of genes encoding the human alpha 4 and beta 2 neuronal nicotinic acetylcholine receptor (nAChR) subunits.

Partial cDNA clones generated by RT-PCR were used as probes to clone the cDNAs encoding the human alpha 4 and beta 2 neuronal nicotinic acetylcholine receptor (nAChR) subunits. The 2.1-kb alpha 4 cDNA shows 84 and 76% identity to the rat and chicken cDNA sequences, respectively. The deduced amino-acid sequence shares 89 and 84% similarity, respectively, with the corresponding rat and chicken proteins, with most of the divergence occurring in the cytoplasmic domain. The 1721-nucleotide beta 2 sequence was identical to the human beta 2 sequence previously reported. Transfection of the alpha 4 and beta 2 clones into HEK293 cells resulted in the formation of binding sites that display high affinity towards [3H] cytisine, a characteristic of the alpha 4 beta 2 subtype produced in vivo.

In vivo transcripts of the S-layer-encoding structural gene of the archaeon Methanococcus voltae.

The 5' region of the S-layer-encoding structural gene (sla) of Methanococcus voltae was sequenced. The sequence information was then used to identify the in vivo transcription products of the gene. We observed three transcripts, and upstream from each transcription start point was a region with similarity to the Box A consensus sequence observed in archaeal promoters. In two of the three cases, two Box A sequences were present in tandem. This arrangement may play a role in the high level of gene expression expected for the sla gene. Presumptive archaeal Box B signatures were also identified.

Nicotine effects on the regulation of amyloid precursor protein splicing, neurotrophin and glucose transporter RNA levels in aged rats.

It has been reported that an inverse relationship exists between nicotine intake and the incidence of Alzheimer's Disease (AD). Although nicotine has been reported to induce c-fos, in the present study it was shown that this induction does not alter the accumulation of a number of transcripts associated with AD. Altered splicing patterns of Amyloid Precursor Protein (APP) and changes in neurotrophin and glucose transporter expression have been implicated in AD and behavioral deficits in rats. The effects of subacute administration of nicotine (12 mg/ml at 2.3 microliters/hr for 14 days) on the abundance levels of APP, glucose transporter (GLUT) and neurotrophin transcripts were determined by rtPCR in the hippocampus, cortex, and striatum of aged (22-24 months) male Wistar rats. No significant differences between saline and nicotine infused rats were detected for APP abundance levels or ratio of the various isoforms. However, both groups had a higher level of APP transcripts containing the Kunitz Protease Inhibitor (KPI) domain in the hippocampus than in either the cortex or striatum. The mean percentages of APP 695 for the two groups were 75% in the hippocampus and 82 and 81% in the cortex and striatum, respectively (P < 0.01). No changes in the abundance of GLUT1, GLUT3, nerve growth factor (NGF) or brain derived neurotrophic factor (BDNF) transcripts were detected. However, since both APP and GLUT1 are thought to be regulated post-transcriptionally, the present results do not rule out a change at the protein level. Further work will be required to determine whether nicotine can influence the expression of these proteins which affect neuronal function.

Similarities between beta amyloid peptides 1-40 and 40-1: effects on aggregation, toxicity in vitro, and injection in young and aged rats.

Peptides corresponding to the first 40 amino acids of beta amyloid peptide (beta 1-40) and the reverse sequence (beta 40-1) were synthesized, purified, and compared for their ability to aggregate and cause toxicity in vitro to human neuroblastoma cells (SH-SY5Y), as well as for effects following injection into young or aged rats. Aggregation of both peptides produced similar sedimentation velocity profiles and resulted in significant toxicity in vitro with no observable differences between beta 1-40 and beta 40-1. In addition, when injected into the cortex of young rats, beta 1-40 was more toxic than beta 40-1 although both resulted in significant lesions. However, in aged rats the two peptides resulted in lesions of similar size. Alz 50 staining and abnormal neurites were associated with both beta 1-40 and beta 40-1 lesions; however, no evidence of plaques or tangles was found in either age group. While both peptides were toxic in vitro, only beta 1-40 elicited Alz 50 staining of SH-SY5Y cells. Electron microscopic examination of beta 1-40 and beta 40-1 aggregates showed that beta 1-40 formed fibrillar structures whereas beta 40-1 resulted in amorphous particles. Thus, although both peptides were toxic to cultured cells and aged rats, the toxicities may have resulted from different mechanisms.

Altered levels and splicing of the amyloid precursor protein in the adult rat hippocampus after treatment with DMSO or retinoic acid.

Alzheimer's disease and cognitive impairment in rats has been associated with an increase in the percentage of amyloid precursor protein (APP) containing the KPI domain. It has recently been reported that retinoic acid (RA) is capable of increasing the levels and altering the splicing ratio of APP in cultured SH-SY5Y cells. The effects of peripherally administered RA (64 or 640 micrograms/kg; i.p.; q.d.) on the abundance of APP, the ratio of the three major isoforms, and the relative abundance of nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) were determined by rtPCR in the hippocampus of aged rats. Corresponding changes in choline acetyltransferase (ChAT) activity were also measured. Vehicle (DMSO) treated rats exhibited a 2 x (P < 0.01) increase in total APP and an 8 x (P < 0.001) decrease in the cyclophilin transcript. In addition, DMSO increased the percentage of APP 695 from 89% in saline treated rats to 94%. Treatment of RA in DMSO decreased the accumulation of total APP relative to cyclophilin at both the low (6.4 x; P < 0.01) and high (8 x; P < 0.05) dosages when compared to DMSO treated rats. Furthermore, the level of APP-695 decreased to 82% with low dosage of RA and 75% at high dosage of the total APP transcripts. No significant change in either NGF, NT-3, or BDNF transcripts were observed following low or high dosage RA administration relative to cyclophilin RNA nor was a change in ChAT activity detected at either of the dosages tested.(ABSTRACT TRUNCATED AT 250 WORDS)