Sustained depolarizing potentials in reticulospinal axons during evoked seizure activity in lamprey spinal cord

1. Intracellular recordings were made from lamprey reticulospinal axons (Müller axons) during seizures evoked by electrical stimulation of the isolated spinal cord in saline containing either 0 Cl or 1 mM picrotoxin. The seizures had tonic and clonic-phases similar to ictal seizures in mammalian brain. 2. During seizures Müller axons were depolarized by 10-15 mV. These seizure-depolarizations were not due to any direct effect of the evoking stimulus on the Müller axons themselves nor were they initiated by an accumulation or extracellular potassium. 3. A decrease in axonal input resistance occurred during a seizure-depolarization. Also, the amplitude of a seizure-depolarization was decreased by depolarizing the axon 5-15 mV with injected current. Further, hyperpolarizing the axon increased the amplitude of the seizure-depolarization, but the growth flattened out beyond 30-40 mV of hyperpolarization. The decrease in input resistance during the seizure-depolarization and the dependence of the response amplitude on axonal membrane potential suggested that the seizure-depolarization was an excitatory synaptic potential. However, the failure of the seizure-depolarization amplitude to continue to grow at membrane potentials greater than 30 mV negative to the resting potential was not consistent with this interpretation. 4. A synaptic conductance change as the cause of the seizure-depolarization was ruled out by setting the axonal membrane potential at different levels with injected current and monitoring the input resistance of the axon before and during seizure-depolarizations. It was found that no change in input resistance occurred during the seizure-depolarization when the axon was hyperpolarized more than approximately 30 mV, the same potential at which the growth in the response amplitude ceased. From analysis of these data and the passive current-voltage properties of Müller axons it is concluded that the seizure-depolarization is not a chemical synaptic potential, but rather the result of the passive injection of depolarizing current into the axons. 5. The source of the depolarizing current which flows into Müller axons during seizures is probably paroxysmal action-potential activity in spinal motoneurons and interneurons, many of which are electrically coupled to Müller axons.     

Stimulation of transmitter release by guanidine derivatives.

Guanidine and its alkylated derivatives stimulate transmitter release in the sciatic nerve-sartorius muscle preparation of the frog, the excitatory nerve-opener muscle preparation of the crayfish cheloped, and the phrenic nerve-diaphragm preparation of the rat. The mechanism underlying this action has been studied. The amplitude of the end-plate potential was increased by guanidine derivatives. Guanidine, methylguanidine, 1,1-dimethylguanidine, ethylguanidine and propylguanidine were almost equally potent with the threshold concentration of 0.1–0.2mM. Formamidine and aminoguanidine were 20–40 times less potent than guanidine. This effect was reversed slowly after washing with normal solution free of drugs. However, the frequency of miniature end-plate potentials was not affected by high concentrations (5–10mM) of guanidine, and no change was observed in membrane potential and input resistance following application of guanidine, methylguanidine and 1,1-dimethylguanidine. The iontophoretically induced acetylcholine potential was suppressed by methylguanidine, 1,1-dimethylguanidine, ethylguanidine and propylguanidine, and was restored quickly upon washing with normal solution. Based on the actions on the end-plate potential and on the iontophoretic acetylcholine potential, the order of the potencies of guanidine derivatives in stimulating transmitter release were estimated as: methylguanidine, 1,1-dimethylguanidine, ethylguanidine, propylguanidine > guanidine > aminoguanidine, formamidine. Amylguanidine and octylguanidine had no presynaptic effect.Increasing external calcium concentration from 1.8 mM to 6 or 12 mM did not prevent the stimulating action of guanidine and 1,1-dimethylguanidine on the end-plate potential. Methylguanidine and 1,1-dimethylguanidine did not seem to affect the time constant of decay of neuromuscular facilitation in the rat. The quantal content of the end-plate potential in the rat preparation was increased by methylguanidine and 1,1-dimethylguanidine, and this increase was due entirely to an increase in the immediately available store of acetylcholine in the nerve terminal. The probability of release remained constant. It is unlikely that this action is due to the inhibition of mitochondrial respiration.     

Ca(2+) channel-mediated currents in retinal glial (Müller) cells of the toad (Bufo marinus)

Whole-cell voltage-clamp recordings were used to detect voltage-gated Ca(2+) channels in freshly isolated retinal glial (Müller) cells of the toad (Bufo marinus). Using Ca(2+) ions (2 mM) as charge carriers (in the presence of 1 mM Mg(2+)), no inwardly directed currents could be observed during the application of depolarizing voltage steps. However, after omitting the divalent cations from the bath solution, large-amplitude inwardly directed currents were evoked that were carried by Na(+) ions, and were mediated by at least two different kinds of Ca(2+) channels, transient low voltage-activated (LVA) channels and sustained high voltage-activated (HVA) channels. While the LVA currents activated at potentials positive to -90 mV and peaked at -40 mV, the HVA currents activated positive to -60 mV and peaked at -20 mV. It is concluded that Müller glial cells of the toad express distinct types of voltage-gated Ca(2+) channels that may be activated, under certain conditions, close to physiological membrane potentials.     

Microelectrode study of spreading depression (SD) in frog retina-Müller cell activity and [K+] during SD--.

By means of K+ microelectrodes, K+ potentials and [K+] were studied in frog retinas conditioned for SD by CL--free Ringer's. A marked increase in [K+]o was observed during SD, the increase being maximal in the inner plexiform layer (up to 50 mM or more) and subsiding towards the distal retina. The extracellular K+ potential change during SD and also the graded K+ potential changes produced locally by iontophoretic injection of SD-stimulant chemicals resembled the membrane potential changes in Müller cells under the same conditions, suggesting that the Müller cells act as K+ electrodes. In retinas conditioned by Cl--free Ringer's Müller cells were swollen. This allowed intracellular recording with K+ electrodes in Müller cells to reveal that upon SD the Müller cells immediately start to cleanse the extracellular space of excess K+ which is probably a product of pathologically enhanced synaptic activity in the inner plexiform layer. The mechanism of SDP, the field potential change associated with SD, is discussed from a proposed model.     

Activity-dependent modulation of axonal excitability in unmyelinated peripheral rat nerve fibers by the 5-HT(3) serotonin receptor

Activity-dependent fluctuations in axonal excitability and changes in interspike intervals modify the conduction of trains of action potentials in unmyelinated peripheral nerve fibers. During inflammation of a nerve trunk, long stretches of axons are exposed to inflammatory mediators such as 5-hydroxytryptamine [5-HT]. In the present study, we have tested the effects of m-chlorophenylbiguanide (mCPBG), an agonist at the 5-HT(3) serotonin receptor, on activity- and potential-dependent variations in membrane threshold and conduction velocity of unmyelinated C-fiber axons of isolated rat sural nerve segments. The increase in axonal excitability during application of mCPBG was much stronger at higher frequencies of action potentials and/or during axonal membrane hyperpolarization. The effects on the postspike recovery cycle also depended on the rate of stimulation. At an action potential frequency of 1 Hz or in hyperpolarized axons, mCPBG produced a loss of superexcitability. In contrast, at 0.33 Hz, a small increase in the postspike subexcitability was observed. Similar effects on excitability changes were found when latency instead of threshold was recorded, but only at higher action potential frequencies: at 1.8 Hz, mCPBG increased conduction velocity and reduced postspike supernormality. The latter effect would increase the interspike interval if pairs of action potentials were conducted along several cm in an inflamed nerve trunk. These data indicate that activation of axonal 5-HT(3) receptors not only enhances membrane excitability but also modulates action potential trains in unmyelinated, including nociceptive, nerve fibers at high impulse rates.     

Nerve growth factor-induced hyperexcitability of rat sensory neuron in culture

Abnormal spontaneous firing of primary sensory neurons is considered to be a cause of neuropathic pain. However, pathogenic mechanisms of hyperexcitable sensory neurons in neuropathic model animals are unclear. We examined effects of chronic treatment of nerve growth factor (NGF), one of candidate mediators for the pathogenesis, on excitability of sensory neurons by voltage-clamped recording in a cell-attached configuration. From rat dorsal root ganglion (DRG) neurons cultured without NGF, only stable holding currents without spontaneous firing activity were recorded. On the other hand, more than 20% neurons cultured in the presence of NGF for more than 3 days showed spontaneous current spikes at frequencies between 0.1 and 5 Hz. Each spikes had an initial inward phase followed by the outward phase, resulted from spontaneous transient depolarization followed by transient hyperpolarization. These spontaneous spikes were abolished by tetrodotoxin, lidocaine and reduction of extracellular concentration of Na+ from 154 mM to 100 mM, in all-or-none fashion, suggesting that spontaneous current spikes reflected spontaneous action potentials. From these results, it became evident that DRG neurons of adult rats had a nature to respond to NGF and obtained the abnormal hyperexcitability to fire spontaneously.     

Synaptic transfer at a vertebrate central nervous system synapse.

1. The relation between presynaptic depolarization and transmitter release was examined at a synapse between a Müller axon and a lateral interneurone in the spinal cord of the lamprey. Two micro-electrodes, one for passing current and the other for recording the resulting voltage change, were placed in the presynaptic axon; a single electrode for recording the post-synaptic potential produced by release of transmitter was placed in the post-synaptic cell. 2. When action potentials were blocked with tetrodotoxin, brief depolarizing pulses in the presynaptic fibre were as effective as the action potential had been in producing transmitter release. 3. The release process had an apparent threshold depolarization of 40-50 mV and saturated at presynaptic depolarizations of the order of 100 mV. Increasing the duration of the presynaptic pulse increased the maximum level of release. 4. Displacing the presynaptic voltage recording electrode from the position of synaptic contact toward the current passing electrode increased the apparent depolarization required to produce a given level of transmitter release. This shift in the input-output relation was consistent in magnitude with the voltage attenuation between the presynaptic recording electrode and the synapse expected from the space constant of the fibre. 5. The effect of conditioning hyperpolarization and depolarization of the presynaptic fibre on subsequent transmitter release by brief depolarizing pulses was examined. No effect was observed when the presynaptic recording electrode was in the region of synaptic contact. When the presynaptic electrode was not so positioned, conditioning effects were observed which depended on electode position and could be attributed to changes in the space constant of the presynaptic fibre. No conditioning effects were observed on transmitter release by the action potential.     

5-Hydroxytryptamine depresses reticulospinal excitatory postsynaptic potentials in motoneurons of the lamprey.

Application of 5-hydroxytryptamine (5-HT) to the lamprey spinal cord in vitro reversibly depressed the chemical component of excitatory post-synaptic potentials recorded intracellularly in motoneurons and evoked by stimulation of single reticulospinal Müller cells. The depression could be produced either by local application of small volumes of 10 mM 5-HT to the surface of the spinal cord or by bath-application of 1 or 10 microM 5-HT. No effect on the input resistance of the postsynaptic cells or their sensitivity to glutamate, the suspected transmitter at this synapse, could be detected, suggesting the possibility of a presynaptic action of 5-HT at this synapse in the lamprey.     

Long-term nerve excitability changes by persistent Na(+) current blocker ranolazine

The persistent Na(+) current (Na(p)) in peripheral axons plays an important functional role in controlling the axonal excitability. Abnormal Na(p) is believed to contribute to neurodegeneration and neuropathic pain, and thus it is an attractive therapeutic target. To assess the chronic behavior of selective Na(p) blockade, axonal excitability testing was performed in vivo in normal male mice exposed to ranolazine by recording the tail sensory nerve action potentials (SNAPs). Seven days after administering ranolazine i.p. (50mg/kg) daily for 1 week, nerve excitability testing showed decreased strength-duration time constant in the ranolazine group in comparison to the control (P<0.03). This change is explained by the long-term effects of ranolazine on Na(p). Importantly, ranolazine showed no effect on other ion channels that influence axonal excitability. Further study is needed to assess the chronic Na(p) blockade as a useful therapy in peripheral nerve diseases associated with abnormal nerve excitability.     

Spontaneous synaptic potentials and quantal release of transmitter in the stellate ganglion of the squid

1. Several kinds of synapses have been studied in the stellate ganglion of the squid.2. A small electric coupling was found between giant fibres in different stellar nerves.3. Post-synaptic potentials recorded from the cells of small axons are composite, indicating that there are converging inputs from several pre-ganglionic fibres.4. Spontaneous miniature synaptic potentials were recorded from all types of synapses. Miniature potentials in the cells of small axons had a slower time course than those in the giant fibre system.5. Tetrodotoxin abolished nerve impulses in the ganglion but did not prevent the spontaneous quantal release of transmitter from the terminals, or its action on the post-synaptic membrane; nor did it prevent the increase in rate of release produced by depolarization of the presynaptic fibre.6. Glutamate depolarized the giant fibre when applied iontophoretically to the synaptic region. Similar doses applied intracellularly were without effect.     

Divalent cations and the action potential of leech Retzius cells

Summary The effects of Sr, Ba, Mn, La and Co on the action potential of the leech Retzius cell were examined using intracellular recording techniques. A previous paper showed that these cells could fire Ca-dependent action potentials in Na-free solution provided TEA was present (Kleinhaus and Prichard, 1975). Under the same conditions Sr 1.5-20 mM was capable of substituting as a current carrier. Ba 2–25 mM added to normal Ringer prolonged the duration and increased the amplitude of the action potential of the Retzius cell, and supported action potentials without requiring TEA in Na-free solutions. The overshoots of the Sr- and Ba-dependent action potentials varied with a slope of 40 mM and 75 mV, respectively per 10-fold change in divalent cation concentration. Mn and La selectively blocked that portion of the action potential resulting from an inward movement of Ca, Sr or Ba without affecting the Na-dependent depolarization. The actions of Ca 1 mM on Sr-dependent action potentials were compatible with reversible competitive antagonism. In conclusion the findings: 1. support the proposition that outward K current must be blocked in order for divalent cations to dominate the Retzius cell's behavior during excitation. 2. characterize the divalent cation conductance channel as pharmacologically distinct from the Na conductance channel in the Retzius cell and similar to those described in several other excitable membranes. 3. suggest that the current carrying divalent cations probably flow through the same channel.     

Early postnatal Müller cell death leads to retinal but not optic nerve degeneration in NSE-Hu-Bcl-2 transgenic mice

Topographically localized over-expression of the human Bcl-2 protein in retinal glial Müller cells of a transgenic mice (line 71) leads to early postnatal apoptotic Müller cell death and retinal degeneration. Morphological, immunohistological and confocal laser microscopic examination of transgenic and wild-type retinas were achieved on paraffin retinal sections, postnatally. Apoptosis occurs two to three days earlier in the internal nuclear layer of transgenic retinae, than in wild-type littermates. In parallel there was a progressive disappearance of transgenic Hu-Bcl-2 over-expression, as well as of the Müller cell markers, cellular retinaldehyde-binding protein and glutamine synthetase. This phenomenon led to retinal dysplasia, photoreceptor apoptosis and then retinal degeneration and proliferation of the retinal pigment epithelium. The optic nerve, however, remains intact. Two complementary observations confirm the pro-apoptotic action of Bcl-2 over-expression in Müller cells: (i) in the peri-papillary and peripheral regions where the transgene Bcl-2 is not expressed, cellular retinaldehyde-binding protein or glutamine synthetase immunostaining persist and Müller glia do not die; and (ii) the retina conserves a normal organisation in these two regions inspite of total retinal degeneration elsewhere.We conclude that retinal dysplasia and degeneration are linked to primary Müller cell disruption. Besides its generally accepted anti-apoptotic function, over-expression of Bcl-2 also exerts a pro-apoptotic action, at least in immature Müller glia. One may suppose that Bcl-2 translocation resulting in its over-expression in retinal Müller cells could be a putative mechanism for early retinal degeneration.     

Identification of interneurons with contralateral, caudal axons in the lamprey spinal cord: synaptic interactions and morphology

1. As part of a continuing investigation of the organization of the spinal cord of the lamprey, propriospinal interneurons with axons projecting contralaterally and caudally (CC interneurons) were surveyed with intracellular recordings. 2. CC interneurons were identified by recording their axon spikes extracellularly in the spinal cord during intracellular stimulation of the cell body. The axon projections of Cc interneurons were confirmed after intracellular injection and development of horseradish peroxidase. 3. Intracellular stimulation of CC interneurons produced synaptic potentials in myotomal motoneurons, lateral interneurons and other CC interneurons that lay caudally on the opposite side of the spinal cord. Most CC interneurons were inhibitory, but some were excitatory. 4. CC interneurons were divided into three classes on the basis of reticulospinal Müller cell inputs. CC1 interneurons were excited by the ipsilateral Müller cell B1 and the contralateral Mauthner cell. CC1 interneurons were inhibitory. They were excited polysynaptically by ipsilateral sensory dorsal cells and were inhibited by contralateral dorsal cells. They were distinguished morphologically by having no rostral axon branch and no contralateral dendrites. CC1 interneurons were phasically active during fictive swimming with their peak depolarizations preceding those of myotomal motoneurons by about 0.15 cycle. 5. CC2 interneurons were also inhibitory, but they were distinguished from CC1 interneurons by their excitation from the ipsilateral Müller cells B2-4 nd by their thin rostral and thicker caudal axonal branches on the contralateral side of the spinal cord. 6. CC3 interneurons were excitatory, and they were inhibited by the ipsilateral Müller cell I1. CC3 interneurons could have contralateral dendrites and bifurcating axons, and they had lower average axonal conduction velocities than CC1 and CC2 interneurons. 7. Inhibitory CC interneurons may be important for motor coordination in the lamprey. Movements of the lamprey body during reflexes and swimming consist of contraction and relaxation of myotomal muscles on opposite sides of the body. By being coactive with ipsilateral myotomal motoneurons, inhibitory CC interneurons could contribute to the inhibition of contralateral motoneurons during these movements.     

A modified minimal hemolymph-like solution, HL3.1, for physiological recordings at the neuromuscular junctions of normal and mutant Drosophila larvae

The hemolymph-like HL3 saline(Stewart et al., 1994)and standard saline(Jan & Jan, 1976)are two widely used bathing solutions for physiological recordings at the Drosophila larval neuromuscular junction. It has been established that longevity of larval preparations is better maintained in HL3 saline. However, HL3 can produce results that are inconsistent with previous findings in standard saline, particularly on temperature sensitivity and membrane excitability phenotypes. In wild-type larvae, the excitatory junctional potentials(EJPs)in standard saline(containing 4 mM Mg(2+)and 1.8 mM Ca(2+))were not blocked by a temperature increase up to 39-40 degrees C, consistent with unimpaired larval locomotion below these temperatures. However, in HL3 saline(containing 20 mM Mg(2+)and 1.5 mM Ca(2+)), EJPs were blocked at 30 degrees C. As for temperature-sensitive mutants nap(ts)and para(ts), the EJP-blocking temperatures were decreased from about 29 and 33 degrees C in standard saline to about 23 and 26 degrees C in HL3, respectively. Compound action potential recordings confirmed that segmental nerve action potentials were more readily blocked by a temperature increase in HL3 than in standard saline. Axonal excitability was suppressed in HL3 even at room temperatures, as evidenced by a lengthened refractory period in wild-type larvae. Similar suppression occurred for the hyper-excitable double mutant eag Sh, which maintained high-frequency spontaneous EJPs in standard saline but showed a rapidly declining EJP frequency in HL3. Application of HL3 saline also strongly suppressed the prolonged transmitter release following removal of repolarization mechanisms by K(+)channel blockers or by the eag Sh mutation previously described in standard saline. These discrepancies suggest that the high divalent cation content in HL3 may confer a surface charge screening effect to suppress nerve membrane excitability. We found that a minimal adjustment of the HL3 saline, decreasing the Mg(2+)ion concentration from 20 to 4 mM, was sufficient to resolve the discrepancies. While retaining the longevity of the larval neuromuscular preparation, the modified HL3 saline(HL3.1)restored the established wild-type EJP properties as well as phenotypes of several widely used temperature-sensitive and hyper-excitable mutants previously documented in standard saline.     

Reticulon3 expression in rat optic and olfactory systems

Reticulon3 (RTN3), which belongs to a reticulon family, is first isolated from the retina, but little is known about its function. We investigated the distribution of RTN3 in rat retina and olfactory bulb by immunohistochemistry. In the retina, Müller cells highly expressed RTN3. The expression level of RTN3 in the optic nerve was high in the embryo, but low in the adult. In the olfactory system, RTN3 was highly expressed in the olfactory nerve both in developmental and adult stages. Further, RTN3 was co-localized with synaptophysin in tubulovesicular structures in the developing axon of cultured cortical neurons. These results suggest that RTN3 may play an important role in the developing axons and also in some glial cells such as Müller cells.     

Model of electroretinogram b-wave generation: a test of the K+ hypothesis

Generation of the electroretinogram b-wave is simulated with a computer model representing a dark-adapted amphibian retina. The simulation tests the K+ hypothesis of b-wave generation, which holds that b-wave currents arise from localized Müller cell depolarizations generated by light-evoked increases in extracellular K+ concentration, [K+]o. The model incorporates the following components and processes quantitatively: 1) two time-dependent K+ sources representing the light-evoked [K+]o increases in the inner and outer plexiform layers, 2) a time- and [K+]o-dependent K+ sink representing the [K+]o decrease in the rod inner segment layer, 3) diffusion of released K+ through extracellular space, 4) active K+ reuptake and passive K+ drift across the Müller cell membrane, 5) spatial variations in the tortuosity factor and the volume fraction of extracellular space, 6) an extraretinal shunt resistance. Müller cells are modeled with 1) cytoplasmic resistance, 2) spatial variations in membrane permeability to K+, and 3) a membrane potential specified by the Nernst equation and transmembrane current flow. For specified K+ source and sink densities, the model computes [K+]o variations in time and retinal depth. Based on these [K+]o distributions, Müller cell potentials, current source-density profiles, and intraretinal and transretinal voltages are calculated. Imposed [K+]o distributions similar to those seen experimentally during the b-wave lead to the generation of a transient b-wave response and to a prolonged Müller response in the model system. These response time courses arise because the b-wave is dominated by the short-lived distal [K+]o increase, while the Müller response primarily reflects the long-lived proximal [K+]o increase. Current source-density distributions and intraretinal voltage profiles that are generated by the model at the peak of the b-wave closely resemble experimental results. The model generates a realistic slow PIII potential in response to prolonged [K+]o decreases in the distal retina and reproduces the K+ ejection results of Yanagida and Tomita (50) accurately. Simulations also suggest that tissue damage caused by K+-selective micropipettes in experimental preparations can lead to an underestimation of the distal [K+]o increase. The simulations demonstrate that the spatiotemporal properties of intraretinal b-wave voltages and currents and Müller cell responses can be generated according to the K+ hypothesis: by passive Müller cell depolarization driven by variations in [K+]o.     

The Negative Chronotropic Effect of Cs<Superscript>+</Superscript> Ions on Generation of Transmembrane Potentials in Mouse Sinoatrial Node Cells

Experiments on spontaneously contracting strips from sinoatrial region of the hearts of 2-month-old albino mice showed that cesium (Cs⁺), a blocker of hyperpolarization-activated I_f current, in a concentration of 1 mM produced the greatest negative chronotropic effect on the duration of diastolic depolarization phase (75%), its rate (59%), and action potential duration (29%). The threshold concentration of Cs⁺ was approximately 0.15 mM. In a concentration of about 8.5 mM, spontaneous generation of action potentials stopped. The effect was reversible. Thus, blockade of I_f current by Cs⁺ reduced the rate of action potential generation in cells of mouse sinoatrial node by ~42% in comparison with controls.     

Long-term effects of synaptic activation at low frequency on excitability of myenteric AH neurons

Intracellular microelectrodes were used to record the effects of extended periods (1-30 min) of synaptic activation on AH neurons in the myenteric ganglia of the guinea-pig ileum. Low-frequency (1 Hz) stimulation gave rise to a slowly developing, sustained increase in excitability of the neurons associated with depolarization and increased input resistance. The increased excitability lasted for up to 3.5 h following the stimulus period. Successive stimulus trains (1-4 min) elicited successively greater increases in excitability. The neurons went through stages of excitation. Before stimulation, 500-ms depolarizing pulses evoked up to three action potentials (phasic response) and anode break action potentials were not observed. As excitability increased, more action potentials were evoked by depolarization (the responses became tonic), anode break action potentials were observed, prolonged after hyperpolarizing potentials that follow multiple action potentials were diminished and, with substantial depolarization of the neurons, invasion by antidromic action potentials was suppressed. It is concluded that a state of elevated excitability is induced in myenteric AH neurons by synaptic activation at low frequency and that changes in excitability can outlast stimulation by several hours.     

Effects of the calcium ionophore A23187 on pancreatic acinar cell membrane potentials and amylase release.

1. The effects of the Ca2+-ionophore A23187 and the non-metabolizable cholinergic agonist bethanechol on acinar cell membrane potentials and amylase release from the superfused mouse pancreas were studied. 2. In the presence of extracellular Ca2+ (2.56 mM), A23187 (10(-5)M) and bethanechol (3 X 10(-5)M) caused an equal increase in the release of amylase. Both stimulants depolarized theacinar cells, A23187 by 6-0 mV and bethanechol by 12-3 mV. 3. When Ca2+ and Mg2+ were removed from the superfusate, the ability of A23187 to increase the rate of amylase release was virtually abolished, while the effect of bethanechol remained unaltered. Similarly, in the absence of these divalent cations, A23187 did not cause depolarization of the acinar cells, while depolarization in response to bethanechol was largely normal. Consequently it is unlikely that cholinergic agonists initiate secretion by activating a Ca2+-ionophore-like mechanism in the cell membrane. 4. When the concentration of Ca2+ in the medium was raised to 10 mM was the only extracellular divalent cation present, the depolarization in response to A23187 was increased to 11-8 mV. When Mg2+ in a concentration of 10 mM was the only extracellular divalent cation, the depolarization was only 2-1 mV. 5. The Ca2+ dependent, A23187-induced depolarization was abolished in the absence of Na+ (Tris substitution). Addition of Na+ to the superfusate caused an immediate depolarization. 6. It is concluded that the Ca2+ dependent depolarization of pancreatic acinar cells induced by A23187 is not directly due to an increased divalent cation conductance. Our findings are consistent with the view that the depolarization is due to an increased influx of Na+ resulting from a Ca2+ mediated increase in Na+ permeability.     

Immunohistochemical demonstration of glial fibrillary acidic protein in normal rat Müller glia and retinal astrocytes

The presence of glial fibrillary acidic protein (GFA)-positive Müller glia and retinal astrocytes were studied immunohistochemically in normal rat retina. Using GFA antiserum both Müller glia and separate star-shaped cells were observed in spread-preparations as well as cryostat sections. The retinal astrocytes were also visualized using two different monoclonal GFA antibodies. These cells were found to be located in the nerve fiber and ganglion cell layers. In contrast, Müller glia were not normally visualized with any of the monoclonal GFA antibodies but could be stained 4 days after an optic nerve crush. Our results demonstrate that normal rat Müller glia expresses GFA-like immunoreactivity.