Biomedical subjects

M Wiemann

Publications and source records attributed to M Wiemann.

At least 19 recordsLinked to original sources

PubMed2002

A reporter-cell assay for the detection of BMP-2 immobilized on porous and nonporous materials.

Human recombinant bone morphogenetic protein-2 (rhBMP-2) immobilized on the surface of metal implants can facilitate osseointegration. Here, we describe a cell reporter assay useful for quantifying small amounts of immobilized rhBMP-2 on various materials. The peptide was dotted and heat-fixed on titanium, 316L stainless steel, nitrocellulose, or glass, and its distribution was monitored by in situ biotinylation followed by detection with the avidin-biotin method. Bioactivity of rhBMP-2 was demonstrated by means of a confluent layer of osteoblastic MC3T3-E1 cells that evenly covered rhBMP-2-free and rhBMP-2-loaded surface areas, as shown with epifluorescence microscopy of calcein acetoxymethyl (AM)-loaded cells. Expression of osteocalcin, fibronectin, actin, and vimentin increased where cells were located on rhBMP-2 dotted areas, but the signal:noise ratio was too low to bioassay the peptide. However, local pronounced expression of alkaline phosphatase was used to quantify BMP-2 in the range of 5-80 ng/dot by means of a cytochemical color reaction for alkaline phosphatase and image analysis of resulting dots. The lower detection limit was in the order nitrocellulose > glass > titanium > 316L steel. We conclude that the cell reporter assay is useful to assess biological activity of rhBMP-2 even after immobilization on three-dimensional implant materials.

Alkaline Phosphatase↗

PubMed2002

[Optimizing histological image data for 3-D reconstruction using an image equalizer].

Bone cells form a wired network within the extracellular bone matrix. To analyse this complex 3D structure, we employed a confocal fluorescence imaging procedure to visualize live bone cells within their native surrounding. By means of newly developed image processing software, the "Image-Equalizer", we aimed to enhanced the contrast and eliminize artefacts in such a way that cell bodies as well as fine interconnecting processes were visible.

Algorithms↗

PubMed2001

A novel inhibitor of the Na+/H+ exchanger type 3 activates the central respiratory CO2 response and lowers the apneic threshold.

Cultured CO2-sensitive neurons from the ventrolateral medulla of newborn rats enhanced their bioelectric activity upon intracellular acidification induced by inhibition of the Na+/H+ exchanger type 3 (NHE3). Now we detected NHE3 also in the medulla oblongata of adult rabbits. Therefore, this animal model was employed to determine whether NHE3 inhibition also affects central respiratory chemosensitivity in vivo. Seven anesthetized (pentobarbital), vagotomized, paralyzed rabbits were artificially ventilated with O2-enriched air. From the phrenic nerve compound discharge, integrated burst amplitude (IPNA), respiratory rate (fR), and phrenic minute activity (IPNA. fR) were taken as measures of central respiratory rhythm and drive. Effects of potent NHE3 inhibition with the novel brain permeant substance S8218 were studied by comparing respiratory characteristics before and after up to 9.2 +/- 1.1 mg/kg cumulative drug application, yielding average plasma concentrations of 0.9 +/- 0.2 microg/ml. In response to S8218, the baseline level of IPNA. fR was significantly enhanced by an average of 51.0 +/- 6.4% (n = 27, p < 0.0001). The influence of NHE3 inhibition on the respiratory CO2 response was studied at plasma concentrations of S8218 maintained in the range of 0.3 microg/ml (10(-6) M). Although the metabolic acid-base status thereby remained widely unchanged, the group mean apneic threshold PaCO2 was significantly lowered by 0.45 +/- 0.11 kPa (n = 7, p < 0.01), whereby in four of seven animals even strong hyperventilation failed to suppress phrenic nerve rhythmicity completely. Likewise, S8218 significantly augmented IPNA. fR, in the range of PaCO2 between 1 and 6 kPa above threshold, by an average of 38.0 +/- 8.5% (n = 35, p < 0.0001). These in vivo results are compatible with the effects of NHE3 inhibition on chemosensitive brainstem neurons in vitro. Moreover, rhythmogenesis is supported through NHE3 inhibition by lowering the threshold PCO2 for central apnea.

Animals↗

PubMed2001

Enhancement of connexin 43 expression increases proliferation and differentiation of an osteoblast-like cell line.

Bone cells form a functional syncytium as they are coupled by gap junctions composed mainly of connexin 43 (Cx43). To further understand the role of Cx43 in bone cell growth and differentiation, we stably transfected Cx45-expressing UMR 106-01 cells with Cx43 using an expression vector containing rat Cx43 cDNA. Three stably transfected clones were analyzed, all of which showed altered expression of Cx43 and/or Cx45 as was obvious from immunocytochemistry and Northern blotting. Double whole-cell patch clamping revealed single-channel conductances of 20 (Cx45) and 60 pS (Cx43). The overexpression of Cx43 led to an increase in dye coupling concomitant with elevated gap-junctional conductance. The phenotype of the transfected clones was characterized by an increased proliferation (4- to 7-fold) compared to controls. Moreover, a transfectant clone with 10- to 12-fold enhanced Cx43 expression showed a significantly increased calcium content of the extracellular matrix and enlarged mineralization nodules, while alkaline phosphatase was moderately increased. We conclude that enhanced gap-junctional coupling via Cx43 significantly promotes proliferation and differentiation of UMR cells.

Alkaline Phosphatase↗

PubMed2001

Ventrolateral neurons of medullary organotypic cultures: intracellular pH regulation and bioelectric activity.

The hypothesized role of the intracellular pH (pH(i)) as a proximate stimulus for central chemosensitive neurons is reviewed on the basis of data obtained from organotypic cultures of the medulla oblongata (obex level) of new born rats (OMC). Within OMC a subset of neurons responds to hypercapnia as do neurons in the same (or similar) brain areas in vivo. Maneuvers altering intra- and/or extracellular pH (pH(o)) such as hypercapnia, bicarbonate-withdrawal, or ammonium pre-pulses, evoked well defined changes of the neuronal pH(i). During hypercapnia (pH(o) 7.0) or bicarbonate-withdrawal (pH(o) 7.4) most ventrolateral neurons adopted a pH(i) which was < or = 0.2 pH units below the steady state pH(i), while signs of pH(i)-regulation occurred only in a small fraction of neurons. During all treatments leading to intracellular acidosis, bioelectric activity of chemosensitive neurons increased and was often indistinguishable from the response to hypercapnia, regardless of whether pH(o) was unchanged, decreased or increased during the treatment. These data strongly suggest that the pH(i) acts as proximate stimulus. The mode of acid extrusion of chemosensitive neurons is, therefore, of major importance for the control of central chemosensitivity. Immunocytochemical data, pH(i) measurements and neuropharmacological studies with novel drugs pointed to the Na(+)/H(+) exchanger subtype 3 (NHE3) as a main acid extruder in ventrolateral chemosensitive neurons. Possible functions and neuropharmacological strategies arising from this very local NHE3 expression are discussed.

Animals↗

PubMed2000

Cutting of living hippocampal slices by a highly pressurised water jet (macromingotome).

Living brain slices are usually cut with razor blades, which compress a ca. 50-microm-thick layer of tissue. This results in cell debris and lesioned cells which, e.g. form diffusion barriers between the bath and living neurons underneath, thereby prolonging response times of neurons to drugs in the bath saline and impeding the experimental access to intact neurons. To avoid such drawbacks, a macromingotome was developed which cuts nervous tissue with water jets. Physiological saline under pressures of 100-1800 bar was ejected through nozzles of 35-100 microm to cut 300-500-microm-thick hippocampal slices. Systematic variations of pressure and nozzle diameter revealed best results at 400-600 bar and with nozzle diameters of 60-80 microm. Under these conditions, intact CA1- and CA3-neurons as well as granule cells were detected with infrared microscopy at less than 10 microm underneath the surface of the slice. Superficial neurons with intact fine structures were also seen when the slices were studied by light-microscopy. Intra- and extracellular recordings from superficial neurons showed normal membrane- and full action potentials and the development of stable epileptiform discharges in 0 Mg(2+)-saline. These results indicate that the macromingotome offers an alternative way of cutting slices which may facilitate electrophysiological/neuropharmacological or fluorometric studies on superficial neurons.

Action Potentials↗

PubMed2000

Moclobemide reduces intracellular pH and neuronal activity of CA3 neurones in guinea-pig hippocampal slices-implication for its neuroprotective properties.

Mechanisms underlying the neuroprotective properties of the weak MAO-A inhibitor moclobemide are not understood. Increasing evidence suggests that a moderate increase in intracellular free protons may contribute to neuroprotective properties due to a proton-mediated decrease in neuronal activity. Therefore, we studied effects of 10-700 microM moclobemide (i) on the intracellular pH (pH(i)) of BCECF-AM loaded CA3 neurones as well as (ii) on spontaneous action potentials and epileptiform activity (induced by bicuculline-methiodide, caffeine, or 4-aminopyridine) of CA3 neurones in the stratum pyramidale. Moclobemide-concentrations of > or = 300 microM reversibly reduced the steady-state pH(i) by up to 0. 25 pH-units within 5-20 min. Simultaneously, the frequency of spontaneous action potentials and epileptiform discharges became depressed. Moclobemide also abolished 4-aminopyridine-induced GABA-mediated hyperpolarisations suggesting that the inhibitory and acidifying effects of moclobemide do not result from an amplification of the GABA system. The stronger MAO-A inhibitors clorgyline or pargyline (both 10 microM) mimicked the moclobemide-effects. Investigating effects on pH(i)-regulation we found that 700 microM moclobemide impaired the recovery from intracellular acidification elicited by an ammonium prepulse which demonstrates an impairment of transmembrane acid extrusion. We suggest that the latter effect is responsible for the moderate decrease in the steady-state pH(i) which in turn reduced neuronal activity. This mechanism may substantially contribute to the neuroprotective properties of moclobemide.

Action Potentials↗

PubMed2000

Alteration of intracellular pH and activity of CA3-pyramidal cells in guinea pig hippocampal slices by inhibition of transmembrane acid extrusion.

Transmembrane acid extruders, such as electroneutral operating Na(+)/H(+)-exchangers (NHE) and Na(+)-dependent Cl(-)/HCO(3)(-)-exchangers (NCHE) are essential for the maintenance and regulation of cell volume and intracellular pH (pH(i)). Both of them are hypothesised to be closely linked to the control of excitability. To get further information about the relation of neuronal pH(i) and activity of cortical neurones we investigated the effect of NHE- and/or NCHE-inhibition on (i) spontaneous action potentials and epileptiform burst-activity (induced by bicuculline-methiodide, caffeine or 4-aminopyridine) and (ii) on pH(i) of CA3-neurones. NHE-inhibition by amiloride (0.25-0.5 mM) or its more potent derivative dimethylamiloride (50 microM) and NCHE-inhibition by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, 0.25-0.5 mM) induced a biphasic alteration of neuronal activity: an initial, up to 30 min lasting, increase in frequency of action potentials and bursts preceded a growing and partially reversible suppression of neuronal activity. In BCECF-loaded neurones the pH(i), however, continuously decreased during either amiloride- or DIDS-treatment and reached its steady-state (DeltapH(i) up to 0.3 pH-units) when the neuronal activity was markedly suppressed. Combined treatment with amiloride (0.5 mM) and DIDS (0.5 mM) or treatment with harmaline alone (0.25-0.5 mM), which also continuously acidified neurones via inhibition of an amiloride-insensitive NHE-subtype, induced a monophasic and partially reversible suppression of neuronal activity. As an initial excitatory period failed to occur during combined NHE/NCHE-inhibition we speculate that its occurrence during amiloride- or DIDS-treatment resulted rather from disturbances in volume- than in pH(i)-regulation. The powerful inhibitory and anticonvulsive properties of NHE- and NCHE-inhibitors, however, very likely based upon intracellular acidification - as derived from our previous findings that a moderate increase in intracellular free protons is sufficient to reduce membrane excitability of CA3-neurones.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2000

Intracellular pH modulates spontaneous and epileptiform bioelectric activity of hippocampal CA3-neurones.

A growing body of evidence hints at intracellular free protons to be involved in the modulation of electric activity of cortical neurones. In this study we demonstrate that application of the weak acid propionate (2.5-20 mM) transiently lowers intracellular pH (pH(i)) of BCECF-AM loaded CA3-neurones in hippocampal slices. The predictability of this acidification prompted us to use propionate as a tool to investigate effects of pH(i) on spontaneous bioelectric activity (SBA) and epileptiform activity (EA, induced by bicuculline, caffeine or low magnesium) of CA3 neurones: SBA and EA were transiently suppressed by 2-20 mM propionate - coinciding with the transient neuronal acidification. As activation of Na(+)/H(+)-exchangers (NHE) is involved in the recovery from neuronal acidosis and NHE-inhibition alone is known to increase the activity of intracellular free protons of hippocampal neurones, we tested the effect of the NHE-blockers amiloride (0.5-1 mM) or HOE642 (200 microM) on SBA and EA of CA3-neurones. Long-term application of NHE-inhibitors alone continuously suppressed SBA and EA, which recovered during additional exposure to the weak base trimethylamine (5-10 mM). Simultaneous administration of propionate and NHE-blockers intensified the inhibition of neuronal activity. Together, these results indicate that intracellular acidification inhibits bioelectric activity of hippocampal CA3-neurones. This supports the hypothesis that pH(i) contributes to the control of cortical excitability.

Animals↗

PubMed2000

Intercellular communication between bone marrow stromal cells and CD34+ haematopoietic progenitor cells is mediated by connexin 43-type gap junctions.

The existence of functional gap junctions between haematopoietic progenitor cells (HPCs) and stromal cells of the haematopoietic microenvironment in the human system is a controversial issue. Primary CD34+ HPCs isolated from leukapheresis products were co-incubated with the human fibroblastoid bone marrow stromal cell line L87/4 in short-term liquid culture. Using the highly sensitive double whole-cell patch-clamp technique, we found that the majority (91%) of CD34+ HPCs are electrically coupled to L87/4 cells. Importantly, efficient coupling was observed within 1 h of the attachment of CD34+ HPCs to plastic adherent L87/4 cells. By comparison, homologous cell pairs formed by L87/4 cells exhibited a significantly higher electric coupling. Analysis of single-channel conductances revealed an electric profile characteristic of connexin 43 (Cx43)-type gap junctions for both homologous and heterologous cell pairs. The Cx phenotype was confirmed using Cx43-specific monoclonal antibodies in a flow cytometric assay and reverse transcription polymerase chain reaction (RT-PCR) for the detection of Cx43 mRNA. Finally, the electrophysiological studies were complemented by dye-transfer experiments using the recently described 'parachute' technique that allows the monitoring of dye diffusion without disruption of the plasma membrane. Taken together, our data indicate that functional Cx43-type gap junctions exist between stromal cells and immature HPCs and, thus, may provide an important regulatory pathway in haematopoiesis.

Antigens, CD34↗

PubMed2000

Different effects of GABAergic anticonvulsants on 4-aminopyridine-induced spontaneous GABAergic hyperpolarizations of hippocampal pyramidal cells--implication for their potency in migraine therapy.

Clinical studies indicate anti-migrane efficacy of the probably GABAergic anticonvulsants valproate and gabapentin. For the GABAergic anticonvulsants vigabatrin and tiagabine, studies about antimigrane efficacy are missing. The aim of this study was to test the GABAergic potency of these drugs in vitro before further clinical studies. Intracellular recordings were obtained from hippocampal pyramidal cells. Spontaneous GABAergic hyperpolarizations (SGH) elicited by 75 microM 4-aminopyridine were used to test the effect of these drugs on GABA-dependent potentials. Tiagabine (0.1 mM) prolonged the duration of SGH. Furthermore, monophasic SGH turned over into triphasic typical GABAergic membrane potential fluctuations within 20 min. In contrast, valproate, gabapentin, and vigabatrin failed to affect SGH up to 60 min of application. The reason for the fast action of tiagabine on SGH may be caused by a faster increase of synaptic GABA levels compared with other drugs. As migraine therapy benefits from an augmentation of GABA activity, we recommend clinical studies of tiagabine as a fast-acting agent in migraine attacks.

4-Aminopyridine↗

PubMed1999

Ammonium prepulse: effects on intracellular pH and bioelectric activity of CA3-neurones in guinea pig hippocampal slices.

The ammonium prepulse technique was used to study influences of intracellular pH (pH(i)) on bioelectric activity of CA3-neurones in hippocampal slices. 60, 180 or 600 s lasting NH(4)Cl (10 mM) pulses led to a transient intracellular alkalosis (DeltapH(i): up to 0.2 pH-units) in about one-half of the neurones loaded with 2', 7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethylester (BCECF-AM). No alkalosis was seen in the remainder cells. The amount of alkalosis depended on the actual pH(i) of each neurone and increased when the pH(i) decreased. Washout of NH(4)Cl induced a fall in pH(i) (DeltapH(i): 0.12-0.54 pH-units) which recovered within <20 min. Frequency of spontaneous action potentials remained unchanged during washin of ammonium (60 or 180 s). However, pre-treatment with low concentrations of bicuculline-methiodide (0. 01 microM) or caffeine (0.1 mM), both of which did not change bioelectric activity per se, permitted a burst-activity to occur during ammonium-washin in about one-half of the neurones. In all neurones, washout of ammonium inhibited spontaneous and epileptiform activity (elicited by 1 mM caffeine, 20-50 microM bicuculline-methiodide, or 50-75 microM 4-aminopyridine) for </=20 min. This inhibition was accompanied by an increased membrane conductance (up to 20%) and a hyperpolarisation of up to 10 mV. We conclude that intracellular alkalosis augments, whereas intracellular acidosis depresses bioelectric activity of CA3-neurones.

Alkalosis↗

PubMed1999

Interference of lead with the calcium release activated calcium flux of osteoblast-like cells.

Lead (Pb(2+)) tends to accumulate in bone from where it is released during bone resorption, thus leading to high local concentrations of Pb(2+) with the risk of cellular toxicity. We investigated the interference of Pb(2+) with the calcium release activated calcium influx (CRAC) of osteoblast-like (OBL) cells. CRAC was elicited by depletion of intracellular Ca(2+) stores with thapsigargin and/or A23187 under Ca(2+)-free conditions and re-addition of extracellular Ca(2+). The fura-2 excitation ratio (R) was used to monitor changes of the free intracellular concentration of Ca(2+) and Pb(2+), the latter being reversible by the heavy metal chelator TPEN. Five or 12. 5 microM Pb(2+) applied simultaneously with re-added Ca(2+) reduced the immediate CRAC of OBL cells to 70% or 37% of control value, respectively. An enlarged influx of Pb(2+) occurred during CRAC, which led to a 2.7-fold faster increase of R. When 1 microM Pb(2+) was added during ongoing CRAC, the Pb(2+)-mediated increase of R correlated with the degree of CRAC (r = 0.83). Inhibitory effects of Pb(2+) on Ca(2+) ATPase activity did not contribute to the aforementioned findings. Our results demonstrated that CRAC channels of OBL cells are blocked as well as permeated by Pb(2+).

Animals↗

PubMed1999

Selective inhibition of the Na+/H+ exchanger type 3 activates CO2/H+-sensitive medullary neurones.

Hypercapnia as well as lowered intracellular pH (pHi) increase the bioelectric activity of CO2/H+-sensitive neurones (VLNcs) of the ventrolateral medulla oblongata. Here we describe that immunoreactive Na+/H+ exchanger (NHE3) is present in ventrolateral neurones from medullary organotypic cultures (obex level). To test whether VLNcs can be acidified and thereby activated by inhibition of NHE3, we used the novel high-affinity NHE3-inhibitors S1611 and S3226. Both drugs raised the firing rates of VLNcs to at least 150% of the control values, and depolarized membrane potential by up to 15 mV at concentrations (0.5-1 micromol/l) suitable for selective inhibition of NHE3. The changes in bioelectric activity strongly resembled the responses to hypercapnia (PCO2: 60-100 mmHg). In BCECF-AM-loaded cultures a subfraction of ventrolateral VLNcs was found to be intracellularly acidified by 0.05-0.1 pH units following treatment with S1611; the time course of this acidification was similar to that evoked by hypercapnia. All drug effects were sustained and readily reversible upon washing. Non-CO2/H+-responsive medullary neurones as well as hippocampal CA3 neurones were unaffected by up to 20 micromol/l S1611. It is concluded that the selective inhibition of NHE3 acidifies and activates CO2/H+-sensitive neurones within the ventrolateral medulla oblongata.

Action Potentials↗

PubMed1999

Matched-pair analysis of hematopoietic progenitor cell mobilization using G-CSF vs. cyclophosphamide, etoposide, and G-CSF: enhanced CD34+ cell collections are not necessarily cost-effective.

Using matched-pair analysis, we compared two popular methods of stem cell mobilization in 24 advanced-stage breast cancer patients who underwent two consecutive mobilizing procedures as part of a tandem transplant protocol. For the first cycle, 10 microg/kg/day granulocyte colony-stimulating factor (G-CSF) was given and apheresis commenced on day 4 and continued for < or =5 days (median 3 days). One week after the first cycle of apheresis, 4000 mg/m2 cyclophosphamide, 400 mg/m2 etoposide, and 10 microg/kg G-CSF were administered for < or =16 days (cycle 2). Apheresis was initiated when the white blood cell (WBC) count exceeded 5000 cells/microL and continued for < or =5 days (median 3 days). Mean values of peripheral blood WBC (31,700+/-3200 vs. 30,700+/-3300/microL) were not significantly different between cycles 1 and 2. Mean number of mononuclear cells (MNC) collected per day was slightly greater with G-CSF mobilization than with the combination of chemotherapy and G-CSF (2.5+/-0.21x10(8) vs. 1.8+/-0.19x10(8) cells/kg). Mean daily CD34+ cell yield, however, was nearly six times higher (12.9+/-4.4 vs. 2.2+/-0.5x10(6)/kg; p = 0.01) with chemotherapy plus G-CSF. With G-CSF alone, 13% of aphereses reached the target dose of 5x10(6) CD34+ cells/kg in one collection vs. 57% with chemotherapy plus G-CSF. Transfusions of red blood cells or platelets were necessary in 18 of 24 patients in cycle 2. Three patients were hospitalized with fever for a median of 3 days after cycle 2. No patients received transfusions or required hospitalization during mobilization with G-CSF alone. Resource utilization (cost of drugs, aphereses, cryopreservation, transfusions, hospitalization) was calculated comparing the median number of collections to obtain a target CD34+ cell dose of 5x10(6) cells/kg: four using G-CSF vs. one using the combination in this data set. Resources for G-CSF mobilization cost $7326 vs. $8693 for the combination, even though more apheresis procedures were performed using G-CSF mobilization. The cost of chemotherapy administration, more doses of G-CSF, transfusions, and hospitalizations caused cyclophosphamide, etoposide, and G-CSF to be more expensive than G-CSF alone. A less toxic and less expensive treatment than cyclophosphamide, etoposide, and G-CSF is needed to be more cost-effective than G-CSF alone for peripheral blood progenitor cell mobilization.

Adult↗

PubMed1999

Autotransplants in men with breast cancer. ABMTR Breast Cancer Working Committee. Autologous Blood and Marrow Transplant Registry.

The purpose of this study was to determine the outcome of high-dose therapy with autologous hematopoietic stem cell support (autotransplants) in men with breast cancer. We studied 13 men receiving autotransplants for breast cancer and reported to the Autologous Blood and Marrow Transplant Registry (ABMTR) by 10 centers. Six men had stage 2 breast cancer, four had stage 3, and three had metastatic breast cancer. Of twelve tumors tested, all were estrogen receptor positive. Median age at transplant was 50 years. The most common conditioning regimen was cyclophosphamide, thiotepa and carboplatin (n = 5); the remaining eight men received other alkylator-based regimens. Three men received bone marrow, eight received blood stem cells, and two received both for hematopoietic support. All patients had hematopoietic recovery. There were no unexpected regimen-related toxicities. Of 10 men receiving autotransplants as adjuvant therapy, three relapsed 3, 5 and 50 months post-transplant and died 16, 19 and 67 months post-transplant. Seven of 10 are disease-free with median follow-up of 23 months (range 6-50 months). Of three men treated for metastatic breast cancer, one had progressive disease and two recurrent disease at 6, 7 and 16 months post-transplant. In conclusion, results of autotransplants for male breast cancer appear similar to those reported for women receiving autotransplants for breast cancer.

Adult↗

PubMed1998

Microcutting of living brain slices by a pulsed ultrafine water jet which allows simultaneous electrophysiological recordings (micromingotome).

Up to now microsurgical dissections in living nervous tissue (e.g. in slices or cell cultures) are performed either by micro-scalpels or by laser beams. As an alternative technique, a device for cutting with an ultrafine pulsed water jet was developed to allow precise, visually controled dissections in neuronal circuits even during electrophysiological recordings. Water is ejected by pressure (20-30 bar) from patch pipettes with tip diameters of 10-12 microm. By means of a piezo-element the pipette and the water jet are forced to oscillate vertically with a frequency of 200-400 Hz with an adjustable amplitude. These oscillations facilitate the transsection of neuronal connections even in thick slice preparations. Best results were obtained when the tip of the pipette was about 500 microm above the surface of the submerged slice tissue. This micromingotome offers the following advantages: (i) histological studies show that the water jet cleans the cutting surface, thus avoiding debris and its uncontrolable effects on cells underneath; (ii) the arrangement enables ongoing electrophysiological recordings from hippocampal slices during the cutting procedure and thus facilitates studies of the functions of neuronal connections; (iii) the device allows even disconnection in cultured nervous tissue overgrowing polyamid grids with 50 microm wide meshes.

Animals↗

PubMed1998

CO2/HCO3(-)-withdrawal from the bath medium of hippocampal slices: biphasic effect on intracellular pH and bioelectric activity of CA3-neurons.

Many studies analyzing interactions of pH and bioelectric activity focus on changes of the extracellular pH, whereas data concerning central neuronal excitability and intracellular pH (pHi) are rare. Here, we report on the spontaneous bioelectric activity and epileptiform activity of CA3-neurons during a procedure which changed pHi. As monitored in BCECF-AM loaded cells, the change from a CO2/HCO3(-)-buffered to a HEPES-buffered medium (CO2/HCO3(-)-withdrawal, hereafter termed W) was associated with a transient intracellular alkalosis (delta pH = 0.2 +/- 0.04) which preceded a sustained intracellular acidosis (delta pH = 0.4 +/- 0.04). Coinciding with this W-induced biphasic shift of pHi a biphasic alteration of spontaneous bioelectric activity was recorded: as a rule, an up to 30 min lasting increase (excitatory phase) preceded a typical sustained suppression (inhibitory phase). This biphasic action was also observed using various in vitro-epilepsy-models (bicuculline, penicillin, caffeine): epileptiform discharges were completely suppressed after an initial increase in frequency. This modulation of bioelectric activity was unlikely due to alterations of the postsynaptic GABA-system as hyperpolarizing GABAA- and GABAB-responses of CA3-neurons were hardly affected. In the majority of the neurons, the initial increase of spontaneous bioelectric activity (excitatory phase) culminated in transient burst periods lasting 5-30 min. These transient burst periods were blocked by NMDA- or AMPA-antagonists: DL-2-amino-5-phosphonovalerate (APV, 50 microM) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 50 microM). The calcium-antagonist verapamil (50 microM) reduced amplitudes of depolarizations and duration of the transient burst periods. The results suggest that the biphasic alteration of pHi modulates the susceptibility of glutamate receptors and voltage-gated calcium-channels, which leads to respective changes of bioelectric activity.

Animals↗