Neonatal Rat Hippocampal Neurons Research Articles

The General Anesthetic Isoflurane Depresses Synaptic Vesicle Exocytosis

General anesthetics have marked effects on synaptic transmission, but the mechanisms of their presynaptic actions are unclear. We used quantitative laser-scanning fluorescence microscopy to analyze the effects of the volatile anesthetic isoflurane on synaptic vesicle cycling in cultured neonatal rat hippocampal neurons monitored using either transfection of a pH-sensitive form of green fluorescent protein fused to the luminal domain of VAMP (vesicle-associated membrane protein), (synapto-pHluorin) or vesicle loading with the fluorescent dye FM 1-43. Isoflurane reversibly inhibited action potential-evoked exocytosis over a range of concentrations, with little effect on vesicle pool size. In contrast, exocytosis evoked by depolarization in response to an elevated extracellular concentration of KCl, which is insensitive to the selective Na+ channel blocker tetrodotoxin, was relatively insensitive to isoflurane. Inhibition of exocytosis by isoflurane was resistant to bicuculline, indicating that this presynaptic effect is not caused by the well known GABA(A) receptor modulation by volatile anesthetics. Depression of exocytosis was mimicked by a reduction in stimulus frequency, suggesting a reduction in action potential initiation, conduction, or coupling to Ca2+ channel activation. There was no evidence for a direct effect on endocytosis. The effects of isoflurane on synaptic transmission are thus caused primarily by inhibition of action potential-evoked synaptic vesicle exocytosis at a site upstream of Ca2+ entry and exocytosis, possibly as a result of Na+ channel blockade and/or K+ channel activation, with the possibility of lesser contributions from Ca2+ channel blockade and/or soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated vesicle fusion.

Inhibition of sustained repetitive firing in cultured hippocampal neurons by an aqueous fraction isolated from Delphinium denudatum

In this report we investigated the effects of the aqueous fraction (AF) isolated from Delphinium denudatum on sustained repetitive firing in cultured neonatal rat hippocampal pyramidal neurons. Blockade of SRF is one of the basic mechanisms of antiepileptic drugs (AED) at the cellular level. The effects of aqueous fraction (0.2–0.6 mg/ml) were compared with the prototype antiepileptic drug, phenytoin (PHT). Using the whole cell current-clamp technique, sustained repetitive firing was elicited in neurons by a depolarizing pulse of 500 ms duration, 0.3 Hz and 0.1–0.6 nA current strength. Similar to phenytoin, aqueous fraction reduced the number of action potentials (AP) per pulse in a concentration-dependent manner until no action potentials were elicited for the remainder of the pulse. There was a corresponding use-dependent reduction in amplitude and V max (velocity of upstroke) of action potentials. The V max and amplitude of the first action potential was not affected by phenytoin, while aqueous fraction exhibited concentration-dependent reduction. At 0.6 mg/ml aqueous fraction reduced V max to 58–63% and amplitude to 16–20% of the control values. The blockade of sustained repetitive firing by aqueous fraction was reversed with hyperpolarization of membrane potential (−65 to −75 mV) while depolarization of membrane potential (−53 to −48 mV) potentiated the block. The results suggest that aqueous fraction blocks sustained repetitive firing in hippocampal neurons in a use-dependent and voltage-dependent manner similar to phenytoin. However, unlike phenytoin, which interacts preferably with the inactive state of the Na + channel, the compounds present in aqueous fraction apparently also interact with the resting state of the Na + channels as suggested by dose-dependent reduction of V max and amplitude of first AP. We conclude that aqueous fraction contains potent anticonvulsant compounds.

Anticonvulsant effect of FS-1 subfraction isolated from roots of Delphinim denudatum on hippocampal pyramidal neurons.

The effects were investigated of a partially purified subfraction (FS-1) isolated from Delphinium denudatum on sustained repetitive firing (SRF) of cultured neonatal rat hippocampal pyramidal neurons. The blockade of sustained repetitive firing is one of the basic mechanisms of antiepileptic drugs at the cellular level. Using the whole cell current-clamp technique, sustained repetitive firing was elicited in pyramidal neurons under study by a depolarizing pulse of 500 ms duration, 0.3 Hz and 0.1-0.6 nA current strength. FS-1 (0.01-0.06 mg/mL) reduced the number of action potentials per pulse in a dose-dependent manner until no action potentials were elicited for the remainder of the pulse. There was a corresponding use-dependent reduction in amplitude and Vmax of action potentials. The Vmax of action potential 1 exhibited a dose-dependent reduction. At a dose of 0.06 mg/mL FS-1 reduced Vmax to 29%-38% and amplitude to 16%-20 % of the control values. The blockade of sustained repetitive firing by FS-1 was reversed by hyperpolarization of the membrane potential (-65 to -75 mV) while depolarization of the membrane potential (-53 mV to -48 mV) potentiated the block. The results suggest that FS-1 blocks sustained repetitive firing in hippocampal neurons in a use-dependent and voltage-dependent manner similar to the prototype anticonvulsant drug, phenytoin. However, unlike phenytoin, which binds preferably to the inactive state, the compounds present in FS-1 also interacted with the resting state of the Na+ channels by reducing Vmax of action potential 1. The results indicate that the partially purified FS-1 subfraction of Delphinium denudatum contains a potent anticonvulsant compound.

Modulation of large conductance calcium-activated potassium channels from rat hippocampal neurons by glutathione

We have investigated the modulation of neuronal large conductance Ca 2+-activated K + channels by glutathione. Single channel recordings were made from cultured neonatal rat hippocampal neurons by using excised inside-out patch clamp method. Glutathione, a physiological sulfhydryl specific reducing reagent, increased channel activities in concentration dependent manner with half activation concentration of 710 μM. Conversely, oxidized form of gluthathione inhibited channel activities with half inhibition concentration of 520 μM. Out results provide direct evidence that when neuronal large conductance Ca 2+-activated K + channels are exposed to reducing or oxidizing environments, channel activities are increased or decreased in opposite directions due to the redox modification. This may constitute an important regulatory mechanism of neuronal Ca 2+-activated K + channel activities.

Facilitation of miniature GABAergic currents by ruthenium red in neonatal rat hippocampal neurons.

The whole cell configuration of the patch-clamp technique was used to study the modulation gamma-aminobutyric acid (GABA)-mediated postsynaptic currents by ruthenium red in CA3 hippocampal neurons in slices obtained from postnatal (P) days P6-P10 old rats. In the presence of kynurenic acid (1 mM), ruthenium red (100 microM) completely blocked stimulus-elicited GABA-mediated postsynaptic currents and reduced by 50% the amplitude of the spontaneous ones. Ruthenium red (100 microM) increased the frequency but not the amplitude of miniature GABAergic currents recorded in the presence of tetrodotoxin (1 microM) and kynurenic acid (1 mM), an effect that was prevented by heparin (100 microM). Ruthenium red did not modify the kinetics of miniature postsynaptic currents and the currents induced by exogenous application of GABA (10 microM) in the presence of tetrodotoxin, suggesting that its action was presynaptic in origin. The effects of ruthenium red on quantal GABA release was independent of external calcium. In a nominally Ca2+-free solution the potentiating effect induced by this polyvalent cation on miniature postsynaptic currents was still present. Intracellular calcium stores were not involved in ruthenium red action, because this polyvalent cation was able to facilitate miniature currents also in the presence of thapsigargin (10-20 microM). These results indicate that ruthenium red has a dual action on GABA release from GABAergic interneurons: it reduces the amplitude of spontaneous events and increases the frequency of miniature currents. The former effect is calcium-dependent, whereas the latter is calcium independent.

The anesthetic steroid (+)-3alpha-hydroxy-5alpha-androstane-17beta-carbonitrile blocks N-, Q-, and R-type, but not L- and P-type, high voltage-activated Ca2+ current in hippocampal and dorsal root ganglion neurons of the rat.

High voltage-activated (HVA) Ca2+ current (ICa) was recorded from neonatal rat hippocampal and adult rat dorsal root ganglion neurons. In both cell types, (+)-3alpha-hydroxy-5alpha-androstane-17beta-carbonitrile [(+)-ACN], a neuroactive steroid, had no effect on nifedipine- (L-type) or omega-agatoxin IVA- (P-type) sensitive ICa. Selective blockade of N-type current with omega-conotoxin GVIA and of Q-type current with omega-conotoxin MVIIC indicated that (+)-ACN inhibits both N- and Q-type current components in both cell types. Current persisting after blockade of all other current components (R-type) was also sensitive to (+)-ACN. Half-blockade of (+)-ACN-sensitive HVA current occurred in the range of 3-25 microM, with N-type current somewhat more sensitive than Q- or R-type. The (+)-ACN enantiomer, (-)-ACN, and pregnanolone were somewhat less effective at inhibiting total HVA current than (+)-ACN, whereas several steroid analogs, including alfaxalone, were relatively ineffective at inhibiting total HVA current. Neither guanosine-5'-O-(2-thio)diphosphate nor guanosine-5'-O-(3-thio)triphosphate altered the ability of (+)-ACN to inhibit HVA current in dorsal root ganglion neurons, indicating that (+)-ACN acts directly on Ca2+ channels. The partial selectivity exhibited by (+)-ACN among different HVA current components suggests that manipulations of steroid analogues may be a useful strategy in the generation of more selective, more potent, small-molecular-weight HVA channel blockers.

Properties and the cytoskeletal control of Ca(++)-independent large conductance K+ channels in neonatal rat hippocampal neurons.

A member of the family of Ca(++)-independent large conductance K+ channels (termed BK channels) was identified in patch clamp experiments with cultured neonatal rat hippocampal neurons. Permeation was characterized (at 5 mmol/l external, 140 mmol/l internal K+; 135 mmol/l external Na+) by a conductance of 107 pS, a ratio PNa/Pk approximately 0.01, and outward rectification near the reversal potential. Channel activity was not voltage-dependent, could not be reduced by internal TEA or by a shift of internal pH from 7.4 to 6.8, i.e., discriminating features within the Ca(++)-independent BK channel family. Cytosolic proteolysis abolished the functional state of hippocampal Ca(++)-independent BK channels, in contrast to the pronase resistance of hippocampal Ca(++)-activated BK channels which suggests structural dissimilarities between these related channels. Cytoskeletal alterations had an activating influence on Ca(++)-independent BK channels and caused a 3-4-fold rise in Po, but patch excision and channel isolation from the natural environment provoked the strongest increase in Po, from 0.07 +/- 0.03 to 0.73 +/- 0.04. This activation process operated slowly, on a minute time scale and can be most easily explained with the loss of a membrane-associated inhibitory particle. Once activated, Ca(++)-independent BK channels reacted sensitively to a Mg-ATP supplemented brain tissue extract with a Po decline, from 0.60 +/- 0.06 to 0.10 +/- 0.05. Heated extracts failed to induce significant channel inhibition, providing evidence for a heat-unstable molecule with reassociates with the internal channel surface to reestablish channel inhibition. A dualistic channel control, by this membrane-associated molecule and by the cytoskeleton seems possible.

Activation of metabotropic glutamate receptors increase the frequency of spontaneous GABAergic currents through protein kinase A in neonatal rat hippocampal neurons.

1. The tight-seal whole cell recording technique was used to study the effects of the metabotropic glutamate receptor (mGluR) agonist, trans-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD) on spontaneous gamma-aminobutyric acid (GABA)-mediated synaptic currents in neonatal rat CA1 hippocampal neurons in slices obtained from postnatal (P) days P6-P12. 2. Bath application of t-ACPD (3-30 microM), in the presence of kynurenic acid, induced a concentration-dependent increase in frequency but not in amplitude of spontaneous GABAergic currents. The mean frequency ratio (t-ACPD 10 microM over control) was 2.6 +/- 1 (mean +/- SD), whereas the mean amplitude ratio was 1.1 +/- 0.3. 3. The effect of t-ACPD was partially antagonized by the mGluR antagonist (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG, 1 mM). 4. t-ACPD (10-30 microM) did not modify the frequency of miniature GABAergic synaptic currents recorded in tetrodotoxin (the mean frequency ratio of t-ACPD over control was 0.7 +/- 0.3). 5. Forskolin (30 microM), but not its analogue 1,9 dideoxyforskolin (30 microM), mimicked the effect of t-ACPD. Similar effects were obtained with 3-isobutyl-1-methylxanthine (IBMX, 200 microM). 6. The potentiating effect of t-ACPD on spontaneous GABAergic currents was prevented by Rp-cAMPS (30 microM), a specific antagonist of protein kinase A. This suggests that mGluRs localized at the soma-dendritic level of GABAergic interneurons and positively coupled to cyclic AMP may modulate GABA release during a critical period of postnatal development.

The influence of membrane potential on chloride channels activated by GABA in rat cultured hippocampal neurons.

Chloride currents were activated by a low concentration of GABA (0.5 microM) in neonatal rat hippocampal neurons cultured for up to 14 days. Currents elicited by 0.5 microM GABA in neurons, voltage-clamped using the whole-cell technique with pipettes containing 149 mM Cl-, reversed close to 0 mV whether pipettes contained 144 mM Na+ or 140 mM Cs+, and were blocked by 100 microM bicuculline. Current-voltage curves showed outward rectification. Single channel currents appeared in cell-attached patches when the pipette tip was perfused with pipette solution containing 0.5 microM GABA and disappeared when a solution containing 100 microM bicuculline plus 0.5 microM GABA was injected into the pipette tip. The channels showed outward rectification and, in some patches, had a much lower probability of opening at hyperpolarized potentials. The average chord conductance in 10 patches hyperpolarized by 80 mV was 7.8 +/- 1.6 pS (SEM) compared with a chord conductance of 34.1 +/- 3.5 pS (SEM) in the same patches depolarized by 80 mV. Similar single channel currents were also activated in cell-free, inside-out patches in symmetrical chloride solutions when 0.5 microM GABA was injected into the pipette tip. The channels showed outward rectification similar to that seen in cell-attached patches, and some channels had a lower probability of opening at hyperpolarized potentials. The average chord conductance in 13 patches hyperpolarized by 80 mV was 11.8 +/- 2.3 pS (SEM) compared with 42.1 +/- 3.1 pS (SEM) in the same patches depolarized by 80 mV.

Developments in three-dimensional stereo brightfield microscopy.

We present recent developments of a widefield computer/microscope system and image reconstruction algorithm for producing three-dimensional (3D) increased depth of field images in the form of brightfield stereo pairs of thick specimens. The theoretical principle of this image reconstruction technique is based on Weiner-type inverse filtering. A number of extensions and refinements to our previous work have included further testing of the system with a broader class of specimens and the implementation of several pragmatic refinements important for future 3D microscopy systems. These refinements include histogram modification routines for improving visualization, a preprocessing routine to eliminate edge artifacts due to circular convolution and other effects, stereo viewing angle optimization, a rule of thumb estimate for the axial sampling rate, and incorporation of a variation of the Fast Fourier Transform and filtering operations that significantly reduce computational time. Images of spyrogyra, neonatal rat hippocampal neurons, and cervical/vaginal cell smears are presented to show the utility of these methods for 3D visualization. The primary advantages of these methods are that they operate with an ordinary transmitted light microscope and are inexpensively implemented on a personal computer with reasonable computation time.

Modulation of GABA-mediated Synaptic Potentials by Glutamatergic Agonists in Neonatal CA3 Rat Hippocampal Neurons.

Intracellular recordings were made from slices of adult and neonatal hippocampal neurons. During the first 2 weeks of life the majority of pyramidal cells exhibited spontaneous gamma-aminobutyric acid (GABA)-mediated synaptic potentials, which were depolarizing at birth and became hyperpolarizing by the end of the first postnatal week. These synaptic potentials were reduced in frequency or blocked by the N-methyl-d-aspartate (NMDA) receptor antagonist d(-)2-amino-5-phosphonovalerate (AP-5, 50 microM) (13/15 cells). The non-NMDA antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 5 - 10 microM) abolished the GABA-mediated synaptic potentials in all the cells tested (n=12), Superfusion of l-glutamate (up to 100 microM) increased the frequency of both depolarizing and hyperpolarizing GABA-mediated synaptic potentials. This effect was reduced by AP-5 or dl-2-amino-7-phosphonoheptanoate (AP-7, 50 microM) and fully blocked by concomitant application of AP-5 (50 microM) and CNQX (5 - 10 microM). NMDA (0.5 - 2 microM) increased the frequency of the GABA-mediated synaptic potentials. These effects were blocked by AP-5 (50 microM) and by bicuculline (10 microM). Quisqualate (100 - 300 nM), (RS)-alpha-amino-3-hydroxy-5-methyl-4-izopropionate (AMPA, 100 - 300 nM) and kainate (100 nM) also increased the frequency of the GABA-mediated synaptic potentials. These effects were blocked by CNQX (5 - 10 microM) and by bicuculline (10 microM) but not by AP-5 (50 microM). In the presence of tetrodotoxin (TTX, 1 microM), quisqualate (up to 300 nM), AMPA (up to 500 nM) and kainate (100 nM) had no effect on membrane potential or input resistance. In conclusion, our experiments suggest that, in early postnatal life, NMDA and non-NMDA receptors located on GABAergic interneurons modulate GABAergic synaptic potentials.

Neonatal hippocampal neurons, retrogradely labeled with granular blue, survive intracerebral grafting and explantation to tissue culture

Previous studies have shown that developing neocortical neurons labeled in situ by retrograde axonal transport of the fluorescent dye Granular Blue can retain this dye for at least 2 months essentially without fading or leakage. In this study, retrograde labeling with Granular Blue was used to label neonatal rat hippocampal neurons prior to intracerebral grafting to uninjected littermates, and explantation as slice cultures. Hippocampal regio inferior and hilar neurons labeled through their developing commissural axons were found to survive axotomy and subsequent grafting and explantation for at least 3 and 8 weeks, respectively. The labeling helped define the developmental differentiation of the neurons at the time of manipulation and provides a new method for identification of a specific population of transplanted or explanted neurons.