Alzheimer's disease proteins in cerebellar and hippocampal synapses during postnatal development and aging of the rat.

Alzheimer's dementia may be considered a synaptic disease of central neurons: the loss of synapses, reflected by early cognitive impairments, precedes the appearance of extra cellular focal deposits of beta-amyloid peptide in the brain of patients. Distinct immunocytochemical patterns of amyloid precursor proteins (APPs) have previously been demonstrated in the synapses by ultrastructural analysis in the cerebellum and hippocampus of adult rats and mice. Now we show that during postnatal development and during aging in these structures, the immunocytochemical expression of APPs increases in the synapses in parallel with the known up-regulation of total APPs brain levels. Interestingly, as shown previously in the adult rodents, the presenilins (PSs) 1 and 2, which intervene in APPs metabolism, exhibit a synaptic distribution pattern similar to that of APPs with parallel quantitative changes throughout life. In the brain tissue, single and double immunocytochemistry at the ultrastructural level shows co-localisation of APPs and PSs in axonal and dendritic synaptic compartments during postnatal synaptogenesis, adulthood and aging. In addition, double-labelling immunocytofluorescence detects these proteins close to synaptophysin at the growth cones of developing cultured neurons. Thusly, the brain expression of APPs and PSs appears to be regulated synchronously during lifespan in the synaptic compartments where the proteins are colocated. This suggests that PS-dependent processing of important synaptic proteins such as APPs could intervene in age-induced adjustments of synaptic relationships between specific types of neurons.

A role for phospholipase D1 in neurotransmitter release.

Phosphatidic acid produced by phospholipase D (PLD) as a result of signaling activity is thought to play a role in membrane vesicle trafficking, either as an intracellular messenger or as a cone-shaped lipid that promotes membrane fusion. We recently described that, in neuroendocrine cells, plasma membrane-associated PLD1 operates at a stage of Ca(2+)-dependent exocytosis subsequent to cytoskeletal-mediated recruitment of secretory granules to exocytotic sites. We show here that PLD1 also plays a crucial role in neurotransmitter release. Using purified rat brain synaptosomes subjected to hypotonic lysis and centrifugation, we found that PLD1 is associated with the particulate fraction containing the plasma membrane. Immunostaining of rat cerebellar granule cells confirmed localization of PLD1 at the neuronal plasma membrane in zones specialized for neurotransmitter release (axonal neurites, varicosities, and growth cone-like structures). To determine the potential involvement of PLD1 in neurotransmitter release, we microinjected catalytically inactive PLD1(K898R) into Aplysia neurons and analyzed its effects on evoked acetylcholine (ACh) release. PLD1(K898R) produced a fast and potent dose-dependent inhibition of ACh release. By analyzing paired-pulse facilitation and postsynaptic responses evoked by high-frequency stimulations, we found that the exocytotic inhibition caused by PLD1(K898R) was not the result of an alteration in stimulus-secretion coupling or in vesicular trafficking. Analysis of the fluctuations in amplitude of the postsynaptic responses revealed that the PLD1(K898R) blocked ACh release by reducing the number of active presynaptic-releasing sites. Our results provide evidence that PLD1 plays a major role in neurotransmission, most likely by controlling the fusogenic status of presynaptic release sites.

High sensitivity of mouse neuronal cells to tetanus toxin requires a GPI-anchored protein.

Tetanus neurotoxin (TeNT) produced by Clostridium tetani specifically cleaves VAMP/synaptobrevin (VAMP) in central neurons, thereby causing inhibition of neurotransmitter release and ensuing spastic paralysis. Although polysialogangliosides act as components of the neurotoxin binding sites on neurons, evidence has accumulated indicating that a protein moiety is implicated as a receptor of TeNT. We have observed that treatment of cultured mouse neuronal cells with the phosphatidylinositol-specific phospholipase C (PIPLC) inhibited TeNT-induced cleavage of VAMP. Also, we have shown that the blocking effects of TeNT on neuroexocytosis can be prevented by incubation of Purkinje cell preparation with PIPLC. In addition, treatment of cultured mouse neuronal cells with cholesterol sequestrating agents such as nystatin and filipin, which disrupt clustering of GPI-anchored proteins in lipid rafts, prevented intraneuronal VAMP cleavage by TeNT. Our results demonstrate that high sensitivity of neurons to TeNT requires rafts and one or more GPI-anchored protein(s) which act(s) as a pivotal receptor for the neurotoxin.

NGF enhances depolarization effects on SNAP-25 expression: induction of SNAP-25b isoform.

The 25 kDa synaptosomal associated protein (SNAP-25), which is implicated in neuronal plasticity and neurosecretion, exists as two isoforms generated by alternative splicing of exons 5a and 5b. The aim of the present study was to characterize factors influencing isoform expression. We report that chronic depolarization of PC12 cells alone or in the presence of NGF induces the expression of isoform-b, in addition to a 1.8- to 3-fold increase in SNAP-25 mRNA and protein as determined by immunoblotting and combined RT-PCR and Southern blot analysis. When cerebellar granule neurons were cultured in elevated K+, the predominant isoform switched from SNAP-25a to SNAP-25b. Taken together these results suggested that chronic depolarization regulates the transcription and processing of SNAP-25 mRNA.

Reversible protein kinase C activation in PC12 cells: effect of NGF treatment.

Although protein kinase C (PKC) is a key enzyme in the signal transduction process, there is little information on the mechanism leading to PKC activation in living cells. Using a new fluorescence imaging method, we studied this mechanism and correlated PKC conformational changes with intracellular Ca2+ concentration. PC12 cells were simultaneously loaded with Fura-2-AM and Fim-1, two fluorescent probes, which recognize Ca2+ and PKC, respectively. KCl and carbachol (an agonist to muscarinic receptors) applications induced dose-dependent increases of fluorescence for both probes. Both Ca2+ and PKC responses were observed within seconds following KCl or carbachol application, and were reversible upon stimulus withdrawal. PKC activation kinetics was slightly more rapid than the Ca2+ response after KCl application. After nerve growth factor (NGF) treatment of the cells, the amplitude of the KCl-induced PKC responses was larger indicating an increase in the activated PKC-pool in these cells. This difference between control and NGF-treated cells was not observed following carbachol application, suggesting the involvement of different PKC pools. While the Ca2+ response uniformly occurred in the cytosol, the PKC response displayed a patch pattern with higher intensities in the peripheral zone near the plasma membrane. This heterogeneous distribution of PKC activation sites was similar to the immunocytological localization of Ca2+-dependent and independent PKC isoforms, which suggested that at least several PKC isoforms interacted with intracellular elements. Upon repeated stimulation, the PKC response rapidly desensitized.

Pervanadate-triggered MAP kinase activation and cell proliferation are not sensitive to PD 98059. Evidence for stimulus-dependent differential PD 98059 inhibition mechanism.

A tight and stable complex with corresponding protein kinases and phosphatases establishes coupling between activators and inactivators. One such example is emerging from the studies of the Ras-dependent MAP kinase cascade signaling pathway. Pervanadate, a potent inhibitor of protein tyrosine phosphatase, stimulates MAP kinase and elicits cell proliferation in cultured mouse fibroblasts which is insensitive to PD 98059, the major inhibitor of upstream MEK, whereas serum- or TPA-triggered proliferation is sensitive to PD 98059. It is suggested that imbalanced coordination between protein kinase and protein phosphatase determines the cellular responses such as cell proliferation. The PD 98059-insensitive cell proliferation upon protein tyrosine phosphatase inhibition is attributed to a MEK bypass pathway.

Polyethylenimine-mediated DNA transfection of peripheral and central neurons in primary culture: probing Ca2+ channel structure and function with antisense oligonucleotides.

To study neuronal ion channel function with antisense oligonucleotides, a reliable method is needed which allows different neuronal cell types to be transfected without artifactual disruptive effects on their electrical properties. Here we report that use of the recently introduced transfecting agent, polyethylenimine, fulfills this requirement. Four days after transfection, in both central and peripheral neurons, an antisense designed to block the synthesis of the Ca2+ channel beta subunits induced a maximal decrease of the Ca2- current amplitude and modification of their kinetics and voltage-dependence. Controls with scrambled oligonucleotides, as well as Na+ current recordings of antisense transfected neurons, confirm both that the transfecting agent does not modify the electrophysiological properties of the neurons and that the effect of the antisense is sequence specific.

Enhancement of NMDA receptor maturation by BDNF in cultured mouse cerebellar granule cells.

The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor, which is essential during organogenesis for neuronal differentiation and formation of synaptic connections in the developing brain, changes its receptor properties during development by differential expression of multiple subunits. Using a combined electrophysiological and pharmacological approach on primary cultures of mouse cerebellar neurones, we investigated the evolution of the NMDA receptor and the potential effect of the neurotrophin BDNF on its expression. We showed that 1) the current density of NMDA responses increased with time of culture; 2) epsilon 1 subunit expression increased with time in culture relative to epsilon 2 subunit expression; and 3) the time course of the increase in NMDA responses was accelerated by about 2 days in the presence of BDNF.

Voltage-activated ionic currents in differentiating rat cerebellar granule neurons cultured from the external germinal layer.

The electrical properties of the precursor cells of the external germinal layer of rat cerebellum were assessed during their differentiation in control medium (Dulbecco's modified Eagle's medium) supplemented or not with either basic fibroblast growth factor (bFGF) or 25 mM potassium chloride (KCl). Resting potential was shown to be -10 mV in all three conditions 3 hours after plating [days in vitro (DIV)0]. By DIV 5, it reached -63 mV for cells cultured in 25 mM KCl but only -28 mV in control and bFGF media. The main voltage-sensitive ionic current measured at DIV 0 under all conditions was a composite IK consisting in a sustained K+ current blocked by tetraethylammonium (IK(TEA)), plus a rapidly activating and inactivating TEA-insensitive IK(A). Both currents increased with time in all conditions, but after 5 days IK(A) became dominant in terms of density. IK(TEA) is likely an IK(Ca), since it was blocked by 67% in 1 mM TEA. On DIV 0, INa and ICa were absent or small in amplitude. By DIV 3, 80% of the cells had currents able to generate a spike. Interestingly, ICa mean amplitude and current density measured at -10 mV in control condition on DIV 1 was significantly larger than those recorded in bFGF and 25 mM KCl. The order of appearance of the ionic currents, IK, ICa, and INa, leads directly to fast spike activity allowing for poor calcium entry. Firing rate likely depends on IK(A), which increased during the first 6 days of development but could be differentially regulated by bFGF.

Ionic control of intracellular pH in rat cerebellar Purkinje cells maintained in culture.

1. Intracellular pH (pHi) was measured in single rat cerebellar Purkinje cells maintained in primary culture using microspectrofluorescence analysis of the intracellularly trapped pH-sensitive dye 2',7'-bis-(2-carboxyethyl)-5 (and -6)-carboxyfluorescein (BCECF). 2. The ratio of the fluorescence signals measured at 530 nm in response to an alternating excitation at 450 and 490 nm was calibrated using the K(+)-H+ ionophore nigericin. This calibration gave a steady-state pHi of 7.06 +/- 0.02 (S.E.M., n = 17) when cells were perfused by a 5% CO2-25 mM-HCO3(-)-buffered solution at an external pH of 7.40 at 37 degrees C. 3. Replacement of external chloride with gluconate in the presence of bicarbonate induced a cytoplasmic alkalinization of about 0.3 pH unit. This alkalinization was independent of external sodium and was greatly reduced by 0.5 mM-DIDS, indicating the presence of a chloride-bicarbonate exchange. 4. In bicarbonate-free (HEPES-buffered) solution the steady-state pHi was 7.37 +/- 0.02 (n = 19), significantly higher than in bicarbonate-buffered solution. Recovery from an intracellular acid load brought about by the ammonium chloride pre-pulse technique was blocked by the removal of external sodium or the addition of 1.5 mM-amiloride, indicating the presence of a sodium-hydrogen exchange. 5. In bicarbonate-buffered solution pHi recovery after an acid load was also completely blocked by addition of 1.5 mM-amiloride indicating the absence of a bicarbonate-dependent acid extrusion mechanism. 6. Addition of 12-O-tetradecanoylphorbol-13-acetate (TPA, 100 nM) induced an amiloride-sensitive alkalinization of about 0.3 pH unit in bicarbonate-buffered solution but had no effect in HEPES-buffered solution. This observation suggests that in cultured Purkinje cells the sodium-hydrogen exchanger could be activated through a protein kinase C pathway only when pHi is maintained at a low physiological value by the activity of the chloride-bicarbonate exchange.

[Effects of subcutaneous administration of sandostatine (SMS 201.995) in 18 cases of thyroid medullary cancer]. / Effets de l'administration sous cutanée de la sandostatine (SMS 201.995) en sous cutané dans 18 cas de cancer médullaire du corps thyroïde.

Recent studies have suggested that somatostatin could reduce calcitonin plasma levels (CT) in normal subjects and in medullary thyroid carcinoma (MTC). The aim of this study was to examine the usefulness of the somatostatin analog, sandostatine (SMS 201.995) in MTC with elevated residual CT levels post-thyroidectomy with or without metastases. 18 patients (17-64 years, 12 men and 8 women) with CT greater than 850 pg/ml (N less than 150 pg/ml) and with metastases in 12 cases, were studied. MTC was sporadic in 11 cases, familial in 4 cases and of undefined form in 3. Initial posology was 300 micrograms/d of sandostatin (3 injections/day). It was then increased by 300 micrograms/d every 9 day till a maximum of 1500 micrograms/d. Treatment duration was 37 days in 11 cases and 60 days in 7 cases. Plasma CT and carcinoembryonic antigen levels (CEA) were measured before treatment and at the end of each dosage plateau. Morphologic evaluation of metastases was done at 0, 30, 60 days. 7/18 patients were reevaluated 2 to 8 months after with drawal of sandostatine. Treatment was well tolerated. Flushes improved in 4 out of 5 cases but diarrhea in only 2 out of 9 patients. Sandostatine was without any effect on plasma CEA. Heterogenous responses were observed for plasma CT levels (CT decreases greater than 20% in 8/18 patients when 900 to 1500 micrograms/day were administered). Patients were subdivised into 3 groups according to CEA levels and presence or absence of metastases. Group A (n = 9) had elevated CEA levels (greater than 10 mg/ml) and metastases.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium currents in rat cerebellar Purkinje cells maintained in culture.

Calcium permeabilities were examined in large cerebellar neurons maintained in culture, and morphologically identified as Purkinje cells. When cells were supplied with a Dulbecco Minimum Eagle's Medium with 10% horse serum added (5-10 days), somatic recordings revealed complex spikes and these were shown to be generated by Na and Ca components, the Na one being tetrodotoxin-sensitive. At the dendritic level, Ca currents were better resolved than at the soma. In dendrites, Ca entry was shown to occur through at least two distinct currents. The first was a low-threshold transient current (elicited above -60 mV from a holding potential of -80 mV) which was reduced by almost 30% by 50 microM cadmium. The second was a high-threshold current (above -20 mV) which gave rise to (1) a transient component exhibiting a steady-state inactivation and so requiring holding potentials at -80 mV, and (2) a sustained component. Both components were suppressed by 50 microns cadmium. We measured a total Ca current at the dendritic level reaching values of up to 1 nA. In another culture medium (Leibovitz medium) known to allow expression of three types of calcium currents in nodose cells we observed the development of the dendritic tree of Purkinje cells but with no simultaneous expression of the high-threshold Ca current.

Potassium currents in rat cerebellar Purkinje neurones maintained in culture in L15 (Leibovitz) medium.

Cerebellar Purkinje cells (PC) can be maintained in culture for one to two weeks in L15, a rich medium known to allow expression of a normal excitability in peripheral neurones. When examined using whole cell recordings, PC proved to be inexcitable in these conditions, and this inexcitability could be related to the presence of large outward K currents. Depolarizing steps of -100 mV revealed a voltage-dependent biphasic K current with a large early transient phase followed by a small plateau phase. The early transient phase could be selectively eliminated by holding the cell at -40 mV or by extracellularly applying 5 mM 4-aminopyridine (4-AP), whereas the plateau was abolished by 15 mM tetraethylammonium (TEA). Hereafter, these currents will be identified as the IA and the delayed current respectively, IA being the predominant current. IA activated between -25 and +65 mV with a midpoint at +3 mV; inactivation occurred between -70 and -20 mV with a midpoint at -57 mV. Current decay followed an exponential time course with a time constant of about 30 ms between -20 and +10 mV. In the cell-attached recording configuration, depolarization elicited openings of two types of K channels, one inactivating and one non-inactivating. The non-inactivating K channel probably corresponded to the delayed K current and had a conductance of 22 pS in a physiological K gradient.

Radioiodinated meta-iodobenzylguanidine uptake in medullary thyroid cancer. A French cooperative study.

Fifty meta-iodobenzylguanidine (MIBG) scintiscans were performed in three groups of medullary thyroid cancer (MTC) patients. Group 1 (n = 11) included treated patients with normal calcitonin levels; Group 2 (n = 24) included patients with elevated calcitonin levels due to sporadic and isolated MTC; Group 3 (n = 15) included patients with elevated calcitonin levels due to familial MTC or multiple endocrine neoplasia Type IIA syndrome (MEN). In Group 1 three pheochromocytoma were depicted by MIBG scintiscan. In Group 2 MTC was seen in a small number of patients (3 of 24). In Group 3, besides adrenal hyperplasia and pheochromocytoma four patients, MIBG scintigraphy showed where MTC had localized and spread in almost half of patients (7 of 15). MIBG uptake occurred in patients with relatively high calcitonin level (greater than 0.6 nmol/l). These data indicate that in patients with familial MTC or MEN syndrome, MIBG scintiscan can be useful not only in detecting associated pheochromocytoma, but also in showing MTC.

Spontaneous and GABA-evoked chloride channels on pituitary intermediate lobe cells and their internal Ca requirements.

On porcine intermediate lobe (IL) endocrine cells, spontaneously opening chloride channels have been studied and compared to GABA-A activated chloride channels. Elementary currents were recorded mainly from outside-out patches excised from IL cells maintained in culture for 1-4 weeks. Spontaneous inward currents were observed in Cs-loaded cells after replacing Na in the extracellular medium by the impermeant ion choline. This activity, at an internal calcium concentration of 10(-8) M corresponded to a channel for chloride ions with a main conductance level of 26 pS, and substates around 11 pS. The sequence of permeabilities to halides was I greater than Br greater than Cl. These conductance characteristics were common to the GABA-operated channels which also showed a main conductance substate of 23-31 pS. The open time of the 26 pS level mostly encountered in spontaneous activity, was distributed along two modes: one, the most frequent, around 1 ms, and the other around 4 ms. This latter mode was the predominant one observed during GABA and isoguvacine applications but in addition a bursting activity of 19 ms duration was also seen. Specific GABA-A receptor antagonists (bicuculline and SR42641, 1 microM) blocked activity evoked by GABA (1-10 microM), but did not affect spontaneous events. These spontaneous Cl events were only observed in a restricted range of internal Ca concentrations, i.e. between 1 nM and 0.1 microM, and were practically abolished at Cai 1 microM. The GABA-induced activity of Cl channels was also Ca-sensitive, being reduced when Cai reached 1 microM.

Postnatal development of the chemosensitivity of rat cerebellar Purkinje cells to excitatory amino acids. An in vitro study.

In vitro sagittal slices of immature rat cerebellum were used to study the development of the sensitivity of Purkinje cells (PCs) to L-aspartate (L-Asp), L-glutamate (L-Glu) and related derivatives. As early as postnatal day 0 all PCs already displayed clear excitatory responses to short iontophoretic applications of L-Asp, L-Glu and quisqualate while in the same conditions no effect of N-methyl-D,L-aspartate (NMDLA) was detected. By postnatal day 5, i.e. after the onset of the synaptogenesis, the sensitivity of PCs to L-Asp, L-Glu and quisqualate significantly increased up to values similar to those recorded in adult rat cerebellum and surprisingly nearly all (87%) the recorded cells now also displayed excitatory responses to NMDLA. Although this sensitivity of PCs to NMDLA was significantly lower than that observed with the other drugs, it persisted until the end of the first postnatal month when the adult type of connectivity is already well established but at this stage only 30 per cent of the tested cells were still sensitive to the agonist. During this period, excitatory responses elicited by NMDLA were selectively antagonized by 2-amino-5-phosphonovalerate (2-APV), suggesting that during postnatal development, NMDA receptor types are transiently expressed on PCs membranes since in the adult, NMDLA no longer had an excitatory effect. Instead, this drug now exerted a preferential antagonistic action on the excitatory response elicited by L-Asp. Also in the adult, no major changes occurred in the sensitivity of PCs to L-Asp, L-Glu and quisqualate when these drugs were ejected at a dendritic site whereas, when ejected at the somatic level, the sensitivity of the cell appeared 2-3 times lower.