Aquatic environment and differentiation of vibrissae: comparison of sinus hair systems of ringed seal, otter and pole cat.

This study investigated the structure and innervation of the vibrissal systems of the pole cat (Mustela putorius), European otter (Lutra lutra) and ringed seal (Phoca hispida) in order to find adaptations to aquatic environment. The number of myelinated nerve fibers of deep vibrissal nerve (DVN) of the entire vibrissal system was considerably greater in the ringed seal (10x, aquatic mammal) and in the otter (4x, semi-aquatic mustelid) compared to the pole cat which is a terrestrial mustelid. Similarly, the number of neural end organs in the vibrissae of ringed seals was about ten times more numerous than in pole cats. The number of the vibrissae in the heads of otters was almost two times more than in pole cats, and all vibrissa groups had similar structures and innervation. The asymmetrically developed carpal vibrissae of otters were, however, more poorly innervated than the vibrissae of the head and had only smooth musculature. In the ringed seal the orientation of lanceolate end organs differed in different vibrissae, indicating the specialization of these vibrissae for different kinds of sensory functions. Ringed seal vibrissae contain structures which obviously are developed as adaptations to an aquatic environment. These include the modified mitochondria of Merkel cells, with Merkel cell-neurite complexes very often associated ciliated cells, liquid filled vesicles or intercellular spaces below the basal cell layer of the outer root sheath at the ring sinus level, a long upper cavernous sinus and a flattened beaded structure of the vibrissa hairs. As the vibrissae of aquatic mammals have analogous functions to the lateral line organ of fishes, we suggest using the term 'vibrissal sense' for the vibrissa system of aquatic mammals.

Status epilepticus alters zolpidem sensitivity of [3H]flunitrazepam binding in the developing rat brain.

GABA, the main inhibitory neurotransmitter in the adult brain, exerts its effects through multiple GABA(A) receptor subtypes with different pharmacological profiles, the alpha subunit variant mainly determining the binding properties of benzodiazepine site on the receptor protein. In adult experimental epileptic animals and in humans with epilepsy, increased excitation, i.e. seizures, alters GABA(A) receptor subunit expression leading to changes in the receptor structure, function, and pharmacology. Whether this also occurs in the developing brain, in which GABA has a trophic, excitatory effect, is not known. We have now applied autoradiography to study properties of GABA(A)/benzodiazepine receptors in 9-day-old rats acutely (6 h) and sub-acutely (7 days) after kainic acid-induced status epilepticus by analyzing displacement of [(3)H]flunitrazepam binding by zolpidem, a ligand selective for the alpha1beta2gamma2 receptor subtype. Regional changes in the binding properties were further corroborated at the cellular level by immunocytochemistry. The results revealed that status epilepticus significantly decreased displacement of [(3)H]flunitrazepam binding by zolpidem 6 h after the kainic acid-treatment in the dentate gyrus of the hippocampus, parietal cortex, and thalamus, and in the hippocampal CA3 and CA1 cell layers 1 week after the treatment. Our results suggest that status epilepticus modifies region-specifically the pharmacological properties of GABA(A) receptors, and may thus disturb the normal, strictly developmentally-regulated maturation of zolpidem-sensitive GABA(A) receptors in the immature rat brain. A part of these changes could be due to alterations in the cell surface expression of receptor subtypes.

A new metabolite contributing to N-acetyl signal in 1H MRS of the brain in Salla disease.

OBJECTIVE: To determine whether N-acetylaspartate (NAA) is reduced in patients with Salla disease, a neurodegenerative disorder. BACKGROUND: 1H MRS allows the brain metabolism to be studied noninvasively in vivo. N-acetyl (NA) is composed primarily of NAA, which is regarded as a neuronal marker. The NA signal in 1H MRS is reduced in several neurodegenerative disorders. Increased NA signal has thus far only been found in Canavan's disease as a result of NAA accumulation in the brain tissue. In Salla disease, an autosomal recessive free sialic acid storage disorder, N-acetylneuraminic acid (NANA), accumulates in lysosomes of brain tissue. METHODS: The authors studied eight patients with Salla disease (age range, 6 to 44 years) and eight age-matched healthy volunteers using quantitative 1H MRS. The spectra were obtained from two selected 8-cm3 volumes of interest localized in the basal ganglia and in the parietal white matter using conventional 1.5-T MRI equipment. The spectral resonance lines of NA groups, creatine and phosphocreatine (Cr), and choline-containing compounds (Cho) were analyzed quantitatively. All MR images were evaluated to verify the state of myelination. RESULTS: 1H MRS from parietal white matter revealed 34% higher NA and 47% higher Cr concentrations, and a 35% lower Cho concentration in the patients with Salla disease compared with the age-matched control subjects. The patients had a 22% higher water content in their parietal white matter, whereas in the basal ganglia the water concentrations did not differ significantly. In the patients' basal ganglia the Cr concentration was 53% higher. CONCLUSIONS: NAA is considered to be a neuronal marker that, except for Canavan's disease, has been found or assumed to be either stable or reduced. However, in Salla disease the high NA signal may have a contribution from accumulated lysosomal NANA, which offsets the possible loss of NAA. The high Cr is in line with the increased glucose uptake found in our earlier 2-fluoro-2-deoxy-D-glucose-PET study, reflecting increased energy demand. It is worth noting that in a conventional 1H MRS ratio-based analysis these underlying abnormalities would have remained undetected. Our study thus emphasizes the importance of a quantitative assessment of metabolite concentrations in 1H MRS for detecting altered brain metabolism.

Focal and global cortical hypometabolism in patients with newly diagnosed infantile spasms.

OBJECTIVE: To evaluate the occurrence and prognostic importance of focal defects in cerebral cortical glucose metabolism in infants with newly diagnosed symptomatic and cryptogenic infantile spasms. PATIENTS AND METHODS: Ten children with symptomatic and seven with cryptogenic infantile spasms underwent MRI, video-EEG, and PET using fluorodeoxyglucose as a tracer within 2 weeks of diagnosis. PET was repeated at 1 year of age in 12 patients. RESULTS: Cortical hypometabolic foci were found in 13 children (77%) with newly diagnosed spasms (six cryptogenic and seven symptomatic). The hypometabolic foci disappeared in seven of nine reexamined at age 1. The occipital foci disappeared in all (n = 6). Focal findings on PET correlated well with focal findings on video-EEG. There was no difference in quantitative cortical or subcortical glucose metabolic rate at the onset of infantile spasms between children with cryptogenic and symptomatic etiology of spasms. The glucose metabolic rate at the onset of spasms or focal lesions in glucose metabolism did not have prognostic value for seizure outcome. CONCLUSIONS: Infantile spasms are often associated with transient cortical, especially occipital, hypometabolic foci that are not necessarily associated with structural lesions and do not indicate a poor prognosis.

Role of central GABAB receptors in physiology and pathology.

There are two major classes of gamma-aminobutyric acid (GABA)-sensitive receptors: GABAA and GABAB. The GABAA receptor, the better known of the two GABA receptors, is a heterooligomeric complex that forms a chloride channel. Multiple subtypes of the GABAA receptor result from the composition of different subunits. In contrast to the GABAA receptor, the GABAB receptor protein has not been isolated and purified to homogeneity. Various effector systems, however, have been identified for the GABAB receptor using a limited GABAB-specific pharmacologic reportoire. In almost all cases, activated GABAB receptors employ a guanosine triphosphate-binding protein to transduce a signal intracellularly. There may be multiple subtypes of the GABAB receptor. Because the responses elicited by activation of GABAB receptors are small in terms of their intensity and are considered to be modulatory, the role these receptors play in the central nervous system (CNS) may not be very obvious. However, it is our view that in a finely tuned instrument such as the brain, treatment with neuromodulators (drugs that produce slight changes in brain neurochemistry) may be safer than most current drugs. Moreover, neuromodulators may have far greater potential as pharmacotherapeutic agents for CNS disorders. Thus, in this article, we will review the pharmacologic characteristics of the GABAB receptor, known physiologic roles that this receptor plays in the CNS, and the importance of this receptor in certain disease states.

Efflux of 45calcium from cultured primary astrocytes: effects of glutamate receptor agonists and antagonists.

The effects of high-K, glutamate and glutamate receptor agonists on the efflux of Ca2+ were studied in cultured primary astrocytes, prepared from the brains of newborn rats. An increase in efflux of 45Ca2+ of produced by a large extracellular concentration of K+ was effectively inhibited by verapamil, a blocker of voltage-gated Ca2+ channels, suggesting that these cells have functional voltage-sensitive Ca2+ channels. Glutamate and its agonists kainate, quisqualate and N-methyl-D-aspartate (NMDA) stimulated the efflux of preloaded 45Ca2+, in a dose-dependent manner. The most effective agonist was quisqualate followed by glutamate, whereas kainate and NMDA were less potent. In the Mg-free medium, the response to NMDA was significantly increased. The quisqualate receptor agonist (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) also stimulated the efflux, about equally effectively as quisqualate. The glutamate-, quisqualate- and kainate-induced efflux of 45Ca2+ was significantly inhibited by L-glutamatediethylester (GDEE) and that of NMDA by DL-2-amino-5-phosphonopentanoic acid (AP5). The kainate-induced efflux was totally inhibited by verapamil, but that of glutamate only partially. No effect of verapamil was observed on the quisqualate-induced efflux of 45Ca2+. The results imply that, in cultured astrocytes, Ca2+ fluxes induced by glutamate agonists, occur partially through voltage-dependent Ca2+ channels. The extensive release of 45Ca2+ caused by quisqualate was mainly due to a release from internal stores.

D-aspartate release from cerebellar astrocytes: modulation of the high K-induced release by neurotransmitter amino acids.

The properties of D-aspartate release were studied in cerebellar astrocytes (14-15 DIV) in primary cultures in the rat. The spontaneous release of D-aspartate from astrocytes was fast, being further enhanced in Na- and Ca-free (EDTA-containing) media. Kainate, quisqualate, D-aspartate and L-glutamate stimulated the release, whereas L-glutamatediethylester was inhibitory. The release was enhanced by veratridine and high K (50 mM). Substitution of chloride by acetate in the experimental medium did not change the basal release but slightly decreased the potassium-induced release, indicating that the high K-induced D-aspartate release is primarily due to depolarization of cells. The K-stimulated release was independent of extracellular Ca2+ and potentiated by kainate and quisqualate. The effect of kainate was reduced by kynurenate, and that of quisqualate by L-glutamatediethylester. Glycine, taurine and GABA were equally effective in depressing the stimulated release of D-aspartate. The inhibition of GABA could be blocked by GABA antagonists. The results suggest that inhibitory amino acids may be involved in the regulation of glutamate release from cerebellar astrocytes. A further implication is that cerebellar astrocytes possess functional glutamate receptors of kainate and quisqualate subtypes.

Neuronal activity regulates GABAA receptor subunit expression in organotypic hippocampal slice cultures.

The postnatal expression of GABA(A) receptor subunit mRNAs in the rat brain, including the hippocampus, exhibits a unique temporal and regional developmental profile in vivo, which may be altered by external stimuli. Using the in situ hybridization technique we have now studied the in vitro expression of alpha1,alpha2, alpha 4, alpha 5, beta 1, beta 3, gamma 2, and gamma 3 subunit mRNAs of GABA(A) receptors in organotypic hippocampal slices cultured for 7 days. To find out whether neuronal activity regulates the subunit expression, a subset of cultures was chronically treated either with a GABA(A) receptor antagonist picrotoxin, or by a non-N-methyl-D-aspartate (non-NMDA)-receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX). In untreated control cultures, the expression pattern of the subunits varied regionally, the most abundantly expressed subunits being alpha 2 and alpha 5 in all subregions. All studied subunits were expressed in CA3a/b and CA1, whereas in CA3c and in granule cells of the dentate gyrus (DG) no signal of alpha 4 and gamma 3 was detected. The drug treatment differently affected the regional subunit expression. In picrotoxin-treated cultures, the expression of alpha1, alpha 5 and gamma 2 mRNAs was significantly increased in pyramidal cell layers, and in DNQX-treated cultures the expression of alpha2 mRNA in CA3c and DG, and that of beta1 in DG. Changes in the expression of GABA(A) receptor subunit mRNAs in treated cultures suggest that neuronal activity can regulate their regional expression in vitro. Since the expression profile in untreated control cultures closely resembled that observed earlier in vivo, organotypic hippocampal slice cultures could serve as a good model system to study the regulatory mechanisms of receptor expression under well-controlled experimental conditions in the developing hippocampus.

Release of taurine from cultured cerebellar granule cells and astrocytes: co-release with glutamate.

The properties of the release of preloaded [3H]taurine and endogenous taurine were studied with cultured cerebellar granule cells (7-8 days in vitro) and astrocytes (14-15 days in vitro) from the rat. The spontaneous release of taurine from both cell types was slow. The release from both neurons and astrocytes was significantly enhanced by 0.1 mM veratridine, the stimulatory effect being more pronounced in granule cells than in astrocytes. No homo or heteroexchange with extracellularly added taurine or its structural analogues could be detected, suggesting that the efflux is probably not mediated via the membrane transport sites. Kainate stimulated the release more from granule cells than from astrocytes, the effect apparently being mediated by kainate-sensitive receptors. Depolarization of cell membranes by 50 mM K+ induced co-release of endogenous taurine and glutamate from both cell types. Preloaded [3H]taurine was readily released from astrocytes by potassium stimulation. Stimulated release occurred from granule cells if they had been cultured for 4 days with the label but not from the cells preloaded for only 15 min.

Glucose deprivation depolarizes plasma membrane of cultured astrocytes and collapses transmembrane potassium and glutamate gradients.

Primary cultures of astrocytes were used to investigate the effects of glucose deprivation on plasma membrane potential, on the respiration and on the energy status of these cells. Plasma membrane potential, as monitored with a cyanine dye, 3,3'-diethylthiadicarbocyanine, hyperpolarized by about 100% when glucose was added to substrate-deprived cells. The effect of glucose was prevented by iodoacetate or ouabain. In the absence of glucose, cellular adenosine triphosphate/adenosine diphosphate ratio was extensively reduced and pyruvate was unable either to restore energy status or to hyperpolarize the plasma membrane of astrocytes, although it was the preferential substrate for mitochondria within the cells. Glucose deprivation and inhibition of glycolysis or respiration in the presence of glucose caused dramatic decrease in transmembrane potassium ion and L-glutamate gradients. The gradients were not restored in the presence of pyruvate. Thus, aerobic glycolysis, rather than oxidation of pyruvate, is required to maintain maximal plasma membrane potential, adenosine triphosphate/adenosine diphosphate ratios as well as K+ and L-glutamate gradients. This evidence, together with the unresponsiveness of astrocyte respiration to ouabain, indicates a functional dissociation between energy dissipation at the plasma membrane and mitochondrial synthesis of adenosine triphosphate. The results are discussed with regard to the vulnerability of glia at low levels of blood glucose and the contribution of glial dysfunction to development of hypoglycaemic encephalopathy.

86Rubidium release from cultured primary astrocytes: effects of excitatory and inhibitory amino acids.

The effects of high K+, glutamate and its analogue, kainate, on K+ release were studied in primary astrocyte cultures prepared from newborn rat brains using 86Rb+ as a tracer for K+. An increase in 86Rb+ release was observed when the extracellular K+ concentration was elevated (10-40 mM). Glutamate and kainate stimulated the release in a dose-dependent manner, 100 microM concentrations being about as equally effective as high K+ (40 mM). Both compounds also caused an increase in the absorbance of the cyanine dye, 3,3'-diethylthiadicarbocyanine, indicating depolarization of the membrane. No significant Na+-dependent uptake of [3H]kainate occurred in the cells, thus excluding the possibility that depolarization was due to electrogenic uptake of amino acid into the cells. GABA and taurine significantly depressed the high K+- and glutamate-induced 86Rb+ release. Taurine itself caused a small increase in 86Rb+ release and the membrane was depolarized, judging from the increase in the absorbance of the cyanine dye, 3,3'-diethylthiadicarbocyanine. No effect of taurine was observed when the Cl- concentration was reduced in the experimental medium. The results suggest that cultured astrocytes respond by membrane depolarization to high external K+ and to glutamate and kainate. The degree of this depolarization can be modified by the inhibitory amino acids GABA, taurine and glycine, the effect of taurine probably being mediated by an increase in Cl- conductance across the cell membrane. The role of functional receptors for amino acid transmitters and the effects observed are discussed.

Increased brain glucose utilization in Salla disease (free sialic acid storage disorder).

UNLABELLED: Salla disease is an autosomal recessive lysosomal free sialic acid storage disorder characterized by psychomotor retardation and ataxia. MRI studies have revealed evidence of dysmyelination, but the biological mechanism of the brain dysfunction is unknown. METHODS: Nine patients with Salla disease (age 2.5 mo-42 y) presenting the disease in varying degrees of severity were studied by PET using 2-fluoro-2-deoxy-D-glucose (FDG) as a tracer. Local cerebral metabolic rates for glucose (LCMRGlc) in individual brain regions were compared with controls. RESULTS: The FDG PET results showed significantly increased LCMRGlc values in the frontal and sensorimotor cortex and especially in the basal ganglia of the patients. Cerebellar hypometabolism was present in all seven patients with marked ataxia, whereas the less severely affected patients without obvious ataxia had normal or even high glucose uptake in the cerebellum. CONCLUSION: The increased cerebral glucose utilization is a constant finding in Salla disease and may reflect the basic defect of the sialic acid metabolism in this disorder. The FDG PET findings in the cerebellum suggest a correlation between glucose uptake and the severity of the clinical symptoms.

Glutamate release from cerebellar granule cells differentiating in culture: Modulation of the K(+)-stimulated release by inhibitory amino acids.

The properties of l-[(3)H]glutamate release with an emphasis on the modulation by inhibitory amino acids of the potassium-induced release were studied with cerebellar granule cells from 7-day-old rats cultured for 7 or 14 days. Spontaneous glutamate release from cells grown for 7 days was fast, being slightly enchanced in Na(+)-free medium. l-Glutamate, kainate and quisqualate stimulated the release whereas N-methyl-d-aspartate and taurine were without any effect. The potassium-evoked glutamate release was Ca(2+)-dependent and potentiated by l-glutamate and quisqualate. Stimulated release was strongly depressed by glutamatediethylester. This inhibition was antagonized by GABA but not by taurine. GABA and its structural analogues taurine, hypotaurine, ?-alanine and glycine were all equally effective in depressing stimulated glutamate release. The inhibition by GABA could be blocked by GABA antagonist. Both K(+)-evoked release and the kainate-induced release of glutamate were significantly greater in 14-day-old than in 7-day-old cultures, but the other properties of release were similar. The demonstration of calcium-dependent and potassium-stimulated glutamate release from cerebellar granule cells is consonant with the proposed neurotransmitter role of glutamate in these cells. The release could be modulated by both glutamatergic substances and inhibitory amino acids, the effect of GABA probably being mediated by GABAergic receptors.

Taurine and hypotaurine transport in neuroblastoma cells: effects of cations.

The cation requirements of [(3)H]taurine and [(35)S]hypotaurine uptake by cultured neuroblastoma C1300 cells were compared in Krebs-Ringer-Hepes-glucose medium. The uptakes were strictly sodium-dependent at both low and high taurine and hypotaurine concentrations. The omission of Ca(2+) or Mg(2+) ions affected uptakes only marginally. The optimal K(+) concentration was equal to the physiological concentration, whereas abnormally high K(+) levels inhibited similarly taurine and hypotaurine uptake. The sodium dependence curves of both uptakes were sigmoidal in character at low and high taurine and hypotaurine concentrations. Hill plots suggest that two Na(+) ions are coupled with the transfer of one taurine or hypotaurine molecule into neuroblastoma cells. With respect to cation requirements taurine and hypotaurine transports are similar in cultured neuroblastoma cells and display features considered typical of the uptake of a neurotransmitter amino acid.

Mechanisms of kainate-induced region-specific neuronal death in immature organotypic hippocampal slice cultures.

Excessive activation of excitatory amino acid receptors has been implicated in neuronal death in a number of central nervous system insults. We have here investigated, the time course and mechanisms of kainate (KA)- induced neuronal death in immature organotypic hippocampal slice cultures (OHCs) using Fluoro-Jade B (FJB) staining as a marker of cell death, and immunoblotting, immunocytochemistry, and electron microscopy as methods to clarify the mechanisms. After 6 KA treatment (5 microM), no significant neuronal death was detected in any hippocampal subregion, whereas the treatment of 12, 24, and 48 h resulted in neuronal death in the CA3 regions, but not in CA1. The 48 h resting period in normal medium after KA-treatment did not rescue the cells but further increased the number of dead neurons in CA3 as compared to the corresponding acute phase. In Western blotting, the expression levels of the active, 17 kDa form of caspase-3, and the 84-85 kDa cleaved fragment of poly(ADP ribose)polymerase (PARP) were not altered from the control levels. Moreover, no active caspase-3 labelled cells were detected in immunocytochemical study 24 h after KA treatment either in the acute or resting groups. Electron microscopy showed non-apoptotic injury in the CA3a/b pyramidal neurons in KA-treated slices. Our results suggest that KA-induced neuronal death in immature OHCs is a strictly region-specific, irreversible, necrotic process.

Properties of single potassium channels in cultured primary astrocytes.

The patch-clamp technique was used to characterize the single channel ion currents in primary cultures of rat astrocytes. The most dominant channel type, which was found in over half of the inside-out membrane patches, was a potassium channel. The measured reversal potential was -67 mV, which is close to the calculated Nernst potential for potassium ions (-80 mV). These potassium channels activated with bursts of very brief openings. Once activated the channels did not inactivate. The measured probabilities of the channels to be closed showed at least 3 different modes of channel behaviour: one voltage-independent and two voltage-dependent modes. During each activity-mode a 'main' conductance level plus two other conductance levels were observed. In some recordings a pronounced outward rectification could also be seen.

Ethanol-induced Cl- flux in rat cerebellar granule cells as measured by a fluorescent probe.

Cl- fluxes through the GABAA receptor gated ion channels in cultured rat cerebellar granule cells were measured using the chloride-sensitive fluorescent probe SPQ (6-methoxy-N-(3-sulphopropyl)quinolinium) incorporated into the cells. The fluorescence of SPQ is quenched by Cl- ions. The cells were bathed in a low Cl- medium so that the Cl- gradient was directed outward. Ethanol increased the SPQ fluorescence indicating a decrease in intracellular Cl- due to Cl- efflux. Picrotoxin inhibited the effect at low concentrations of ethanol (less than 50 mM) in a concentration dependent manner. The effects of ethanol were potentiated at low concentrations (less than 10 microM) of gamma-aminobutyric acid (GABA), but inhibited at higher concentrations (0.3-2.0 mM). The results support the hypothesis that ethanol may act via the GABAA receptor gated ion channel. The results also suggest that SPQ is a suitable probe for measuring GABAA receptor-coupled Cl- fluxes through the GABAA receptor-gated channels in living cells.

The effect of K+ and glutamate receptor agonists on the membrane potential of suspensions of primary cultures of rat astrocytes as measured with a cyanine dye, DiS-C2-(5).

The cyanine dye DiS-C2-(5) was used to investigate the effect of K+ and glutamate receptor agonists on the membrane potential of whole populations of primary rat astrocytes in suspension. Increasing the external K+ concentration from 5 to 40 mM caused a depolarization of the cells. Ba2+ blocked the response to K+, whereas 4-aminopyridine had no effect on the depolarization. The effect of added external K+ was enhanced by the addition of the neutral K+ ionophore valinomycin. This supports the view that the membrane potential of primary astrocytes is dependent of the K+ gradient, and suggests that the membrane is not ideally permeable to K+ ions. Glutamate caused a depolarization of the cells which was not affected by Ba2+. In the presence of veratridine and ouabain no effect of glutamate was seen. The cells were also depolarized by the glutamate receptor agonists quisqualate, kainate and N-methyl-D-aspartate (NMDA). The response to kainate was blocked by kynurenate, which also diminished the depolarization caused by glutamate. NMDA was effective when added after kainate. The effect of the glutamate receptor agonists tested was generally smaller than that of glutamate itself, and a prior addition of one of the agonists diminished the response to glutamate. The results obtained suggest that cyanine dyes are well suited for investigating the behavior of whole populations of cultured primary astrocytes.

Alpha-receptor and cholinergic receptor-linked changes in cytosolic Ca2+ and membrane potential in primary rat astrocytes.

Both phenylephrine and carbachol caused a sustained increase in Ca2+ influx and intracellular free Ca2+ of primary astrocytes as measured with 45Ca2+ and fura-2. The responses to phenylephrine and carbachol were additive, suggesting that they use different releasable pools of Ca2+. If extracellular Ca2+ was removed by EGTA only a transient rise in cytosolic Ca2+ was seen upon application of the agonists. Both compounds caused depolarization of the astrocyte membrane as determined with the optical probe 3,3-diethylthiadicarboxyamineiodide. Activation of protein kinase C with 12-tetradecanoylphorbol myristate acetate (TPA) or the diacylglycerol analogue dioctanoylglycerol (DiC8) also depolarized the cells. A prior activation of protein kinase C with TPA or DiC8 abolished the depolarizing effect of phenylephrine suggesting that they act through the same mediators. If the cells were made ideally permeable to K+ with the ionophore valinomycin, or the K+ channels had been blocked with Ba2+, neither TPA nor phenylephrine had any significant effect on the membrane potential. Neither TPA nor phenylephrine had any effect on the 86Rb+ equilibrium potential across the cell membrane. The results suggest that the depolarizing effect of these substances could be through a blocking of K+ channels.

Single-channel and whole-cell currents in rat cerebellar granule cells.

The patch-clamp technique was used to study both whole-cell and single-channel currents in cultured rat cerebellar granule cells. In whole-cell recordings under voltage-clamp conditions 3 types of current were found; a transient inward sodium current and a transient and a sustained outward potassium current. Single-channel currents were recorded from both inside-out and outside-out membrane patches. Three types of potassium channel were identified; two non-inactivating channels with unit conductances of 160 and 60 pS and one inactivating channel of 20 pS. No calcium currents were detected.