Involvement of protein kinase C and protein kinase A in the enhancement of L-type calcium current by GABAB receptor activation in neonatal hippocampus.

In the early neonatal period activation of GABAB receptors attenuates calcium current through N-type calcium channels while enhancing current through L-type calcium channels in rat hippocampal neurons. The attenuation of N-type calcium current has been previously demonstrated to occur through direct interactions of the ßγ subunits of Gi/o G-proteins, but the signal transduction pathway for the enhancement of L-type calcium channels in mammalian neurons remains unknown. In the present study, calcium currents were elicited in acute cultures from postnatal day 6-8 rat hippocampi in the presence of various modulators of protein kinase A (PKA) and protein kinase C (PKC) pathways. Overnight treatment with an inhibitor of Gi/o (pertussis toxin, 200 ng/ml) abolished the attenuation of calcium current by the GABAB agonist, baclofen (10 µM) with no effect on the enhancement of calcium current. These data indicate that while the attenuation of N-type calcium current is mediated by the Gi/o subtype of G-protein, the enhancement of L-type calcium current requires activation of a different G-protein. The enhancement of the sustained component of calcium current by baclofen was blocked by PKC inhibitors, GF-109203X (500 nM), chelerythrine chloride (5 µM), and PKC fragment 19-36 (2 µM) and mimicked by the PKC activator phorbol-12-myristate-13-acetate (1 µM). The enhancement of the sustained component of calcium current was blocked by PKA inhibitors H-89 (1 µM) and PKA fragment 6-22 (500 nM) but not Rp-cAMPS (30 µM) and it was not mimicked by the PKA activator, 8-Br-cAMP (500 µM-1 mM). The data suggest that activation of PKC alone is sufficient to enhance L-type calcium current but that PKA may also be involved in the GABAB receptor mediated effect.

Painful neuropathy decreases membrane calcium current in mammalian primary afferent neurons.

Hyperexcitability of the primary afferent neuron leads to neuropathic pain following injury to peripheral axons. Changes in calcium channel function of sensory neurons following injury have not been directly examined at the channel level, even though calcium is a primary second messenger-regulating neuronal function. We compared calcium currents (I(Ca)) in 101 acutely isolated dorsal root ganglion neurons from 31 rats with neuropathic pain following chronic constriction injury (CCI) of the sciatic nerve, to cells from 25 rats with normal sensory function following sham surgery. Cells projecting to the sciatic nerve were identified with a fluorescent label applied at the CCI site. Membrane function was determined using patch-clamp techniques in current clamp mode, and in voltage-clamp mode using solutions and conditions designed to isolate I(Ca). Somata of peripheral sensory neurons from hyperalgesic rats demonstrated decreased I(Ca). Peak calcium channel current density was diminished by injury from 3.06+/-0.30 pS/pF to 2. 22+/-0.26 pS/pF in medium neurons, and from 3.93+/-0.38 pS/pF to 2. 99+/-0.40 pS/pF in large neurons. Under these voltage and pharmacologic conditions, medium-sized neuropathic cells lacked obvious T-type calcium currents which were present in 25% of medium-sized cells from control animals. Altered Ca(2+) signalling in injured sensory neurons may contribute to hyperexcitability leading to neuropathic pain.

Identification of different putative neuronal subtypes in cultures of the superior region of the hippocampus using electrophysiological parameters.

Cultured neurons offer many advantages over a slice preparation for whole-cell patch-clamp studies, such as better control over the environment and space clamp control. However, heterogeneous cultures of neurons present problems in distinguishing the cell type from which recordings are made. The present study uses correlations with data obtained in the hippocampal slice preparation to determine the feasibility of "identifying" different neuronal subtypes in cultures obtained from the superior region of postnatal two- to 13-day-old rat hippocampus. Whole-cell patch-clamp recording in the current-clamp mode after 24-96 h in culture was used to determine if the action potential duration would be a useful criterion in distinguishing cell types. Single action potentials were elicited by a 0.1-0.2 ms, 2-4 nA depolarizing pulse. The average membrane potential and input resistance were -46.8+/-1.2 mV (n = 58) and 576+/-56 Mohms (n = 57), respectively. A frequency distribution of the action potential duration measured at half-maximal amplitude showed four distinct groups of neurons (group 1, 1.36+/-0.03 ms, n = 17; group 2, 2.19+/-0.05 ms, n = 20; group 3, 3.17+/-0.10 ms, n = 16; group 4, 4.36+/-0.13, n = 5). Based on correlations with previous studies using intracellular recording in identified cells in slices, the data suggest that group 1 represents basket cells, group 2 represents vertical cells, group 3 represents a combination of stellate cells and pyramidal cells, and group 4 represents another unidentified class of cells. Further analysis of the fast afterhyperpolarization allows distinction between pyramidal cells and stellate cells in group 3. In contrast to the interneurons in a slice preparation, these cells offer good voltage control and environmental control. Future studies will record from these cells in current-clamp mode to quickly characterize the action potential before switching to voltage-clamp recording to characterize the currents present in the different types of interneurons.

Dissociation of postnatal hippocampal neurons for short term culture.

A method is described to isolate hippocampal neurons from postnatal day 7 to postnatal day 21 rats for use in short term cultures using a combination of enzymatic and mechanical dissociation. A significant fraction of the cells (37.5 +/- 2.3%, n = 10 cultures) were labelled with anti-GABA antibodies and the neurons survived a minimum of 8 days in culture. Patch-clamp recording in the current clamp mode revealed an average membrane potential and input resistance of - 47.6 +/- 2.5 mV and 737 +/- 147 M omega respectively, after 24 h in culture. The cells exhibited normal excitability and fired multiple action potentials in response to a depolarizing pulse. The technique described here provides a good alternative to either the use of embryonic brain tissue for cultures used in electrophysiological studies which may not exhibit the mature phenotype or use of acutely isolated cells which may be unstable or have their channels and receptors modified by enzymatic treatment.

Lambert-Eaton sera reduce low-voltage and high-voltage activated Ca2+ currents in murine dorsal root ganglion neurons.

Voltage-gated Ca2+ channels are categorized as either high-voltage activated (HVA) or low-voltage activated (LVA), and a subtype (or subtypes) of HVA Ca2+ channels link the presynaptic depolarization to rapid neuro-transmitter release. Reductions in transmitter release are characteristic of the autoimmune disorder, Lambert-Eaton syndrome (LES). Because antibodies from LES patients reduce Ca2+ influx in a variety of cell types and disrupt the intramembrane organization of active zones at neuromuscular synapses, specificity of LES antibodies for the Ca2+ channels that control transmitter release has been suggested as the mechanism for disease. We tested sera from four patients with LES. Serum samples from three of the four patients reduced both the maximal LVA and HVA Ca2+ conductances in murine dorsal root ganglion neurons. Thus, even though LES is expressed as a neuromuscular and autonomic disorder, our studies suggest that Ca2+ channels may be broadly affected in LES patients. To account for the specificity of disease expression, we suggest that incapacitation of only a fraction of the Ca2+ channels clustered at active zones would severely depress transmitter release. In particular, if several Ca2+ channels in a cluster are normally required to open simultaneously before transmitter release becomes likely, the loss of a few active zone Ca2+ channels would exponentially reduce the probability of transmitter release. This model may explain why LES is expressed as a neuromuscular disorder and can account for a clinical hallmark of LES, facilitation of neuromuscular transmission produced by vigorous voluntary effort.

Adenosine acting at an A1 receptor decreases N-type calcium current in mouse motoneurons.

The neuromodulator adenosine is known to decrease neurotransmitter release at the neuromuscular junction by activation of an A1 adenosine receptor coupled to a pertussis toxin-sensitive G protein. Among the mechanisms that could contribute to the depression of neurotransmitter release is reduced entry of calcium through channels located in the presynaptic terminal. In the present study, we have examined the effects of adenosine on high-voltage-activated (HVA) calcium currents in motoneurons, the presynaptic cells of the neuromuscular junction. The motoneurons were isolated from embryonic mice, placed in primary tissue culture for 16 hr, and analyzed by means of the whole-cell patch-clamp technique. Adenosine (40 microM) reduced both transient and sustained components of HVA calcium current. This effect was blocked by the A1 antagonist 8-cyclopentyltheophylline (CPT; 100 nM) and was mimicked by the A1 agonist N6-cyclohexyladenosine (CHA; 50 nM to 10 microM) but not by the A2a agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido adenosine (CGS-21680; 1 micron). Pretreatment with pertussis toxin (200 ng/ml, > 16 hr) abolished the depression of HVA calcium current by adenosine receptor activation. Brief (3 min) exposure of the cells to 10 microM omega-conotoxin GVIA irreversibly blocked a part of the HVA current, which can therefore be attributed to N-type channels; the remaining current was unaffected by adenosine receptor activation. Hence, it appears that adenosine decreases only the N-current portion of HVA current and that this inhibition occurs via an A1 receptor linked to a pertussis toxin-sensitive G protein. Other investigators have shown that N-type channels do not play a primary role in eliciting transmitter release at the mammalian neuromuscular junction. Thus, it is uncertain what motoneuronal functions are influenced by adenosine modulation of N-type channels.

Primary structure and functional expression of the omega-conotoxin-sensitive N-type calcium channel from rabbit brain.

The complete amino acid sequence of a rabbit brain calcium channel (BIII) has been deduced by cloning and sequencing the cDNA. The open reading frame encodes 2339 amino acids, which corresponds to an M(r) of 261,167. A phylogenetic tree representing evolutionary relationships indicates that BIII is grouped together with the other rabbit brain calcium channels, BI and BII, into a subfamily that is distinct from the dihydropyridine-sensitive L-type subfamily. Transient expression in cultured skeletal muscle myotubes derived from muscular dysgenic mice demonstrates that the BIII channel mediates an omega-conotoxin-sensitive calcium current with kinetics and voltage dependence like those previously reported for whole-cell N-type current. Cell-attached patch recordings, with isotonic barium as the charge carrier, revealed distinct single channels with an average slope conductance of 14.3 pS.

Developmental expression of voltage-dependent calcium currents in identified mouse motoneurons.

Previous work has shown that during chick embryonic development, large changes occur in the density of specific, motoneuronal calcium currents just prior to the period of naturally occurring motoneuron cell death. Here we report on calcium currents in mouse motoneurons isolated from embryos at the time of peak cell death and also during a subsequent developmental stage when supernumerary synapses are being eliminated. In mouse motoneurons, the density of high-voltage-activated calcium current increases significantly after the phase of cell death, during the period of synapse elimination.

Characterization of voltage-dependent calcium currents in mouse motoneurons.

1. Calcium channel currents were measured with the whole-cell patch clamp technique in cultured, identified mouse motoneurons. Three components of current were operationally defined on the basis of voltage dependence, kinetics, and pharmacology. 2. Test potentials to -50 mV or greater (10 mM external Ca2+) elicited a low-voltage activated T-type current that was transient (decaying to baseline in less than 200 ms) and had a relatively slow time to peak (20-50 ms). A 1-s prepulse to -45 mV produced approximately half-maximal inactivation of this T current. 3. Two high-voltage activated (HVA) components of current (1 transient and 1 sustained) were activated by test potentials to -20 mV or greater (10 mM external Ca2+). A 1-s prepulse to -35 mV produced approximately half-maximal inactivation of the transient component without affecting the sustained component. 4. When Ba2+ was substituted for Ca2+ as the charge carrier, activation of the HVA components was shifted in the hyperpolarizing direction, and the relative amplitude of the transient HVA component was reduced. 5. Amiloride (1-2 mM) caused a reversible, partial block of the T current without affecting the HVA components. 6. The dihydropyridine agonist isopropyl 4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5-nitro-3- pyridine-carboxylate [(+)-SDZ 202-791, 100 nM-1 microM)] shifted the activation of the sustained component of HVA current to more negative potentials and increased its maximal amplitude. Additionally, (+)-SDZ 202-791 caused the appearance of a slowed component of tail current.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophysiological analysis of synaptic transmission between intraocular hippocampus/locus coeruleus co-transplants.

We have examined the establishment of functional connectivity between hippocampal and locus coeruleus co-transplants in oculo. Co-transplants were allowed to mature in oculo for 8-54 weeks following grafting and were subsequently removed from the anterior eye chamber for in vitro electrophysiological studies. Single hippocampal transplants in oculo have been shown to exhibit prolonged synaptic responses to local electrical stimulation, and similar responses were observed in hippocampal neurons following stimulation of the hippocampal portion of hippocampus-locus coeruleus co-transplants. Electrical stimulation of the locus coeruleus attenuated the afterhyperpolarization in hippocampal neurons elicited by the injection of depolarizing current, an effect that has been described previously in hippocampal slices following direct application of norepinephrine, and this effect was antagonized by pretreatment with the beta-adrenergic antagonist timolol. Stimulation of the locus coeruleus also produced both hyperpolarizing and depolarizing changes in the resting membrane potential in hippocampal neurons in 2- and 6-month-old co-transplants. In the 2-month-old co-transplants the responses were primarily depolarizing, and appeared to be mediated by a beta-adrenergic receptor, whereas in the 6-month-old co-transplants the responses were more varied. The results suggest that functional alpha- as well as beta-adrenergic receptors develop in oculo, and that the release of norepinephrine at nerve terminals in double grafts produces effects in the hippocampal neurons which are similar to those observed during superfusion of the hippocampal slice preparation with exogenous norepinephrine.

Electrochemical characterization of stimulated norepinephrine overflow in locus coeruleus-hippocampus double brain grafts grown in oculo.

Rat locus coeruleus-hippocampus double in oculo brain grafts were studied with high-speed electrochemical techniques to characterize stimulus-evoked overflow of norepinephrine. Local pressure ejections of KCl into the locus coeruleus (LC) portion of the double graft elicited electrochemical signals in the hippocampal portion that had NE-like reduction/oxidation current ratios. In contrast, electrical stimulation of the LC part of the double graft did not lead to consistently detectable signals in the hippocampal portion of the transplant. These data demonstrate that LC graft neurons are able to release NE in target tissues when stimulated in an appropriate manner, and that the time course and magnitude of the overflow of NE can be detected with sensitive high-speed electrochemical recording techniques.

Regulation of adrenergic receptors in intraocular hippocampal transplants: role of noradrenergic innervation.

Hippocampal tissue transplanted into the anterior chamber of the eye offers a unique system in which development can be studied in the absence of the noradrenergic innervation. This system was used to determine the extent to which noradrenergic innervation regulates the development of adrenergic receptors. In addition to examining single denervated transplants, transplants grown with innervation from the superior cervical ganglia of the host rat or from locus coeruleus cotransplants were also examined to determine whether the source of norepinephrine and extent of innervation in oculo regulate the development and density of adrenergic receptors. In vitro autoradiographic analysis of ligand binding to both alpha 1- and beta-adrenergic receptors with 125I-BE 2254 and 125I-pindolol, respectively, was used to characterize adrenergic receptors in the intraocular transplants. Quantitative analysis of the receptors showed an up-regulation of both alpha 1- and beta-adrenergic receptors in tissue grown in the absence of norepinephrine, but in general there was not a high degree of correlation between norepinephrine content and receptor density. Although high-performance liquid chromatography (HPLC) analysis of catecholamines revealed higher than normal amounts of norepinephrine in hippocampal transplants innervated by the superior cervical ganglia or a locus coeruleus cotransplant, the density of alpha 1 and beta receptors was quite comparable with values found in the literature for normal adult hippocampus. These results suggest that the relationship between receptor number and density of innervation may differ significantly from what is observed in response to pharmacological manipulation of norepinephrine systems in the adult brain.

In vitro electrophysiological analysis of mature rat hippocampal transplants in oculo.

We have investigated the maturation of isolated rat hippocampus grafted into the anterior chamber of the eye. Electrophysiological responses from transplants were compared to those recorded from the in vitro hippocampal slice preparation. Intracellular recording demonstrated that the passive membrane characteristics of intraocular hippocampal neurons were similar to those of the CA1 pyramidal cells in the in vitro slice preparation. However, the slow after-hyperpolarization which normally follows depolarization-induced action potentials was reduced or completely absent in the intraocular transplants, and the excitatory postsynaptic potential (EPSP) evoked by local stimulation was prolonged. The duration of the EPSP was reduced by perfusion with D-aminophosphonovaleric acid (2.5-50 microM), an N-methyl-D-aspartate receptor antagonist. Normal levels of glutamate decarboxylase (a marker for gamma-aminobutyric acidergic neurons) were found in the transplants, and responses to adenosine, bicuculline, and norepinephrine were similar in the in oculo transplants and in vitro slices. The data suggest that although many properties of hippocampal neurons are intrinsically determined, other aspects of the physiology of mature hippocampus either fail to develop, or develop abnormally in the absence of external inputs in oculo.

Noradrenergic depression of synaptic responses in hippocampus of rat: evidence for mediation by alpha 1-receptors.

Norepinephrine (NE) has been shown to have a biphasic effect on evoked potentials in the CA1 region of the hippocampus of the rat in vitro, with a beta receptor mediating an increase and an alpha receptor eliciting a decrease in the amplitude of the population spike. The purpose of this study was to use selective alpha-adrenergic agonists and antagonists to determine the subtype of receptor mediating the depressant response of NE. The present investigations demonstrated that the selective alpha 1 agonist, phenylephrine (2-50 microM) elicited a dose-dependent depression of the amplitude of the population spike. Clonidine, a relatively selective alpha 2-agonist, also depressed the amplitude of the population spike, but only at concentrations (10 microM) that were inconsistent with a selective action upon alpha 2-receptors. Another alpha 2-agonist, alpha-methylnorepinephrine (100-400 nM) did not depress the amplitude of the population spike. The depressant effect of NE was antagonized by the nonselective alpha antagonist, phentolamine (0.5-50 microM) and the alpha 1-selective antagonist, prazosin (1 microM), but not by the alpha 2-selective antagonist, idazoxan (1-10 microM). Phentolamine and prazosin antagonized the response to phenylephrine but not to clonidine. The depressant effect of NE was not antagonized by the antagonist of serotonin and dopamine, spiperone (100 nM); conversely, the effect of 8-hydroxy-2-(di-n-propylamine) tetralin (50 microM), a 5-HT1A receptor-selective agonist, which also depresses the amplitude of the population spike, was not antagonized by phentolamine (5 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Facilitatory action of etomidate and pentobarbital on recurrent inhibition in rat hippocampal pyramidal neurons.

Biochemical studies have shown that the non-barbiturate anesthetic etomidate can interact in a stereoselective, barbiturate-like fashion with the GABA/benzodiazepine receptor complex, enhancing both benzodiazepine and GABA binding, but its electrophysiological effects upon the mammalian CNS are largely unknown. The present investigations were designed to characterize the electrophysiological effects of etomidate on the recurrent GABAergic inhibitory pathway in the CA1 region of the rat in vitro hippocampal slice and to compare the actions of etomidate to those of pentobarbital. Electrical stimulation of the alveus elicited a biphasic hyperpolarizing response, consisting of an initial bicuculline-sensitive GABAergic IPSP. This was followed by a second component, termed the late hyperpolarizing potential (LHP), which is thought to reflect an increase in potassium conductance. Both pentobarbital (100 microM) and (+)-etomidate (10 microM) markedly increased the duration of the initial GABA-mediated IPSP, and frequently increased its amplitude as well. However, no significant effects of either of these drugs were observed on the LHP. Together with previous biochemical findings, our data suggest that the depressant effects of etomidate and barbiturates on the nervous system may reflect a common action upon a stereoselective receptor site intimately associated with bicuculline-sensitive GABA receptors and the chloride ion channel.

Interaction of monoclonal antibodies to electroplaque acetylcholine receptors with the alpha-bungarotoxin binding site of goldfish brain.

Polyclonal and monoclonal antibodies raised against acetylcholine receptors from Torpedo californica and Electrophorus electricus electroplaque were tested for interaction with the [125I]alpha-bungarotoxin binding protein of goldfish brain. A subset of monoclonal antibodies which recognize the main immunogenic region of the alpha subunit of the Electrophorus acetylcholine receptor interacted at high affinity with the [125I]alpha-bungarotoxin binding protein. Using immunofluorescence, these antibodies were shown to label the same layers of the optic tectum as [125I]alpha-bungarotoxin.