Calcium influx through N-methyl-D-aspartate receptors triggers GABA release at interneuron-Purkinje cell synapse in rat cerebellum.

Ca(2+)-dependent neurotransmitter release was originally thought to occur only following activation of presynaptic voltage-gated calcium channels after a presynaptic action potential. Recent evidence suggests that not only opening of voltage-gated but also ligand-gated ion channels, such as neurotransmitter receptors, can trigger exocytosis, as well as Ca(2+) release from intracellular Ca(2+) stores. It was shown that activation of N-methyl-d-aspartate (NMDA) receptors on presynaptic interneurons led to increases in GABA release from these neurons onto postsynaptic Purkinje cells in rat cerebellum in the presence of tetrodotoxin (TTX), suggesting a presynaptic location for the underlying NMDA receptors. However, the mechanism for the NMDA-induced increase in GABA release remained unclear. The present study addresses the question whether Ca(2+) influx through presynaptic NMDA receptors alone is sufficient to trigger presynaptic GABA release at this synapse or whether activation of presynaptic NMDA receptors leads to opening of voltage-gated Ca(2+) channels, thereby increasing exocytosis. The results suggest that the NMDA-induced increase in presynaptic GABA release neither requires activation of presynaptic voltage-gated Ca(2+) channels nor Ca(2+) release from presynaptic Ca(2+) stores. It is concluded that Ca(2+) influx through the NMDA receptor alone is sufficient to drive presynaptic GABA release at the rat interneuron-Purkinje cell synapse.

Changes in TRPC channel expression during postnatal development of cerebellar neurons.

In the brain, classical (canonical) transient receptor potential (TRPC) channels are thought to be involved in different aspects of neuronal development. We investigated the developmental expression profile of TRPC channels in rat cerebellum during the first 6 weeks after birth. TRPC3 expression is significantly up-regulated whereas TRPC4 and TRPC6 expression are significantly down-regulated over this period of time. TRPC3 expression is mainly found on Purkinje cells and their dendrites, suggesting that the increase in TRPC3 expression reflects development of the dendritic tree of Purkinje cells. TRPC4 expression was restricted to granule and their precursor cells. TRPC6 expression is found on Purkinje cell bodies, on mature granule cells in the internal granule cell layer (but not their precursors) and interneurons in the molecular layer. The decrease in TRPC4 expression suggests that it is required for proper granule cell development whereas the decrease in TRPC6 expression is presumably correlated with interneuron development. Moreover, we demonstrate the presence of functional TRPC channels on Purkinje cell dendrites that are activated following stimulation of metabotropic glutamate receptors. Our results reveal cell-specific expression patterns for different TRPC proteins and suggest that developmental changes in TRPC protein expression may be required for proper postnatal cerebellar development.

Evidence that glutamate acting on presynaptic type-II metabotropic glutamate receptors alone does not fully account for the phenomenon of depolarisation-induced suppression of inhibition in cerebellar Purkinje cells.

Depolarisation-induced suppression of inhibition (DSI) is a form of short-term synaptic plasticity at gamma-aminobutyric-acid-(GABA)ergic synapses between principal neurons and interneurons in both the cerebellum and the hippocampus. The induction of DSI involves an intracellular calcium-dependent release of a retrograde messenger from the postsynaptic principal neuron (Purkinje cell/pyramidal cell in cerebellum/hippocampus) onto presynaptic interneurons, where it is thought to bind to guanine nucleotide-binding protein (G protein)-coupled receptors and subsequently reducing GABA release from these interneurons onto the postsynaptic principal neuron. Pharmacological studies have indicated that glutamate might be a retrograde messenger in both cerebellum and hippocampus, where, in the former at least, it seems to activate type-II metabotropic glutamate receptors (mGluRs). Using LY-341495, a recently described, highly specific and potent antagonist of type-II mGluRs, to block these receptors reduced DSI slightly, but significantly, in spite of the fact that this antagonist completely suppressed the effects of stimulating type-II mGluRs with a specific agonist. Activation of type II mGluRs alone thus cannot account fully for DSI in cerebellum and hence other mechanisms are involved in its induction. Such mechanisms probably involve an additional retrograde signal.

An examination of the secretion-like coupling model for the activation of the Ca2+ release-activated Ca2+ current I(CRAC) in RBL-1 cells.

One popular model for the activation of store-operated Ca2+ influx is the secretion-like coupling mechanism, in which peripheral endoplasmic reticulum moves to the plasma membrane upon store depletion thereby enabling inositol 1,4,5-trisphosphate (InsP3) receptors on the stores to bind to, and thus activate, store-operated Ca2+ channels. This movement is regulated by the underlying cytoskeleton. We have examined the validity of this mechanism for the activation of I(CRAC), the most widely distributed and best characterised store-operated Ca2+ current, in a model system, the RBL-1 rat basophilic cell line. Stabilisation of the peripheral cytoskeleton, disassembly of actin microfilaments and disaggregation of microtubules all consistently failed to alter the rate or extent of activation of I(CRAC). Rhodamine-phalloidin labelling was used wherever possible, and revealed that the cytoskeleton had been significantly modified by drug treatment. Interference with the cytoskeleton also failed to affect the intracellular calcium signal that occurred when external calcium was re-admitted to cells in which the calcium stores had been previously depleted by exposure to thapsigargin/ionomycin in calcium-free external solution. Application of positive pressure through the patch pipette separated the plasma membrane from underlying structures (cell ballooning). However, I(CRAC) was unaffected irrespective of whether cell ballooning occurred before or after depletion of stores. Pre-treatment with the membrane-permeable InsP3 receptor antagonist 2-APB blocked the activation of I(CRAC). However, intracellular dialysis with 2-APB failed to prevent I(CRAC) from activating, even at higher concentrations than those used extracellularly to achieve full block. Local application of 2-APB, once I(CRAC) had been activated, resulted in a rapid loss of the current at a rate similar to that seen with the rapid channel blocker La3+. Studies with the more conventional InsP3 receptor antagonist heparin revealed that occupation of the intracellular InsP3-sensitive receptors was not necessary for the activation or maintenance of I(CRAC). Similarly, the InsP3 receptor inhibitor caffeine failed to alter the rate or extent of activation of I(CRAC). Exposure to Li+, which reduces InsP3 levels by interfering with inositol monophosphatase, also failed to alter I(CRAC). Caffeine and Li+ did not affect the size of the intracellular Ca2+ signal that arose when external Ca2+ was re-admitted to cells which had been pre-exposed to thapsigargin/ionomycin in Ca2+-free external solution. Our findings demonstrate that the cytoskeleton does not seem to regulate calcium influx and that functional InsP3 receptors are not required for activation of I(CRAC). If the secretion-like coupling model indeed accounts for the activation of I(CRAC) in RBL-1 cells, then it needs to be revised significantly. Possible modifications to the model are discussed.

The retrograde inhibition of IPSCs in rat cerebellar purkinje cells is highly sensitive to intracellular Ca2+.

The Ca2+-dependent retrograde inhibition of inhibitory postsynaptic currents (depolarization-induced-suppression of inhibition; DSI) was investigated using fura-2 Ca2+ measurements and whole-cell patch-clamp recordings in rat cerebellar Purkinje cells. DSI was studied in cells loaded with different concentrations of the Ca2+ chelators BAPTA and EGTA. A concentration of 40 mM BAPTA was required to significantly interfere with DSI, whereas 10 mM BAPTA was almost ineffective. 40 mM EGTA reduced DSI, but was less effective than 40 mM BAPTA. Ratiometric Ca2+ measurements indicated that the extent of DSI depended critically on the changes in intracellular calcium ([Ca2+]i). The relationship between DSI and peak Delta[Ca2+]i could be approximated by a hyperbolic function, with apparent half-saturation concentrations of 200 and 40 nM for dendritic and somatic [Ca2+]i, respectively. It is suggested that DSI is due to somatodendritic exocytosis of a retrograde messenger, and that this exocytosis is highly sensitive to [Ca2+]i.

Ca2+ store dynamics determines the pattern of activation of the store-operated Ca2+ current I(CRAC) in response to InsP3 in rat basophilic leukaemia cells.

1. The relationship between the amplitude of the store-operated Ca2+ ICR AC and intracellular inositol 1,4,5-triphosphate (InsP3) concentration is complex. In rat basophilic leukaemia (RBL-1) cells dialysed with high intracellular Ca2+ buffer, the relationship is supra-linear with a Hill coefficient of 12 and resembles an apparent 'all-or-none' phenomenon. The non-linearity seems to arise from InsP3 metabolism. However, it is not clear which InsP3-metabolising pathway engenders the non-linear behaviour nor whether ICRAC is always activated to its maximal extent by InsP3. 2. Using the whole-cell patch clamp technique, we dialysed RBL-1 cells with different concentrations of the InsP3 analogue InsP3-F. InsP3-F is broken down by Ins(1,4,5)P3 5-phosphatase but is not a substrate for Ins(1,4,5)P3 3-kinase. The relationship between InsP3-F and ICRAC amplitude was supra-linear and very similar to that with InsP3 but was distinct from the graded relationship seen with the non-metabolisable analogue Ins2,4,5P3. 3. In the presence of high intracellular Ca2+ buffer, InsP3-F activated ICRAC to its maximal extent. With moderate Ca2+ buffer, however, sub-maximal ICRAC could be obtained to a maximal InsP3-F concentration. Nevertheless, the relationship between the amplitude of ICRAC and InsP3-F concentration was still supra-linear. 4. Submaximal ICRAC in response to InsP3-F in the presence of moderate Ca2+ buffer was due to partial depletion of the stores, because the size of the current could be increased by thapsigargin. 5. The data suggest that first Ins(1,4,5)P3 5-phosphatase is an important factor which contributes to the non-linear relationship between InsP3 concentration and the amplitude of ICRAC and second, InsP3 does not always activate ICRAC to its maximal extent. At moderate buffer strengths, submaximal ICRAC is evoked by maximal InsP3. However, the supra-linear relationship between InsP3 concentration and amplitude of the current still holds.

Presynaptic effects of NMDA in cerebellar Purkinje cells and interneurons.

NMDA receptors (NMDARs) are generally believed to mediate exclusively postsynaptic effects at brain synapses. Here we searched for presynaptic effects of NMDA at inhibitory synapses in rat cerebellar slices. In Purkinje cells, application of NMDA enhanced the frequency of miniature IPSCs (mIPSCs) but not that of miniature EPSCs (mEPSCs). This increase in frequency was dependent on the external Mg2+ concentration. In basket and stellate cells, NMDA induced an even larger mIPSC frequency increase than in Purkinje cells, whereas mEPSCs were again not affected. Moreover, NMDA induced an inward current in both types of interneuron, which translated into a small depolarization (approximately 10 mV for 30 microM NMDA) under current-clamp conditions. In paired recordings of connected basket cell-Purkinje cell synapses, depolarizations of 10-30 mV applied to the basket cell soma enhanced the frequency of postsynaptic mIPSCs, suggesting that somatic depolarization was partially transmitted to the terminals in the presence of tetrodotoxin. However, this effect was small and unlikely to account fully for the effects of NMDA on mIPSCs. Consistent with a small number of dendritic NMDARs, evoked EPSCs in interneurons had a remarkably small NMDA component. Evoked IPSCs at interneuron-interneuron synapses were inhibited by NMDA, and the rate of failures was increased, indicating again a presynaptic site of action. We conclude that activation of NMDARs in interneurons exerts complex presynaptic effects, and that the corresponding receptors are most likely located in the axonal domain of the cell.

Glutamate as a candidate retrograde messenger at interneurone-Purkinje cell synapses of rat cerebellum.

1. Depolarization-induced suppression of inhibition (DSI) is a form of synaptic plasticity which involves a retrograde messenger. We have performed experiments in Purkinje cells of rat cerebellar slices to determine the nature of this messenger. 2. DSI is mimicked by 2-(2,3-dicarboxycyclopropyl)-glycine (DCG-IV), a specific agonist of group II metabotropic glutamate receptors (mGluRs). 3. DSI is reduced if transmitter release is inhibited by saturating doses of DCG-IV. 4. Both DSI and DCG-IV-induced inhibition are inhibited by L-2-amino-3-phosphonopropionic acid (L-AP3), a drug which interferes with several subtypes of mGluRs. 5. DSI is reduced if synaptic activity is enhanced by application of forskolin. 6. We propose that glutamate or a glutamate-like substance is the retrograde messenger implicated in DSI, and that the inhibition resulting from presynaptic glutamate binding is mediated by a decrease in the presynaptic concentration of cAMP.

Functional characterization of two 5-HT3 receptor splice variants isolated from a mouse hippocampal cell line.

Two splice variants of the ligand-gated 5-hydroxytryptamine or serotonin 5-HT3 receptor that differ in a six-amino-acid deletion were cloned by polymerase chain reaction from the hippocampus x neuroblastoma cell line HN9.10e. When expressed in Xenopus oocytes, both variants individually formed 5-HT3 receptors that revealed no significant differences in current responses to the agonists 5-HT and 1-phenylbiguanide and block by the specific antagonist LY-278, 584-maleate. For both receptors, the monovalent cations Na+, K+, Rb+ and Li+ showed the same relative permeability; NH4(+)permeated approximately 2.7 times better than Na+, and Tris+ was only poorly permeable. In contrast to other reports, the receptors were completely and reversibly blocked by extracellular Cs+ in both oocytes and native HN9.10 cells. Moreover, Ca2+ was not permeant and exhibited a concentration-dependent decrease (0.9-18 mM) of the 5-HT-induced currents without affecting the inward rectification of the current/voltage relation. The two receptors were reversibly inhibited by nanomolar concentrations of the specific inhibitor of protein kinase C (PKC) bisindolylmaleimide, but not by the equipotent and less specific inhibitor staurosporine. A regulatory effect on both 5-HT3 receptor subunits by PKC-mediated protein phosphorylation might be possible, however, a functional role of the two splice variants present in one cell remains to be determined.

Cyclic AMP-regulated GABA release at inhibitory synapses in rat cerebellar slices.

We have investigated the effects of agents interfering with the cAMP pathway on the rate of miniature IPSCs in cerebellar slices. Noradrenaline and group II glutamate metabotropic receptor agonists respectively enhance and reduce the rate of miniature IPSCs, presumably because they respectively increase and decrease the presynaptic concentration of cAMP.

[Pregnancy after malignant melanoma. A follow-up of 23 patients]. / Schwangerschaft nach malignem Melanom. Verlaufsbeobachtung bei 23 Patientinnen.

There is little known on the risks associated with pregnancy occurring after primary therapy of a malignant melanoma. Out of a total of 2850 melanoma patients there were 23 women (20 in stage I, 3 in stage II) in whom 33 pregnancies occurred in the course of the follow-up. No metastases developed in 18 patients with a total of 25 pregnancies whereas a relapse occurred in 3 patients in stage I and 2 patients in stage II. The induction of metastases by the pregnancy in 3 of these patients is unlikely owing to the absence of a temporal relationship or because of an existing stage II and only in 2 patients did the temporal relationship between spreading of the tumor and pregnancy suggest an influence of pregnancy on malignant melanoma.