BDNF enhances neuronal growth and synaptic activity in hippocampal cell cultures.

Brain-derived neurotrophic factor (BDNF) (20 ng/ml) significantly enhanced the growth of the somata of GABA-immunoreactive neurones in primary cultures of hippocampal neurones from postnatal rats after only 24h. Whole-cell patch-clamp experiments showed an increase in spontaneous synaptic activity between neurones with time in culture. After 10 days in culture, 90% of neurones sampled in control cultures showed spontaneous synaptic activity, whereas in cultures treated with BDNF, 100% of neurones had synaptic inputs after only 6 days. This difference in spontaneous activity was not due to the lack of synaptic inputs as KCl-induced synaptic activity was equally effective in BDNF and control cultures. These experiments demonstrate the rapid rate at which BDNF can promote neuronal growth and also show that BDNF can promote long term synaptic activity.

Electrophysiological consequences of ligand binding to the desensitized 5-HT3 receptor in mammalian NG108-15 cells.

1. Using the whole-cell variation of the patch-clamp technique to record from mammalian NG108-15 cells, we have studied the ligand-gated ion channel current activated by a high concentration (100 microM) of local pressure-applied 5-hydroxytryptamine (5-HT). The response was induced at intervals of at least 90-120 s, which allowed the receptor to fully recover between activations. 2. The rapid inward current induced by pressure-applied 5-HT was reproducibly inhibited by the superfusion of low concentrations of 5-HT which evoked little or no detectable inward current alone (0.01-0.3 microM). This inhibitory effect was most likely to be due to a direct action on the 5-HT3 receptor as it could be recorded using intracellular solutions with or without adenosine triphosphate (ATP) and guanosine triphosphate (GTP). 3. The maximum inhibitory effect of a given concentration of 5-HT was not dependent on its superfusion time but on the number of activations of the receptor by pressure-applied 5-HT. This activation dependence was clearly evident, since the first inward current in the presence of 0.1 microM 5-HT was often unaffected in amplitude. 4. The inhibitory effect of 5-HT was evident at holding potentials of +60 and -60 mV; with the calcium chelator BAPTA in the recording pipette and with the nominal removal of extracellular calcium and magnesium ions. 5. The inhibitory effect was concentration dependent, with 50% inhibition of the inward current amplitude occurring at approximately 50 nM 5-HT. The slope factor of the inhibition curve was 1.3. The effect was mimicked by two other 5-HT3 receptor agonists, 2-methyl-5-HT and m-chlorophenylbiguanide (mCPBG) which gave 50% inhibition at approximately 600 nM and approximately 20 nM, respectively. These values are similar to the affinity values for these ligands determined in radioligand binding assays. 6. The 5-HT3 receptor "antagonists' (+)-tubocurarine and quipazine (both at 3 nM) reduced the inward current amplitude by approximately 50%. The rate of onset of the inhibitory effect of bath-applied 5-HT was slowed in the presence of (+)-tubocurarine but not in the presence of quipazine. This difference might be explained by the agonist properties seen only with quipazine. 7. The inhibition of the 5-HT3 receptor mediated inward current by low concentrations of bath-applied 5-HT3 receptor agonists is compatible with the cyclic model of receptor activation and desensitization. We conclude that we have been studying the high-affinity binding of agonists to the desensitized form of the 5-HT3 receptor.

Characterization of the 5-hydroxytryptamine2A receptor-activated cascade in rat C6 glioma cells.

We have investigated the identity and intracellular cascade of responses resulting from activation of the endogenous 5-hydroxytryptamine receptor in the C6 rat glioma cell line. Sequence analysis of reverse transcription-polymerase chain reaction products derived from C6 glioma cell messenger RNA revealed complete homology with a portion of the rat 5-hydroxytryptamine2A receptor. The binding of [3H]ketanserin to cell membranes demonstrated a significant correlation with the 5-hydroxytryptamine2A receptor in rat frontal cortex. On intact cells, 5-hydroxytryptamine stimulated a concentration-dependent increase in phosphatidyl inositide turnover and intracellular [Ca2+] mediated by 5-hydroxytryptamine2A receptors. In whole-cell patch-clamp recordings, 5-hydroxytryptamine induced an outward current mediated predominantly by K+ ions (reversal potential = -80 mV). Using caged molecules containing Ca2+ or inositol 1,4,5-trisphosphate in the patch electrode solution, we found that rapid photolytic release of Ca2+ and particularly inositol 1,4,5-trisphosphate within the cytosol induced an outward current with characteristics similar to those seen after application of 5-hydroxytryptamine. Comparison between differentiated and undifferentiated cells revealed significantly higher receptor density and maximal phosphoinositide response to 5-hydroxytryptamine in undifferentiated cells but the associated rise in [Ca2+]i and activation of an outward current was observed more frequently in differentiated cells. Prolonged exposure of the cells to 5-hydroxytryptamine led to a decrease in all responses and to the down-regulation of receptor number. We conclude that the rat C6 glioma cell expresses a 5-hydroxytryptamine2A receptor identical to that found in rat brain and that stimulation of the receptor in C6 cells leads to the activation of Ca2+ activated K+ channels via phosphoinositide hydrolysis and subsequent rise in cytosolic Ca2+ ion concentration. However, the contrasting effects of differentiation on receptor number and phosphoinositide response to 5-hydroxytryptamine compared to Ca2+ release and conductance change indicate that a complex relationship exists between the component parts of the receptor-activated cascade.

5-HT2A receptor-mediated outward current in C6 glioma cells is mimicked by intracellular IP3 release.

The C6 glioma cell line possesses 5-HT2A receptors that have been shown to increase intracellular calcium levels. We have studied the electrophysiological response of these cells to 5-HT using the whole-cell recording method. Under voltage-clamp, 5-hydroxytryptamine (5-HT) produced an outward current in these cells which was inhibited by extracellularly applied ketanserin and spiperone and by EGTA (10 mM) in the recording electrode. The 5-HT induced response could be mimicked by intracellular photolytic release of inositol (1,4,5) trisphosphate (IP3) from caged molecules. The reversal potentials for the IP3- and 5-HT-induced responses were closely matched. The data indicates that the outward current is likely to be mediated by 5-HT2A receptors stimulating IP3 production which increases intracellular calcium leading to the opening of calcium-activated potassium channels.

Changes in adenosine sensitivity in the hippocampus of rats with streptozotocin-induced diabetes.

1. Hippocampal slices have been used to assess the sensitivity of the CNS to adenosine and gamma-aminobutyric acid (GABA) in diabetes. The effects of adenosine, 2-chloroadenosine, GABA, muscimol and baclofen were studied on orthodromic synaptic potentials recorded in the CA1 region of slices taken from normal rats or animals made diabetic by the injection of streptozotocin. 2. In diabetic animals the sensitivity to adenosine was increased 4 fold compared with normal rats. The potency of 2-chloroadenosine was unchanged. 3. The nucleoside transport inhibitor, hydroxynitrobenzylthioinosine (HNBTI), increased the potency of adenosine in slices from normal rats but not in slices from diabetic rats. 4. No change was observed in the potency of GABA or muscimol, although a small but significant decrease was detected in the EC50 value for baclofen. 5. Treatment of diabetic animals with insulin restored the potency of adenosine to control levels. 6. It is concluded that the diabetic state is accompanied by substantial changes of adenosine sensitivity due to the loss of nucleoside uptake processes. Secondary neurochemical changes following from this in human diabetic patients may contribute to the reported behavioural changes.

Depression of purine induced inhibition during NMDA receptor mediated activation of hippocampal pyramidal cells--an iontophoretic study.

Single pyramidal cells in the rat hippocampal slice preparation were stimulated by iontophoretic application of excitatory amino acids and acetylcholine. The purine adenosine 5'-monophosphate (AMP), applied iontophoretically, readily depressed acetylcholine stimulated cell firing, was less effective on quisqualic acid stimulated cells and virtually ineffective during stimulation by N-methyl-D,L-aspartate (NMA). Inhibition could be restored if the AMP ejection current was increased 3-fold. In contrast, the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) exerted a comparable level of inhibition under all 3 neuroexcitants. These data support previously published results which suggest that purine mediated inhibition may be reduced during NMDA receptor channel activation. This may have important implications for the action of adenosine during seizures and ischaemic events as well as neuronal phenomena such as long term potentiation.

Activation of NMDA receptor-coupled channels suppresses the inhibitory action of adenosine on hippocampal slices.

It has previously been found that the sensitivity of hippocampal slices to adenosine depends on magnesium ions. The addition of 2-amino-5-phosphonopentanoic acid (2AP5) in magnesium-free medium, thus blocking receptors for N-methyl-D-aspartate (NMDA) did not modify this dependence. Here it is shown that if 2AP5 is present before the removal of magnesium ions the sensitivity to adenosine is maintained. The channel blocker MK-801 also restores adenosine responsiveness. Superfusing slices with NMDA itself directly suppresses adenosine responses and can reverse adenosine inhibition of synaptic potentials. Increasing neuronal excitability non-selectively with 8.5 mM potassium in the presence of 2AP5 has little effect on adenosine sensitivity, while removing the 2AP5 causes a loss of sensitivity. The results indicate that activation of NMDA receptor-coupled channels causes a reduction of adenosine sensitivity. This sequence of events may be viewed as a positive feedback and may be of fundamental importance in phenomena such as long-term potentiation, seizure generation and kindling.

Dihydropyridines alter adenosine sensitivity in the rat hippocampal slice.

1. The effects of adenosine and a range of adenosine analogues, which are resistant to uptake processes, were studied in the presence of dihydropyridines and verapamil on the population spike potential recorded from the CA1 area of the hippocampal slice. 2. Nifedipine and Bay K 8644, a calcium channel antagonist and activator respectively, enhanced the inhibitory action of adenosine in a concentration-dependent manner. This was in contrast to their effect on adenosine analogues where the inhibition of the population potential was significantly attenuated. Similar interactions between the adenosine compounds and the dihydropyridines were also displayed in studies on spontaneous epileptiform activity in the CA3 region. 3. This effect of nifedipine and Bay K 8644 was not shown by the dihydropyridines, nimodipine or nitrendipine, or by the phenylalkylamine, verapamil. 4. Addition of the adenosine uptake blocker dipyridamole reversed the action of nifedipine on adenosine, so that inhibition by adenosine was now attenuated by nifedipine in a similar manner to that observed with the adenosine analogues. 5. These results can be explained with reference to binding studies that show displacement of adenosine analogues from the adenosine receptor by dihydropyridines. An action at the adenosine uptake site by the dihydropyridines explains the enhancement of adenosine inhibition. 6. The possible sites for this interaction are discussed.

Inhibition of adenosine responses of rat hippocampal neurones by nifedipine and BAYK 8644.

The application of adenosine to hippocampal slices caused a suppression of evoked population spikes in the CA1 region. This effect was enhanced by nifedipine and BAYK 8644 in control slices but was reduced by these dihydropyridines in the presence of dipyridamole. Several analogues of adenosine which are not substrates for the uptake system also depressed the population spikes in the CA1 region but these responses were inhibited by nifedipine and BAYK 8644. Other dihydropyridines including nimodipine and nitrendipine did not affect sensitivity to adenosine or its analogues. It is concluded that some agonist and antagonist dihydropyridines can inhibit adenosine uptake and thus potentiate its effects but can also antagonise receptor activation. Structural features of nifedipine and BAYK 8644 may be specific for a population of dihydropyridine receptors closely linked functionally with the adenosine receptor.

NMDA-receptor-independent effects of low magnesium: involvement of adenosine.

Perfusion of hippocampal slices with magnesium-free media elicits epileptiform activity attributable partly to the activity of N-methyl-D-aspartate (NMDA) receptors, but recent reports have documented an NMDA-independent enhancement of orthodromic potentials by moderate reductions in magnesium concentration. The present experiments indicate that this enhancement is comparable with that produced by perfusion with an adenosine antagonist, 8-phenyltheophylline, and that superfusion with this compound or adenosine deaminase precludes any enhancement of potential size in low magnesium solutions. The low magnesium enhancement is probably attributable to the recently described magnesium dependency of presynaptic inhibition by adenosine.

Interactions of adenosine and magnesium on rat hippocampal slices.

The potency of adenosine in reducing orthodromically evoked population potentials elicited in area CA1 of rat hippocampal slices was greatly reduced by removal of magnesium from the bathing medium. Removal of magnesium increased cell excitability, but this did not account for the loss of potency as the addition of (+/-)-2-amino-5-phosphonovaleric acid (2-APV), or a reduction in extracellular calcium failed to restore adenosine inhibition. However, addition of cobalt (500 microM) did restore inhibition. The results may indicate a requirement for magnesium, or a similar divalent cation, at the A1-inhibitory adenosine receptor. The observation of excitation by adenosine in zero magnesium/low calcium may suggest an A2-excitatory receptor interaction in the absence of adenosine inhibition.

Presynaptic actions of adenosine are magnesium-dependent.

Reducing the extracellular magnesium concentration of medium bathing a rat hippocampal slice preparation greatly reduced the ability of adenosine to inhibit the synaptically evoked population potential. This loss of potency does not appear to be mediated by increased NMDA receptor activity or increased calcium influx. The results suggest that magnesium is required at the A1 adenosine receptor for its interaction with adenosine.