Cultured Mouse Hippocampal Neurons Research Articles

Regulation ofN-Methyl-d-Aspartate Receptor Function by Constitutively Active Protein Kinase C

The ability of the constitutively active fragment of protein kinase C (PKM) to modulate N-methyl-D-aspartate (NMDA)-activated currents in cultured mouse hippocampal neurons and acutely isolated CA1 hippocampal neurons from postnatal rats was studied using patch-clamp techniques. The responses of two heterodimeric combinations of recombinant NMDA receptors (NR1a/NR2A and NR1a/NR2B) expressed in human embryonic kidney 293 cells were also examined. Intracellular applications of PKM potentiated NMDA-evoked currents in cultured and isolated CA1 hippocampal neurons. This potentiation was observed in the absence or presence of extracellular Ca2+ and was prevented by the coapplication of the inhibitory peptide protein kinase inhibitor(19-36). Furthermore, the PKM-induced potentiation was not a consequence of a reduction in the sensitivity of the currents to voltage-dependent blockade by extracellular Mg2+. We also found different sensitivities of the responses of recombinant NMDA receptors to the intracellular application of PKM. Some potentiation was observed with the NR1a/NR2A subunits, but none was observed with the NR1a/NR2B combination. Applications of PKM to inside-out patches taken from cultured neurons increased the probability of channel opening without changing single-channel current amplitudes or channel open times. Thus, the activation of protein kinase C is associated with potentiation of NMDA receptor function in hippocampal neurons largely through an increase in the probability of channel opening.

Block of NMDA receptors by a newly synthesized analog of milnacipran in cultured mouse hippocampal neurons

Immature hippocampal neurons with high input resistances (Rin) are vulnerable to hyperexcitable or epileptogenic conditions. This phenomenon has been suggested to explain the neuroprotective roles of hyperpolarization-activated cation channels (Ih channels) to regulate membrane Rin. In the present study, we tried to electrophysiologically clarify the relationship between membrane Rin and Ih channels and determine the neuroprotective roles of these channels in development. The CA1 neurons from rats (within 3 postnatal weeks) were classified into two groups based on the onset time (shorter or longer than 20 ms) to fire the first action potential (AP) in response to a current injection (100 pA, 800 ms). Neurons with a shorter onset time (Short-OsT), exhibited higher Rin, while neurons with longer onset times (Long-OsT) revealed lower Rin. Unexpectedly, Short-OsT neurons with higher Rin exhibited larger amplitudes of Ih compared with Long-OsT neurons. Furthermore, the application of temporal depolarization stimulus (TDS, −14 mV holding for 150 s) significantly enhanced suprathreshold excitabilities of repetitive APs in Long-OsT but not Short-OsT neurons, suggesting a protective role of Ih channels under high Rin conditions. In the presence of the specific hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker ZD7288, TDS also enhanced the excitability of Short-OsT neurons, suggesting that young CA1 neurons regulate Ih channel expression for neuroprotective modulation against epileptogenic conditions.

Increase of intracellular Ca 2+in cultured mouse hippocampal neurons by trachynilysin, a protein toxin extracted from stonefish (Synanceia trachynis) venom

Increase of intracellular Ca 2+in cultured mouse hippocampal neurons by trachynilysin, a protein toxin extracted from stonefish (Synanceia trachynis) venom

Multiple mechanisms of ketamine blockade of N-methyl-D-aspartate receptors.

The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor is blocked by ketamine, and this action likely contributes to ketamine's anesthetic and analgesic properties. Previous studies suggest that ketamine occludes the open channel by binding to a site located within the channel pore. This hypothesis was examined by investigating the effects of ketamine on single-channel currents from NMDA receptors. The cell-attached and outside-out configurations of the patch clamp technique were used to study NMDA-activated currents recorded from cultured mouse hippocampal neurons. In cell-attached patches, NMDA evoked currents that had an apparent mean open time (tau o) of 3.26 ms. The probability of at least one channel being open (Po') was 0.058. The addition of ketamine (0.1 microM or 1 microM) to the pipette solution decreased Po' to 53% and 24% of control values, respectively. At 1 microM ketamine, this reduction was due to a decrease in both the frequency of channel opening and the mean open time (44% and 68% of control values, respectively). Ketamine did not influence channel conductance and no new components were required to fit the open- or closed-duration distributions. Ketamine (50 microM), applied outside the recording pipette, reduced the opening frequency of channels recorded in the cell attached configuration. This observation suggests that ketamine gained access to a binding site by diffusing across the hydrophobic cell membrane. In outside-out patches, ketamine potency was lower than that observed in cell-attached patches: 1 microM and 10 microM ketamine reduced Po' to 63% and 34% of control values, respectively, and this reduction was due primarily to a decrease in the frequency of channel opening with little change in mean open time. These observations are consistent with a model whereby ketamine inhibits the NMDA receptor by two distinct mechanisms: (1) Ketamine blocks the open channel and thereby reduces channel mean open time, and (2) ketamine decreases the frequency of channel opening by an allosteric mechanism.

Nitric oxide donors enhance neurotrophin-induced neurite outgrowth through a cGMP-dependent mechanism

Nitric oxide (NO), a diffusible and unstable gas, has been implicated in inter- and intra-cellular communication in the nervous system. NO also plays a role in neural development, plasticity and alterations of synaptic function such as long-term potentiation and long-term depression (Gally et al.: Proc NY Acad Sci, 87: 354-355, 1990; Zhuo et al.: Science 260:1946-1950, 1993; Schuman and Madison.: Science 254:1503-1506, 1991; Bruhwyler et al.: Neurosci Biobehav Rev 17:373-384, 1993) some of which likely involve growth and remodelling of neurites. Some actions of NO are mediated directly by protein modification (e.g., nitrosylation) and others by activation of soluble guanylyl cyclase (soluble GC), which increases intracellular levels of guanosine 3',5'-cyclic monophosphate (cGMP). NO is synthesized by the enzyme nitric oxide synthase (NOS), which is induced by treatment of CNS neurons (Holtzman et al.: Neurobiol Disease 1:51-60, 1994) or pheochromocytoma PC12 cells (Hirsch et al.: Curr Biol 3:749-754, 1993) with NGF. NO has been proposed to mediate some of the effects of NGF on PC12 cells by inhibiting cell division (Peunova and Enikolopov: Nature 374:68-73, 1995). In addition, NO can substitute for NGF by delaying the death of trophic factor-deprived PC12 cells through a mechanism that does not involve a cytostatic action (Farinelli et al.: J Neurosci 16:2325-2334, 1996). We investigated whether NO stimulated neurite outgrowth from hippocampal neurons and PC12 cells. Primary cultures of E17 mouse hippocampal neurons co-cultured with neopallial astrocytes were exposed to the NO donors sodium nitrite (100 microM) or sodium nitroprusside (100 nM). After 48 hr, NO donor-treated cultures contained a greater proportion of cells bearing neurites and neurites that were much longer than those found in control cultures. In cultures of PC12 cells, NO donors also enhanced the neuritogenic effects of NGF. The proportion of PC12 cells with neurites 48 hr after exposure to NO donors sodium nitrite (100 microM-10mM) or sodium nitroprusside (100 nM-1 micro M) plus 2.5S nerve growth factor (NGF) was approximately twice the proportion of cells with neurites in sister cultures grown in NGF alone. Neither of the NO donors elicited neurites from the PC12 cells in the absence of NGF. The effects of the NO donors were likely mediated by release of NO since their effects were antagonized by addition of hemoglobin, which avidly binds NO, to the culture medium. The enhancement by NO of NGF-mediated neurite outgrowth in PC12 cells appeared to occur through a cGMP-dependent mechanism. The NO donors stimulated a prompt increase in intracellular cGMP in PC12 cells. Moreover their action was mimicked by addition of the membrane-permeant cGMP analogs 8-Bromo-cGMP (8-Br-cGMP) and para (chlorophenylthio)-cGMP (pCPT-cGMP) to the culture medium and by atrial natriuretic factor which stimulates particulate guanylyl cyclase. The neuritogenic activity of the NO donors was inhibited by LY83583 and methylene blue, inhibitors of guanylyl cyclase. These data imply that NO may act alone or with other growth factors to regulate synapse formation and maintenance by stimulating neurite outgrowth.

Single-channel and whole-cell analysis of ethanol inhibition of NMDA-activated currents in cultured mouse cortical and hippocampal neurons

The effects of 0.1 to 500 mM ethanol on NMDA-activated currents were studied in primary cultures of mouse cortical and hippocampal neurons. In whole-cell recordings the IC 50S for inhibition of NMDA-activated currents by ethanol were 129 mM ± 20 mM in hippocampal neurons and 126 ± 18 mM in cortical neurons. In single-channel recordings from excised outside-out patches of cortical neurons, ethanol inhibited total charge per minute with an IC 50 of 174 ± 23 mM, which was not significantly different from the IC 50S for inhibition of whole-cell current. The reduction in mean open channel lifetime by ethanol was fit by the logistic equation with an apparent IC 50 of 340 ± 28 mM. Analysis of single-channel data indicated that ethanol inhibition of NMDA currents did not involve substantial changes in fast closed state kinetics, changes in open channel conductance, or block of the open channel. At the whole-cell IC 50, of ethanol, mean open channel lifetime would decrease by 28% and frequency of opening would decline by 31% to account for the reduction in current. Single-channel data were consistent with ethanol being an allosteric modulator of gating which reduces agonist efficacy.

Sr2+ and quantal events at excitatory synapses between mouse hippocampal neurons in culture.

1. Whole-cell recording from pairs of adjacent mouse hippocampal neurons in culture was used to study the quantal properties of action potential-evoked excitatory synaptic transmission and to demonstrate the use of Sr2+ in quantifying those properties. 2. In the presence of extracellular Sr2+, excitatory postsynaptic currents (EPSCs) were followed by an after-discharge of miniature excitatory postsynaptic currents (mEPSCs) lasting 1-2 s and generated by evoked asynchronous release of presynaptic quanta of transmitter. Like the EPSC of which it is thought to be an extension, the after-discharge was modulated by procedures expected to modulate Sr2+ influx into the nerve terminal. The number of mEPSCs in the after-discharge was decreased by increasing extracellular [Mg2+], and increased by increasing extracellular [Sr2+] or increasing the number of action potentials used to evoke the after-discharge. 3. EPSCs recorded in media containing either 1 mM Ca2+ or 6 mM Sr2+ were of similar amplitude. Adding Sr2+ to low-Ca2+ media increased EPSC amplitude, while adding Sr2+ to high-Ca2+ media lowered EPSC amplitude. These results suggest that extracellular Sr2+ is less effective than Ca2+ in supporting quantal release. 4. The levels of extracellular Ca2+, Mg2+ and Sr2+ were adjusted so that most after-discharge mEPSCs were discrete and comparable in numbers to the quantal events that contributed to the corresponding evoked EPSCs. In a series of twenty-five pairs of neurons, the mean amplitude of mEPSCs recorded at -80 mV was 35 +/- 10 pA and the mean coefficient of variation was 0.50 +/- 0.10 (range, 0.26-0.62). The mEPSC amplitude histogram was positively skewed. 5. In ten pairs of neurons, the mean and variance of EPSCs and mEPSCs and quantal content were determined from samples of more than 100 evoked events (in superfusion solutions containing (mM): 0.5 Ca2+, 2 Sr2+ and 10 Mg2+) and mean quantal content was determined from the ratio of amplitudes of the mean EPSC and mEPSC. A binomial quantal analysis produced values of 2-12 for Napp (apparent number of independent synapses) and 0.25-0.75 for Papp (apparent probability of releasing a quantum at one of those synapses). These parameters predicted the number of observed failures. The observed coefficient of variation for quantal content predicted the observed coefficient of variation of the EPSC amplitude when the coefficient of variability of quantal amplitude of after-discharge mEPSCs was taken into account. 6. In six pairs of neurons, where more than 250 evoked events were recorded, the observed amplitude histogram for EPSCs could be approximated by a predicted amplitude distribution generated from the estimated binomial parameters and an empirical function describing the amplitude distribution of after-discharge mEPSCs. 7. The observation that parameters derived from mEPSCs that contribute to the Sr(2+)-generated after-discharge can predict the shape of the EPSC amplitude distribution and a quantal content consistent with the observed failure rate and EPSC amplitude variance, suggests that this subset of mEPSCs has the same properties as the quantal events released around the time of the peak of the corresponding EPSCs. The use of Sr2+ to evoke after-discharges of mEPSCs should allow unambiguous determination of the extent to which modification of synaptic strength is pre- or postsynaptic.

Glutamate as a hippocampal neuron survival factor: an inherited defect in the trisomy 16 mouse.

The survival of cultured mouse hippocampal neurons was found to be greatly enhanced by micromolar concentrations of the excitatory neurotransmitter glutamate. Blockade of kainate/AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) glutamate receptors increased the rate of neuron death, suggesting that endogenous glutamate in the cultures promotes survival. Addition of glutamate (0.5-1 microM) further increased neuron survival, whereas glutamate in excess of 20 microM resulted in increased death. Thus, the survival vs. glutamate dose-response relation is bell-shaped with an optimal glutamate concentration near 1 microM. We found that hippocampal neurons from mice with the genetic defect trisomy 16 (Ts16) died 2-3 times faster than normal (euploid) neurons. Moreover, glutamate, at all concentrations tested, failed to increase survival of Ts16 neurons. In contrast, the neurotrophic polypeptide basic fibroblast growth factor did increase the survival of Ts16 and euploid neurons. Ts16 is a naturally occurring mouse genetic abnormality, the human analog of which (Down syndrome) leads to altered brain development and Alzheimer disease. These results demonstrate that the Ts16 genotype confers a defect in the glutamate-mediated survival response of hippocampal neurons and that this defect can contribute to their accelerated death.

Effects of aluminium chloride on mouse hippocampal neurons in culture

Effects of aluminium chloride on mouse hippocampal neurons in culture

Inositol 1,4,5-trisphosphate and ryanodine receptor distributions and patterns of acetylcholine- and caffeine-induced calcium release in cultured mouse hippocampal neurons

The distributions of inositol 1,4,5-trisphosphate and ryanodine receptors (InsP3R and RyR) and the patterns of increase in intracellular calcium ion concentration ([Ca2+]i) elicited by their activation were compared in cultured hippocampal neurons. InsP3R and RyR were labeled using specific antibodies and formed small aggregations in the somata and dendrites of pyramidally shaped neurons. Both receptors were densest in somata. In dendrites the InsP3R and RyR were not distributed homogeneously; InsP3R was found in all regions, while RyR was least dense in fine processes. Increases in [Ca2+]i elicited by acetylcholine (to activate InsP3 receptors via muscarinic receptors) and caffeine (to stimulate ryanodine receptors) were measured in dendrites using Ca(2+)-sensitive fluorescent dyes and confocal microscopy. Ca2+ responses to acetylcholine were transient and observed in proximal and distal dendritic regions. In contrast, caffeine-induced responses were sustained and restricted to proximal dendrites. Thus the patterns of calcium release in fine dendrites mirrored the distributions of InsP3R and RyR. Calcium responses to both acetylcholine and caffeine were observed in the absence of external calcium and thus were dependent on Ca2+ release. Ca2+ responses showed localized fluctuations and variations in response delay times. Sequential activation of InsP3R and RyR in somata resulted in mutual occlusion of Ca2+ release. The existence of InsP3-gated and Ca(2+)-induced Ca2+ release as spatially distinct, but mutually interacting, mechanisms may be important in the generation of oscillations and propagating Ca2+ waves in somata and dendrites of hippocampal neurons.

Single channel properties of the 5-HT 3 subtype of serotonin receptor in primary cultures of rodent hippocampus

Whole cell and outside-out patch clamp recordings were obtained from primary cultures of rat and mouse hippocampal neurons. Serotonin (5-HT) evoked short-latency fast inward currents in about 5% of neurons. These currents reversed near 0 mV, showed inward rectification, and were inhibited by the 5-HT 3 receptor antagonists ICS 205–930, ondansetron and tubocurare. 5-HT activated single channel currents in 14 of 24 membrane patches obtained from neurons which showed 5-HT 3-activated whole cell currents; mean amplitude of channel openings was 0.95 ± 0.09 pA at −100 mV. Chord conductances measured at −80 and −160 mV were 8.3 and 10.5 pS, respectively. 5-HT-induced unitary currents were blocked reversibly by tubocurare (1 μM), ICS 205–930 (30 mM) and ondansetron (100 nM). Thus, single-channel properties of 5-HT 3 receptors in hippocampal neurons are similar to those present in peripheral neurons and do not exhibit solely the sub-picosiemen conductance characteristic of the neuroblastoma and neuroblastoma-derived cloned 5-HT 3 receptor.

Perturbation of intracellular calcium and hydrogen ion regulation in cultured mouse hippocampal neurons by reduction of the sodium ion concentration gradient

Na(+)-Ca2+ exchange has been identified as a mechanism for regulation of intracellular Ca ion concentration ([Ca2+]i) in neurons of invertebrates and vertebrates, but for mammalian central neurons its role in restoration of resting [Ca2+]i after transient increases induced by stimulation has been less clear. We have examined the recovery of [Ca2+]i following K+ depolarization and glutamate receptor activation of cultured mouse hippocampal neurons using the Ca(2+)-sensitive dye Fura-2. Reduction of the transmembrane Na+ gradient by removal of external Na+ slowed the recovery of neurons from imposed Ca2+ loads. We observed that [Ca2+]i regulation was disrupted more severely when N-methyl-D-glucamine (N-MG), Tris, or choline rather than Li+ replaced external Na+. Additional disruption of intracellular pH regulation by substitutes other than Li+ may account for this difference. Measurement of [Ca2+]i and [H+]i (using the H(+)-sensitive dye BCECF) during glutamate receptor activation indicated that Ca2+ influx resulted in production of intracellular H+, and that Li+ but not N-MG could prevent cytoplasmic acidification on removal of external Na+. We also observed that intracellular acidification alone was sufficient to slow recovery from Ca2+ load. We conclude, therefore, that Na(+)-Ca2+ exchange contributes to recovery of [Ca2+]i after stimulation leading to Ca2+ entry into hippocampal neurons, and that Na(+)-H+ exchange limits the acidification (and secondary increase in [Ca2+]i) that accompanies Ca2+ influx. We suggest that because both Na(+)-Ca2+ and Na(+)-H+ exchangers will be compromised during ischemia and hypoglycemia, increased intracellular H+ may synergize with cytoplasmic Ca2+ to potentiate excitotoxic neuronal death.

A heparin-binding domain in the amyloid protein precursor of Alzheimer's disease is involved in the regulation of neurite outgrowth

The amyloid protein precursor (APP) of Alzheimer's disease is synthesized as an integral transmembrane protein that is released from cells in culture following proteolytic cleavage. The function of released APP is not known, although there is evidence that the protein may bind to components of the extracellular matrix (ECM). In the present study, substratum-bound APP stimulated neurite outgrowth in cultures of chick sympathetic and mouse hippocampal neurons. This effect was dependent upon the presence of substratum-bound heparan sulfate proteoglycans (HSPG). The effect of APP on neurite outgrowth was comparable to that of laminin. A 14 K N-terminal fragment of APP was found to bind heparin and a region close to the N-terminus of APP (residues 96-110) identified as a potential heparin-binding domain based on secondary structure predictions and molecular modeling. Mutagenesis of three basic residues (lysine-99, arginine-100, and arginine-102) resulted in a recombinant protein (APPhep) with decreased heparin-binding capacity. A peptide homologous to the heparin-binding domain was synthesized and found to bind strongly to heparin and to inhibit binding of 125I-labeled APP to heparin (IC50 approximately 10(-7) M). The peptide blocked the effect of APP on neurite outgrowth (IC50 approximately 10(-7) M), whereas two other peptides homologous to other domains in APP had no effect. The results indicate that the binding of APP to HSPG in the ECM may stimulate the effects of APP on neurite outgrowth.

Astroglial modulation of transient potassium current development in cultured mouse hippocampal neurons

Hippocampal neurons exhibit three voltage-gated potassium currents, two transient currents and a delayed rectifier, that influence numerous aspects of electrogenesis including action potential duration and accommodation to sustained depolarization. These currents, termed A-, D-, and K-currents, respectively, can be distinguished based on kinetics, steady state inactivation characteristics, and sensitivity to 4-aminopyridine (see Wu and Barish, 1992b). We have compared the voltage-gated potassium currents in voltage-clamped pyramidally shaped cultured hippocampal neurons growing on or touching glial fibrillary acidic protein-expressing astroglia (termed on-glia or touching-glia neurons, respectively) with those in similar neurons growing directly on a coated glass substrate (termed off-glia neurons). We observed differences in the wave forms of total potassium current that correlated with the extent of astroglial contact. After 5-7 d in culture, A-current amplitude in off-glial neurons was approximately 19% of that of neurons growing in the normal (for culture) on-glia configuration. D-current amplitude tended to be larger in these off-glia neurons. Neurons in contact with astroglia had greater membrane area than off-glia neurons. Comparison of current densities (current at a fixed voltage normalized to capacitance and expressed in units of pA/pF) indicated that A-currents were the major component of transient potassium current in on- and touching-glia neurons, while D-currents were more dominant in off-glia neurons. Astroglia influenced membrane currents by a surface- or extracellular matrix-associated mechanism, rather than by free diffusion of a soluble factor, as differences were observed between closely adjacent neurons on the same coverslip. Living glia were required, as potassium currents in neurons grown on dried or methanol-fixed glia resembled those of off-glia neurons. On-glia neurons in cultures treated with an RNA synthesis inhibitor [DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole)] for 5-7 d had reduced whole-cell capacitance and A-current amplitude. This effect was localized to DRB actions on underlying astroglia, not on the neurons. Action potentials elicited by current injection varied with astroglial contact. In on-glia neurons with relatively larger A-currents a delay was seen in the onset of firing after depolarization. In contrast, action potentials in off-glia neurons rose smoothly after initiation of depolarization. We conclude that astrocytes modulate the appearance of transient potassium currents in hippocampal pyramidal neurons by inducing development of A-current.(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of phenytoin on cytoskeletal organization and cell viability of immortalized mouse hippocampal neurons

Phenytoin (PHT) is a commonly used anticonvulsant drug; several side effects have been described, including morphological changes in brain cortex and cerebellar neurons and teratogenic lesions in infants of epileptic mothers. Evidence of other authors indicate that PHT may exert its action through the modification of phosphorylation patterns of cytoskeletal polypeptides. We have studied the influence of the anticonvulsant drug phenytoin on immortalized mouse hippocampal neurons in culture. This was done by means of MTT-assays, immunocytochemical and immunoblot analyses, measurements of cell metabolism, measurements of the length of neuronal processes, and electron microscopy. A distinct and pronounced effect of PHT could be characterized with regard to the formation of neuronal processes, involving malfunction of an assembly-mechanism of cytoskeletal constituents. These accumulated within appendages (blebs) or cytoplasmic condensations, instead of forming normally organized processes. However, PHT did not interfere with bulk synthesis of cell proteins and specific cytoskeletal components.

Haloperidol interacts with the strychnine-insensitive glycine site at the NMDA receptor in cultured mouse hippocampal neurones

N-Methyl-D-aspartate (NMDA)-evoked responses in voltage-clamped hippocampal neurones in culture were reversibly, but not completely, attenuated on superfusion with micromolar concentrations (0.1–100 μM) of haloperidol with an IC 50 (± S.E.M.) value of 1.9 ± 0.2 μM (n = 7). In contrast, kainate- and (RS)-α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-evoked responses were relatively unaffected on application of 50 μM haloperidol. The NMDA receptor antagonist action of haloperidol was neither competitive in nature nor voltage-dependent but was reduced upon elevation of the extracellular concentration of glycine. Furthermore, in the absence of added glycine haloperidol (at 0.1 μM) frequently potentiated NMDA-evoked responses. Haloperidol thus appears to be a partial agonist for the strychnine-insensitive glycine site associated with the NMDA receptor-channel complex.

The ES-242s, novel N-methyl-D-aspartate antagonists of microbial origin, interact with both the neurotransmitter recognition site and the ion channel domain.

ES-242-1 approximately 5 are novel microbial bioxanthracenes which do not contain nitrogen. The ES-242s inhibited the binding of [3H]TCP and [3H]CGS19755 to the N-methyl-D-aspartate (NMDA) receptor complex. They had no effect on the binding of the specific ligands for the non-NMDA receptor. The biochemical and pharmacological properties of ES-242-1 were fully examined since it is the most potent of the five compounds. ES-242-1 is highly specific for the NMDA receptor; it has no effect on other receptors. Kinetic analyses indicated that ES-242-1 inhibited the binding of [3H]TCP and [3H]CGS19755 in a competitive manner, respectively, suggesting that ES-242-1 interacts with both the transmitter recognition site and the channel domain. ES-242-1 selectively inhibited NMDA-induced Ca2+ influx in primary cultures of mouse hippocampal neurons. ES-242-1 also specifically blocked the increase in cyclic GMP accumulation induced by NMDA or L-glutamate in rat cerebellar slices. In a concentration range of 0.1-1.0 microM, ES-242-1 was as potent as MK-801 in preventing glutamate-induced neurotoxicity in primary cultures of mouse hippocampal neurons. These results show that ES-242-1 is a potent and specific antagonist for the NMDA receptor. The antagonistic properties of the ES-242s appear to be due to a novel mechanism of action at the NMDA receptor.

Development of intracellular calcium responses to depolarization and to kainate and [formula omitted] in cultured mouse hippocampal neurons

We have investigated the initial appearance of voltage-gated Ca channels and kainate- and NMDA-type glutamate receptors in cultured embryonic mouse hippocampal neurons. The Ca-dependent fluorescence change of the dye fura-2 was used as a sensitive assay for the presence of functional channels and receptors. Expression of functional NMDA receptors was observed on some hippocampal neurons as early as E14. By the equivalent of E15–16, 40–50% of cells responded to K o-depolarization (50 mM), indicating the presence of functional voltage-gated Ca channels, approximately 20% of cells responded to kainate (50 μM), and just under 20% responded to NMDA (50 μM; in the presence of glycine and strychnine). By the equivalent of the end of the embryonic period 70–80% of cells responded to all 3 stimuli. As approximately 20% of cells in these cultures are glia, these data indicate that by the time of birth close to 100% of neurons express functioning kainate and NMDA receptors, and voltage-gated Ca channels. Increases in [Ca 2+] i in embryonic neurons after application of NMDA were sensitive to APV and to external Mg, as are responses in mature neurons. The IC 50 for block by external Mg of the [Ca 2+] i increase induced by NMDA was 130 μM, and there was a slight positive correlation between the amplitude of the response to NMDA and sensitivity to external Mg.

Actions of ketamine, phencyclidine and MK‐801 on NMDA receptor currents in cultured mouse hippocampal neurones.

1. Stable N-methyl-D-aspartic acid (NMDA) receptor-mediated currents in cultured mouse hippocampal neurons were evoked by 20 ms pressure pulse applications of L-aspartate, repeatedly applied at 30 or 40 s intervals, to the cell body region of the neurone. We have characterized the voltage- and use-dependent blockade of the currents by three dissociative anaesthetics: ketamine, phencyclidine (PCP) and MK-801 in mouse hippocampal neurones grown in dissociated tissue culture. 2. We have used a simple model of the blockade, based on the 'guarded receptor hypothesis' to interpret our data. The model assumes that receptors are maximally activated at the peak of the response with an open probability (Po) approaching 1, that there is no desensitization and that the blocking drug only associates with, or dissociates from, receptor channels which have been activated by agonist (e.g. open channels). 3. The model allows us to estimate forward and reverse rate constants for binding of the blockers to open channels from measurements of the steady-state level of blockade and the rate of change of the current amplitude per pulse during onset and offset of blockade. As predicted by the model, the estimated reverse rate was independent of blocker concentration while the forward rate increased with concentration. Changing the level of positively charged ketamine (pKa 7.5) tenfold by changing pH from 6.5 to 8.5 caused a corresponding change in the forward rate while having no effect on the reverse rate. Most of the voltage dependence of the blockade could be accounted for by reduction of the reverse rate by depolarization. 4. Estimated forward rate constants for ketamine, PCP and MK-801 were similar to one another when measured under similar conditions and were 3 x 10(4) - 3 x 10(5) M-1 S-1. Most of the differences in potency of the three blockers could be accounted for by differences in the reverse rate constants which were approximately 0.2, 0.03 and 0.003 s-1 for ketamine, PCP and MK-801, respectively. The estimated rate constants actually are the product of the rate constants and 1/Po. Suggestions that maximum Po is much less than 1 for NMDA channels imply that both forward and reverse rate constants of blockade may in fact be larger than we have calculated. However, their magnitudes, relative to one another, are unaffected by this consideration. 5. The reverse rate constant of blockade increased at positive potentials. This increase was prevented when the neurone was loaded with N-methyl-D-glucamine, an impermeant cation which prevented outward currents.(ABSTRACT TRUNCATED AT 400 WORDS)

Automated image analysis for counting unstained cultured neurones

A fully automated image analysis technique was developed for counting the number of live or fixed, unstained neurones present in a representative region of a cell culture dish. A dish containing cultured mouse hippocampal neurones was placed on the motorized stage of an inverted microscope, and the neurones were visualized using Hoffman modulation contrast optics. The resulting image was digitized, and processed by subtracting the background illumination, low pass filtering, thresholding, then deleting objects whose areas fell outside a specified range. Two threshold levels were used, each with its own area range, and the two resulting binary images were combined. The number of objects in the combined image was counted. The number of cells in each field was also counted manually, and the processing was repeated on a series of 100 fields covering a representative region of the dish. The automated counts were highly correlated with the manual counts for each of the 12 culture dishes examined in this study. The correlation coefficient was calculated for the manual and automated counts from each dish, and the values ranged from 0.91 to 0.97. Six of the dishes were treated with the envelope protein of the human immunodeficiency virus (gp120), which reduces survival of neurones in this system. The six treated dishes were found to have significantly fewer neurones than the six control dishes, using either manual or automated counting techniques.