scispace - formally typeset
Search or ask a question

Showing papers in "Cellular and Molecular Neurobiology in 1983"


Journal ArticleDOI
TL;DR: Caffeine and other methylxanthines antagonize effectively both A1- and A2-adenosine receptors in brain perparations, while 1-Methylxanthine is more potent than caffeine in rat cerebral cortical membranes, while 3-methylXanthine and 7-methyl Xanthine are less potent than caffeine.
Abstract: 1. The potencies of caffeine and related methylxanthines as adenosine antagonists were assessed with respect to three apparent subtypes of adenosine receptors in rat brain preparations: (i) the A1-adenosine receptor which binds with a very high affinity the ligand [3H]cyclohexyladenosine (KD, 1 nM) in rat brain membranes; (ii) a ubiquitous low-affinity A2-adenosine receptor which activates cyclic AMP accumulation in rat brain slices—this A2-adenosine system exhibits an EC50 for 2-chloroadenosine of about 20µM; and (iii) a relatively high-affinity A2-adenosine receptor which activates adenylate cyclase in rat striatal membranes—this A2-adenosine system exhibits an EC50 for 2-chloroadenosine of about 0.5µM and is present in striatal but not in cerebral cortical membranes. 2. The rank order of potency for methylxanthines versus binding of 1 nM [3H]cyclohexyladenosine in membranes from eight rat brain regions is theophylline (IC50, 20–30µM) > paraxanthine (IC50, 40–65µM) > caffeine (IC50, 90–110µM) > theobromine (IC50, 210–280µM). There thus appears to be little difference in A1-receptors in different brain regions in terms of interaction with these methylxanthines. 1-Methylxanthine is more potent than caffeine in rat cerebral cortical membranes, while 3-methylxanthine and 7-methylxanthine are less potent than caffeine. 3. The rank order of potency for methylxanthines versus activation of cyclic AMP accumulation by 50µM 2-chloroadenosine in rat striatal slices is theophylline (IC50, 60µM) > paraxanthine (IC50, 90µM) > caffeine (IC50, 120µM) » theobromine (IC50, > 1000µM). Similar potencies pertain in cerebral cortical slices. 4. The rank order of potency of methylxanthines versus activation of adenylate cyclase by 1µM 2-chloroadenosine in rat striatal membranes is theophylline (IC50, 20µM) > paraxanthine (IC50, 40µM) > caffeine (IC50, 80µM) » theobromine (IC50, > 1000µM). 5. Caffeine and other methylxanthines, thus, antagonize effectively both A1- and A2-adenosine receptors in brain perparations. Theobromine appears less effective versus A2-receptors than versus A1-receptors. Caffeine exhibits aKi value of about 50µM at the very high-affinity A1-binding sites, aKi value of about 30µM at the low-affinity A2-adenosine site in brain slices, and aKi value of about 27µM at the high-affinity A2-adenosine site in striatal membranes. The functional significance of antagonism of such adenosine receptors by caffeinein situ will depend both on the local levels of adenosine and on the affinity for adenosine for the receptor, since antagonism by xanthines is competitive in nature. In addition, the functional significance of xanthine action will depend on the degree of inhibition of adenosine input which is required to alter the output signal. For a stimulatory input to adenylate cyclase via an A2-adenosine receptor, profound antagonism by methylxanthines is probably required to alter the cyclic AMP-mediated output signal, while for inhibitory input to adenylate cyclase via an A1-adenosine receptor, presumably a lesser degree of antagonism by methylxanthines may be required to alter the cyclic AMP-mediated output signal.

403 citations


Journal ArticleDOI
TL;DR: Studies in culture support work in vivo that astroglia may have an important role in neuronal development, and show that astrocytes in primary culture show uptake of and receptors for a number of transmitters, properties which have wide-ranging implications.
Abstract: 1. Morphological studies have established the ubiquitous nature of astrocytes in the CNS. Their processes surround capillaries and synapses, form the subpial and subependymal layers, and seemingly invest every neuronal surface not covered by other neuronal surfaces or oligodendroglial membranes. Although such interrelationships have long suggested that astrocytes may play many critical roles, there still remains relatively little experimental information on the functions and properties of these cells. 2. About a decade ago it became evident that primary cultures from neonatal rodent brains can consist predominantly of normal astrocytes. Based on these findings there is now an increasing number of studies in which such primary cultures are being used to help unravel the continuing enigma of the properties and functions of astrocytes. Aspects of this work are reviewed in this article. Such work has already shown that astrocytes in primary culture exhibit the basic electrophysiological characteristics which had been the only functional property well established for these cellsin situ. 3. Further studies of the electrophysiological properties of these cells, which can be correlated with ion transport studies, are beginning to show that astrocytes may have more complex electrophysiological properties than had previously been supposed, as well as a number of important electrically silent ion fluxes. In addition, astrocytes in primary culture show uptake of and receptors for a number of transmitters, properties which have wide-ranging implications. 4. Studies in culture also support workin vivo that astroglia may have an important role in neuronal development.

164 citations


Journal ArticleDOI
TL;DR: All of the agents which excite area postrema neurons, with the exception of serotonin and norepinephrine, are emetic, while none of the three agents without excitatory effects is known to be emetic.
Abstract: 1. The responses of 122 neurons in the area postrema of anesthetized dogs to 17 common transmitters and peptides were determined. Recordings were made from one barrel of a seven-barrel ionophoretic electrode. 2. All neurons were silent at rest, but most could be detected and excited by the application of glutamate. The glutamate response was a brief, high-frequency response of less than 1-sec duration. 3. Excitatory responses were also found to histamine, norepinephrine, serotonin, dopamine, apomorphine, angiotensin II, neurotensin, leucine enkephalin, vasoactive intestinal polypeptide, thyrotropin releasing hormone, gastrin, vasopressin, and substance P. While most neurons tested were excited by dopamine and apomorphine, approximately half of those studied were also excited by each of the other substances. Inhibitory responses were found to norepinephrine (6 of 15 cells) and histamine (3 of 45 cells). No responses were found to acetylcholine, somatostatin, or cholecystokinin. 4. The responses to all 13 excitatory substances other than glutamate were similar. Typically these responses had a latency of 2–20 sec and lasted for 30 sec to 5 min on their first application. The frequency of discharge was usually low (~0.5 Hz). Multiple applications of these agents often induced a maintained spontaneous discharge of low frequency. Each application also induced a transient incremental discharge at a frequency that rarely exceeded 2 Hz. 5. The area postrema has been proposed to be the “chemoreceptor trigger zone” for emesis (Borison and Wang, 1953). All of the agents which excite area postrema neurons, with the exception of serotonin and norepinephrine, are emetic, while none of the three agents without excitatory effects is known to be emetic. Thus these results provide strong support for the central role of the area postrema in emesis. 6. The similarity of response to so many substances on small neurons suggests a common ionic and/or metabolic mechanism underlying the response. The prolonged nature of the response to brief administration of these agents would seem to be appropriate for neurons which subserve a sensation and behavior such as nausea and vomiting.

107 citations


Journal ArticleDOI
TL;DR: Glutamate-promoted inactivation, its enhancement by ATP, and the opposition to inactivation by pyridoxal-P and Pi appear to be important in the regulation of glutamate decarboxylase.
Abstract: 1. The substrate-promoted inactivation of glutamate decarboxylase from hog brain was studied. 2. Inactivation was a slow process that was dependent on the concentration of glutamate. 3. Glutamate-dependent inactivation was not first order but was best described as the sum of two exponential decay processes. At 10 mM glutamate, the half-lives at 30°C were about 6 min for the fast component and 70 min for the slow component. 4. Glutamate-dependent inactivation appeared to be due to the formation of apoenzyme since the rate and extent of inactivation were greatly reduced by the presence of pyridoxal 5′-phosphate (the cofactor, pyridoxal-P). Also, inactivated enzyme could be reactivated by adding pyridoxal-P (Meeley and Martin, 1983). 5. Micromolar concentrations of ATP enhanced glutamate-promoted inactivation in the absence of pyridoxal-P. ATP also enhanced inactivation in the presence of 10µM pyridoxal-P, but somewhat higher concentrations were required for an equal effect. ATP had little or no direct effect on the enzyme in the absence of glutamate. 6. In the absence of pyridoxal-P, Pi reduced the enhancement of inactivation by 10µM but not by 750µM ATP. 7. Glutamate-promoted inactivation, its enhancement by ATP, and the opposition to inactivation by pyridoxal-P and Pi appear to be important in the regulation of glutamate decarboxylase.

44 citations


Journal ArticleDOI
TL;DR: A reduction of excitability and synaptic transmission as well as no effect on posttetanic potentiation in the rat hippocampal slice by carbamazepine agrees with previous studies on spinal cord and peripheral nerve.
Abstract: 1. Carbamazepine at therapeutic concentrations has a strong inhibitory effect on the spontaneous field bursts of the CA 1 region of rat hippocampal slices in low-Ca2+, high-Mg2+ solution. 2. A reduction of excitability and synaptic transmission as well as no effect on posttetanic potentiation in the rat hippocampal slice by carbamazepine agrees with previous studies on spinal cord and peripheral nerve. 3. Carbamazepine left synaptic inhibition and hyperpolarizing afterpotentials unaltered while these inhibitory processes were markedly enhanced by pentobarbital and adenosine, respectively. 4. The spontaneous field bursts are, at least in part, synchronized by ephaptic transmission and may serve as a model of epilepsy and trigeminal neuralgia. The clinical effectiveness of carbamazepine in these two ailments may be explained by a suppression of this pathological synchronization.

40 citations


Journal ArticleDOI
TL;DR: Low-affinity (micromolar)3H-dopamine binding was measured under conditions which permitted dopamine activation and opiate inhibition of adenylate cyclase in rat striatal membranes, suggesting that the dopamine D1 receptor in striatum may be modulated by the opiate delta receptor through a shared guanine nucleotide binding subunit.
Abstract: 1. Low-affinity (micromolar)3H-dopamine binding was measured under conditions which permitted dopamine activation and opiate inhibition of adenylate cyclase in rat striatal membranes. Opiate drugs and peptides inhibited the dopamine binding in the presence of both GTP5 and Gpp(NH)p. Opiate inhibition of adenylate cyclase was, however, observed only in the presence of GTP. 2. It is suggested that the dopamine D1 receptor in striatum may be modulated by the opiate delta receptor through a shared guanine nucleotide binding subunit.

29 citations


Journal ArticleDOI
TL;DR: Four factors control a cycle of inactivation and reactivation that appears to be important in the regulation of brain glutamate decarboxylase, possibly by competition of ATP for the cofactor binding site.
Abstract: 1. The effects of ATP and inorganic phosphate (Pi) on the reactivation of glutamate apodecarboxylase by its cofactor pyridoxal-5′-phosphate (pyridoxal-P) was studied. Apoenzyme was prepared by preincubation with glutamate. 2. Apoenzyme prepared with glutamate alone was reactivated slowly and incompletely by adding a saturating concentration of pyridoxal-P (20µM). Reactivation was slightly enhanced by 1–10 mM Pi. 3. Reactivation by pyridoxal-P plus Pi was greatly enhanced by the presence of low concentrations (<100µM) of ATP during the preparation of apoenzyme with glutamate. Reactivation was much lower if Pi was omitted. 4. Enhancement of reactivation by ATP was due to its effect during apoenzyme formation, since ATP did not enhance reactivation if added only during reactivation and since the enhancing effect persisted after the removal of free ATP by chromatography on Sephadex G-25 after apoenzyme preparation and before reactivation. 5. Reactivation was inhibited by high concentrations of ATP (>100µM), possibly by competition of ATP for the cofactor binding site. 6. Four factors (glutamate, pyridoxal-P, ATP, and Pi) control a cycle of inactivation and reactivation that appears to be important in the regulation of brain glutamate decarboxylase.

28 citations


Journal ArticleDOI
TL;DR: It is proposed that elevation of cyclic AMP mediates an increased sensitivity of theIK(Ca) channel to Ca ions, which is connected to the Ca-dependent potassium current in Aplysia neurons.
Abstract: 1. The effect on the Ca-dependent potassium current,IK(Ca), of procedures that increase intracellular cAMP levels was studied inAplysia neurons using three different pharmacological approaches. 2. Exposure to cAMP analogues which were either resistant to or protected from phosphodiesterase hydrolysis caused an increase inIK(Ca) from 30 to 50% in 10 min. The degree of reversibility of this effect varied from complete with db cAMP to very little with pcpt cAMP. 3. Exposure to cholera toxin, which stimulates the synthesis of endogenous cAMP, increasedIK(Ca) 25% in 10 min and the effect was not reversible. Both approaches were effective in all seven neuron types studied. 4. Application of serotonin plus phosphodiesterase inhibitor caused an increase inIK(Ca) in neuron R15 but not in the other neuron types. Application of pentylene tetrazole (PTZ) led to a decrease inIK(Ca). 5. It is proposed that elevation of cyclic AMP mediates an increased sensitivity of theIK(Ca) channel to Ca ions.

27 citations


Journal ArticleDOI
TL;DR: The observation of insulin immunoreactivity in neurons suggests that insulin may be a neuromodulator in the central nervous system.
Abstract: 1. Immunocytochemical studies have been performed which establish the presence of immunoreactive (ir-) insulin in primary cultures from fetal rat brain. Approximately 1% of the identified neurons in culture contained insulin immunoreactivity. Typically, a diffuse staining pattern was observed in the perikaryia and fiber profiles of peroxidase-anti-peroxidase-positive neurons. Varicosity-like structures containing ir-insulin were found on the majority of positively stained fiber profiles. A small number of the neurons appeared to have cytoplasmic granules that stained densely for insulin. No significant staining was observed in the background monolayer of cells predominantly of glial origin. 2. The substitution of primary antiserum absorbed with excess insulin or preimmune serum eliminated staining in the neurons. The observation of insulin immunoreactivity in neurons suggests that insulin may be a neuromodulator in the central nervous system.

25 citations


Journal ArticleDOI
TL;DR: Saturable taurine binding was displaceable by some convulsants and anticonvulsants but its specificity still remains to be established.
Abstract: 1. Saturable sodium-independent taurine binding to mouse and rat brain synaptic membranes was exposed after two freezing-thawing cycles combined with Triton X-100 treatments. 2. The amount of saturable taurine binding was fairly low but was enhanced after depletion of brain taurine. 3. Saturable taurine binding was displaceable by some convulsants and anticonvulsants but its specificity still remains to be established.

24 citations


Journal ArticleDOI
TL;DR: It is concluded that the Ca2+ regulatory system binds Ca2- most strongly compared to the other ions tested, and there is a variable distribution of buffering machinery within the nerve soma, with increased buffer capacity near the plasma membrane of the cell.
Abstract: 1. Absorbance changes associated with divalent cation binding to arsenazo III were used to measure changes in Ca2+, Sr2+, and Ba2+ concentrations under a variety of experimental conditions. 2. The rate of the falling phase of an absorbance change signal, measured in nerve cell bodies injected with arsenazo III and under membrane potential control, was taken as an index of divalent cation buffering. With influx of ions through the membrane or with ionophoretic injection, we found the buffering, i.e., the dye-absorbance signal's falling rate, to be greatest for Ca2+ ions: the sequence was Ca2+ > Sr2+ ≫ Ba2+. 3. Injecting Ca2+ or Sr2+ into the center of a nerve cell produced a significantly greater amplitude of arsenazo III signal than the same injection near the cell membrane. We did not find this to be the case for Ba2+ or Mg2+ injections. 4. We conclude that the Ca2+ regulatory system binds Ca2+ most strongly compared to the other ions tested, and there is a variable distribution of buffering machinery within the nerve soma, with increased buffer capacity near the plasma membrane of the cell. A preliminary report of some of the results presented in this paper has appeared previously (Tillotson and Gorman, 1980).

Journal ArticleDOI
TL;DR: The segregation of ACh receptors to a discrete portion of the cell suggests that they are functional receptors and may be part of a physiologic arrangement which directs different afferent inputs to different portions of the dendritic tree.
Abstract: 1. The responses of pyramidal neurons of rat prepyriform cortex to ionophoretic application of acetylcholine (ACh) were studied in a submerged, perfused brain slice. 2. ACh excited some neurons but only if applied to an area near to the cut surface of the slice. This area contained the basal dendrites of the pyramidal cells and some cell bodies. No excitation was seen if ACh was applied at depths of 250µm or more from the cut surface, an area which contained only apical dendrites, although the apical dendrites were very sensitive to excitatory amino acids such as aspartate (Asp) and glutamate (Glu). 3. On all neurons which did not discharge to ionophoretic application of ACh, ACh potentiated the response to Glu and Asp. No potentiation of amino acid responses was obtained on apical dendrites. The potentiation had a time course similar to that of the discharge of neurons which fired to ACh. This observation suggests that pyramidal neurons have receptors for ACh on basal but not apical dendrites. 4. The ACh response in the basal dendrite-soma region was elicted by pilocarpine and blocked by atropine but not curare. This was true whether the response studied was direct excitation or potentiation of the response to an amino acid. 5. The ACh response was associated with a voltage-dependent increase in membrane resistance which had a slow time course and appeared to be due to a turning off of an M current, as described by Brown and Adams (1980) in sympathetic ganglion cells. The effects of ACh were minimal at the resting potential but increased with depolarization. ACh had no effect on the current-voltage relation of the cell, except at depolarized potentials of less than -60 mV. 6. Ionophoretic application of Ba2+ to the basal dendritic region resulted in potentiation of the amino acid responses and sometimes induced a discharge similar to that of ACh. Since Ba2+ mimics the ACh response, presumably by a direct blockade of the M channel, the effects of Ba2+ on apical dendrites were tested to determine whether these dendrites contain M channels associated with a transmitter receptor other than ACh. However, Ba2+ did not induce potentiation in apical dendrites, suggesting that M channels are also restricted to the basal dendrites or cell bodies. 7. The potentiation of a response to a depolarizing constant-current pulse was comparable in degree and time course to that of amino acid responses, indicating that the ACh potentiation of the amino acid responses is a predictable augmentation of any input which brings the membrane potential into the range of M-channel activation. 8. The segregation of ACh receptors to a discrete portion of the cell suggests that they are functional receptors and may be part of a physiologic arrangement which directs different afferent inputs to different portions of the dendritic tree. Such segregation of receptors may also occur on other neurons with distributed inputs.

Journal ArticleDOI
TL;DR: It is concluded that recovery of Ca channels depends upon both Ca influx and membrane potential and is modulated by agents which affect Ca metabolism.
Abstract: 1. Ca currents were examined with regard to their recovery from inactivation. The experiments were done on isolated nerve cell bodies ofHelix aspersa using a combined suction pippet, microelectrode method for voltage clamp, and internal perfusion. Ca currents were separated by suppressing K and Na currents. 2. The time course of recovery was determined by applying a test pulse at intervals ranging from 1 msec to 20 sec after prepulses varying from 20 to 3000 msec in duration. Each pair of pulses was preceded by a control pulse to ensure that the Ca currents had recovered before the next test pair was applied. Ba and Ca currents were compared and the effects of intracellular perfusion with EGTA, ATP, and vanadate were examined. 3. Ba currents recovered in two stages and this time course was well fit by a sum of two exponentials with amplitudes and time constants given byA1 andτ1 for the fast component andA2 andτ2 for the slow component. In Ba the time constants were unchanged when prepulse durations were prolonged from 70 to 700 msec, although the initial amplitudesA1 andA2, particularlyA2, were increased. 4. Comparable influxes of Ca during the prepulse caused much more inactivation, but interestingly the recovery occurred at the same rate. The time course of Ca current recovery was also fit by a sum of two exponentials, the time constants of which were both smaller than the time constants of Ba current recovery. However, the time constants of Ca current recovery were increased markedly when prepulse durations were prolonged. Increasing the extracellular Ca concentration had a similar effect. 5. Increasing the Ba influx had no effect on the recovery time constants, and the Ba results are consistent with reversible inactivation gating of potential-dependent membrane Ca channels. The Ca results show that Ca influx enhances inactivation. Intracellular perfusion with EGTA resulted in less inactivation in the cast of Ca but it had no effect on Ba currents. Intracellular ATP increased the rate of recovery of Ca currents, and intracellular vanadate inhibited recovery. It is concluded that recovery of Ca channels depends upon both Ca influx and membrane potential and is modulated by agents which affect Ca metabolism.

Journal ArticleDOI
TL;DR: An investigation into two well-known electrostatic properties of dielectrics, electrostriction and piezoelectricity, in the context of the nerve membrane is undertaken, which predicts a few percent change in axon dimensions for voltage- and space-clamped axons.
Abstract: 1. The electromechanical transduction mechanisms operating in nerve membranes are considered theoretically. 2. For mechanical-to-electrical transduction (mechanical generator potentials), a model is proposed in which the surface charge on the membrane mediates stress-induced changes in the intramembrane electric field, thus opening transmembrane ion conductance channels or reducing the ion selectivity of the membrane via leak conductance pathways. 3. For electrical-to-mechanical transduction (axon diameter change with excitation), an investigation into two well-known electrostatic properties of dielectrics, electrostriction and piezoelectricity, in the context of the nerve membrane is undertaken which predicts a few percent change in axon dimensions for voltage- and space-clamped axons.

Journal ArticleDOI
TL;DR: The combining of thestoned mutation withtan in thetan, stoned double mutant results in the loss of the jump behavior as well as the partial restoration of the off-transient to an otherwisetan-like ERG.
Abstract: 1. Mutations at thestoned locus ofDrosophila melanogaster produce a reversible temperature-sensitive debilitation. At permissive temperatures they also exhibit an unusual jump response to a light-off stimulus. 2. An increase in the amplitude of the off-transient of the electroretinogram (ERG) is associated with this abnormal jump. Both the jump response and the increased amplitude of the off-transient are shown to be dependent on the duration of the light pulse prior to the light-off stimulus. 3. Instoned flies which are light adapted, the jump response, as measured by recording from the indirect flight muscles, is seen to habituate with increasing light-off frequency. This habituation corresponds to the decrease in the amplitude of the off-transient that also occurs with high-frequency stimulation. 4. Another visual mutanttan, removes the off and on-transients of the ERG. 5. The combining of thestoned mutation withtan in thetan, stoned double mutant results in the loss of the jump behavior as well as the partial restoration of the off-transient to an otherwisetan-like ERG. The relationship between the increase in the amplitude of the off-transient instoned flies and the eliciting of the jump response is discussed.

Journal ArticleDOI
TL;DR: A possible common mechanism of action of antidepressant drug therapy on presynaptic release of norepinephrine from nerve terminals is suggested.
Abstract: 1. The effect of repeated electroconvulsive shock (ECS) treatment and chronic LiC1 feeding on calcium-dependent, K+-evoked release of [3H] norepinephrine from rat cortical vesicular preparation was studied. 2. There was no significant effect of either acute or repeated ECS treatment on [3H]norepinephrine release in cortical vesicles obtained from animals treated for either 1 or 10 days. Release of norepinephrine was examined over a range of CaCl2 concentrations. 3. Clonidine effectively inhibited release of [3H]norepinephrine in cortical vesicles obtained from control and ECS-treated animals. 4. K+-evoked release of [3H]norepinephrine at low (0.2 mM) and high (1.0 mM) CaCl2 concentrations was significantly increased in cortical vesicles obtained from LiCl-treated animals. Clonidine effectively inhibited release of [3H]norepinephrine in cortical vesicles obtained from both control and LiCl-fed animals. 5. These results suggest a possible common mechanism of action of antidepressant drug therapy on presynaptic release of norepinephrine from nerve terminals.

Journal ArticleDOI
TL;DR: The results suggest that D-600 acts to antagonize the acetylcholine response through a blockade of the open state of the transmitter-activated cation channel by voltage-jump relaxation analysis.
Abstract: 1. The effects of the calcium channel blocker D-600 on the cation channels activated by acetylcholine (ACh) was studied in voltage-clampedAplysia neurons by voltage-jump relaxation analysis. 2. D-600 blocked the steady-state ACh current in a highly voltage-dependent manner, the degree of antagonism increasing with membrane hyperpolarization. 3. In the presence of D-600 the current relaxations following hyperpolarizing command steps became biphasic. The time constants of ACh-induced current relaxations (τf), which approximate the mean channel lifetime, were reduced in a voltage-dependent manner, the degree of reduction ofτf increasing with increasing membrane potential. 4. In addition to the acceleration ofτf, a slow, inverse kinetic component (τs) of the relaxation appeared in the presence of D-600. The rate of this inverse kinetic component was accelerated either by increasing the agonist or antagonist dose or by increasing the membrane potential. 5. These results suggest that D-600 acts to antagonize the acetylcholine response through a blockade of the open state of the transmitter-activated cation channel. Possible kinetic schemes for this interaction are discussed.

Journal ArticleDOI
TL;DR: Intracellular recordings were made from distal photoreceptor cells of the file clamLima scabra in order to examine the ionic and spectral mechanisms which underly the response to light decrement, providing an explanation for the persistent discharge of action potentials which occurs on termination of light in these cells as well as the visual cells of other gastropod mollusks.
Abstract: 1. Intracellular recordings were made from distal photoreceptor cells of the file clamLima scabra in order to examine the ionic and spectral mechanisms which underly the response to light decrement. These receptors are primary sensory neurons that generate nerve impulses in the optic nerve upon light termination without benefit of synaptic interconnections between photoreceptor cells. Microelectrode measurements were made on these cells. Membrane conductance changes were assessed by measuring membrane voltage changes elicited under different conditions while passing constant-current pulses through the microelectrode from an active bridge amplifier. 2. Responses of membrane potential in light and darkness in different concentrations of external potassium ions were fitted to a simplified form of the constant field equation. This analysis allowed an estimation of internal potassium activity (281 mM) as well as changes inPNa/PK in darkness and light.PNa/PK changed from 0.09 in darkness to 0.03 at the peak of the light response. 3. A persistent decrease in membrane conductance at the termination of light is associated with a depolarization that overshoots the dark resting membrane potential. This transient depolarization is dependent on the intensity and duration of the preceding period of light. 4. The amplitude of the dark-dependent depolarization is related to the absorbance of light during the preceding period of light by a long wavelength intermediate of rhodopsin bleaching (metarhodopsin). 5. The frequency of discharge of action potentials with rapid kinetics which occurs following light is proportional to the amplitude of the after depolarizing response. The delay to onset of the discharge is inversely proportional to the amplitude of the after depolarizing response. 6. The sensitivity (response/photon) of distal cells can be modified by background light which passes through a screening pigment found in cells that surround the eye. 7. These data, taken together, provide an explanation for the persistent discharge of action potentials which occurs on termination of light in these cells as well as the visual cells of other gastropod mollusks.

Journal ArticleDOI
TL;DR: The results suggest the possibility that S-100 may be involved in the regulation of some membrane activities, in analogy with previous observations with SYN.
Abstract: 1. The125I-labeled S-100 specific binding to a Triton X-100 (TX-100) extract of synaptosomal particulate fractions (SYN) was investigated. 2. The results indicate that (a) S-100 binding to the TX-100 extract is partially irreversible after a critical association time at 37°C, while it is fully reversible after any association time at 4°C; (b) trypsin and phospholipase C partially reverse the S-100 binding, while phospholipase D enhances the interaction to some extent, in a dose-dependent way; (c) EDTA and high concentrations of NaC1 or KC1 are more efficient as inhibitors of the S-100 binding to the TX-100 extract than as125I-labeled S-100 dissociating agents, in analogy with previous observations with SYN; and (d) two main populations of solubilized S-100 binding sites can be evidenced by gel filtration and sucrose gradient centrifugation when low amounts of the TX-100 extract are processed and/or low S-100 concentrations are used, while two additional molecular species are separated when greater amounts of either factors are tested. 3. These results suggest the possibility that S-100 may be involved in the regulation of some membrane activities.

Journal ArticleDOI
TL;DR: The data presented here suggest that the phosphorylation of protein IV, and perhaps D, may play a role in the early processing of visual information in the fly.
Abstract: 1. Protein IV from synaptosomal fractions ofDrosophila heads is phosphorylatedin vitro by an endogenous cyclic adenosine monophosphate (cAMP)-dependent protein kinase. Thein vivo phosphorylation of this protein is affected by light. 2. Two visual mutants,tan andstoned, exhibit altered levels ofin vivo phosphorylation of protein IV. Thetan strain shows depressedin vivo levels of phosphorylation of protein IV, whereasstoned shows an increase in thein vivo level of phosphorylation of this same protein. 3. Protein D is phosphorylatedin vitro by an endogenous Ca2+/calmodulindependent protein kinase and has a molecular weight identical to that of protein IV. Thestoned mutant strain shows an increase in thein vivo level of phosphorylation of protein D. 4. The data presented here suggest that the phosphorylation of protein IV, and perhaps D, may play a role in the early processing of visual information in the fly.

Journal ArticleDOI
TL;DR: It is suggested that Io1 may be a factor in determining the nonspiking behavior of the dendrites and that Io2 may limit the stretch-induced depolarization in the dendedrite to a value more negative than that at which the maximum rate of transmitter release occurs.
Abstract: 1. Segments from the nonspiking peripheral dendrites of a crustacean coxal receptor (T fiber) were studied using the voltage clamp technique. The peripheral endings of the T fiber are sensitive to stretch applied to a specialized receptor muscle by rotation of the coxa. The intraganglionary portion of the T fiber is presynaptic to the motor neurons innervating the coxal muscle. 2. Depolarizing commands activated three separate fast channels: (i) a transient inward sodium current,INa, which is blocked by tetrodotoxin (TTX); (ii) a transient outward current,Io1, having the same voltage-dependent characteristics asINa; and (iii) a second, longer-lasting, outward current,Io2. 3. BothINa andIo1 were inactivated when segments were clamped at voltages more positive than -50 mV, whereasIo2 could be activated at voltages more positive than -50 mV. 4. Io1 andIo2 were blocked by 4-aminopyridine (4-AP) and by tetraethylammonium (TEA), althoughIo2 shows a greater sensitivity to TEA thanIo1. 5. It is suggested thatIo1 may be a factor in determining the nonspiking behavior of the dendrites and thatIo2 may limit the stretch-induced depolarization in the dendrite to a value more negative than that at which the maximum rate of transmitter release occurs. 6. In addition to the three fast currents, the presence of a slow inward and slow outward current could also be demonstrated. The effects of the slow currents were longer in segments cut from the proximal part of the dendrites.

Journal ArticleDOI
TL;DR: Incubation of intact synaptosomes in the presence of 3–6 mM 4-aminopyridine (4-AP) caused a markedly enhanced phosphorylation of high molecular weight proteins of 90, 100, 130, and 180 kd, with no increase in the 50 or 38 kd proteins.
Abstract: 1. When intact synaptosomes were incubated with [γ-32P]ATP, maximal protein phosphorylation was attained 2 min after the start of incubation. 2. Protein phosphorylation under basal conditions was dependent on external Ca2+, and the dominant peak of phosphorylation was a 50-kd protein. 3. Incubation of intact synaptosomes in the presence of 3–6 mM 4-aminopyridine (4-AP) caused a markedly enhanced phosphorylation of high molecular weight proteins of 90, 100, 130, and 180 kd, with no increase in the 50 or 38 kd proteins. 4. This effect of 4-AP was dependent on external calcium ions in the incubation medium. 5. The 4-AP effect on the high molecular weight proteins was also found in synaptosomal plasma membranes isolated from the synaptosomes. 6. Tetraethylammonium (TEA) ions did not produce this enhancement of phosphorylation.

Journal ArticleDOI
TL;DR: These examples illustrate how principles of cellular neurophysiology can be applied to questions of clinical relevance in retinal pigment epithelial physiology.
Abstract: This review of retinal pigment epithelial (RPE) physiology pays tribute to Anthony L. F. Gorman, who introduced the author to the giant neuron of Anisodoris nobilis (the sea lemon) and cellular neurobiology. The RPE is an epithelial monolayer with tight junctions, which controls the environment of the photoreceptor outer segments. The apical and basal membranes have different electrical properties and generate a standing potential across the eye. The RPE helps maintain adhesion between the retina and the wall of the eye. Adhesion is weakened by cyanide, low pH or low calcium, but enhanced by ouabain or acetazolamide. The RPE transports water from the subretinal space toward the choroid. This water movement is inhibited by hypoxia or cyanide but enhanced by ouabain or acetazolamide. The c-wave of the electroretinogram is a composite of a cornea-positive wave produced by hyperpolarization of the apical RPE membrane and a cornea-negative wave produced by the Muller cells, both in response to the fall in extracellular potassium that follows illumination of the photoreceptors. The "light response" of the standing potential is produced by depolarization of the basal membrane of the RPE. These examples illustrate how principles of cellular neurophysiology can be applied to questions of clinical relevance.

Journal ArticleDOI
TL;DR: It is suggested that the increase of Ca2+ inward current at negative potentials and the prolonged activation of the Ca2-activated K+ current play a major role in causing the bursting discharge behavior in normally beating cells.
Abstract: 1. External quinidine converts the pacemaker neurone L-11, found in theAplysia abdominal ganglion, from spontaneously “beating” to “bursting” discharge activity. 2. Quinidine-induced bursting ceased when entry of Ca2+ ions into the cells was blocked in a Ca2+-free, Co2+-containing solution or if internal Ca2+ accumulation was prevented by the injection of EGTA. 3. The analysis of membrane currents from voltage clamp experiments showed that quinidine blocks the Ca2+ inward current in a dose- and time-dependent manner. In addition, the currents were displaced to the left on the voltage axis, causing an increase of the inward current at negative membrane potentials. 4. External quinidine suppresses the Ca2+-activated K+ current induced by intracellular Ca2+ injections and acts to prolong its decay phase. The slowing of the decay phase of the Ca2+-activated K+ current by quinidine was prevented after intracellular injection of EGTA, indicating that Ca2+ removal is impaired by the drug. 5. It is suggested that the increase of Ca2+ inward current at negative potentials and the prolonged activation of the Ca2+-activated K+ current play a major role in causing the bursting discharge behavior in normally beating cells.

Journal ArticleDOI
TL;DR: It is concluded that the two transmitters tested are not localized within single cells and that morphology alone is inadequate to identify functional cell classes in this area.
Abstract: 1. The purpose of this study was to investigate the coexistence ofγ-aminobutyric acid (GABA) and leu-enkephalin in single neurons from the corpus striatum. 2. Monolayer cell cultures, started from newborn rat corpus striatum, were grown in serum-free medium and examined using GABA autoradiography and leu-enkephalin immunocytochemistry in a double-label protocol. 3. Examples of cells were found which were positive for one or the other neurotransmitter or for neither transmitter, but not for both. Furthermore, cells which appear similar by morphological criteria alone differed in transmitter specificity. 4. We conclude that the two transmitters tested are not localized within single cells and that morphology alone is inadequate to identify functional cell classes in this area.

Journal ArticleDOI
TL;DR: These studies show that primary chick muscle cells are capable of producing the A12 form of AChE if cultured in NE-supplemented medium, and production of the A 12 form does not require activity or innervation.
Abstract: 1. Asymmetric forms of AChE have generally not been detected in cultured chick skeletal muscle cells in the absence of cocultured neurons. To explore further neurotrophic effects of adult peripheral nerve extracts (NE) on musclein vitro, we reexamined the appearance of various molecular forms of AChE in cultured chick muscle cells in the presence of NE. 2. The various molecular forms of AChE were distinguished by sucrose gradient sedimentation and radioenzymatic techniques. 3. In the presence of NE, cells proliferated during the first 48 hr of culture, then fused and formed spontaneously contracting myotubes by 6–8 days in culture. 4. Total AChE, 5.4 S, and 11.5 S molecular forms reached activity plateaus by 8 days in culture which persisted until cultures were terminated at day 20. 5. Betweeen 1 and 6 days in culture, 19.5 S AChE (A12) was not detected. The A12 form was first observed at 7 days reaching a maximum of 11.3% of the total AChE at 14 days and then gradually declined to a level of 1% at day 20. 6. Since the A12 form declined in older cultures but comprised 25% in embryonic muscle tissue, we examined the possible requirement of neurons in culture to attain higher levels of A12 AChE. Spinal cord neurons were plated onto 6-day muscle cultures and AChE activities were measured between 8 and 20 days. 7. The results showed that 19.5 S AChE activity in the presence of both spinal cord neurons and NE was no greater than that found in the presence of NE alone. 8. To suppress spontaneous contraction, 0.6µM tetrodotoxin (TTX) or 15µMd-tubocurarine (dTC) were added to 5-day-old muscle cultures at a time when myotubes were differentiated but contractile activity had not begun. 9. TTX had cytotoxic effects and inhibited further development of myotubes. In contrast, dTC had no deleterious effect on morphological development, eliminated contraction, but did not interfere with the appearance of any forms of AChE including the A12 form. 10. These studies show that primary chick muscle cells are capable of producing the A12 form of AChE if cultured in NE-supplemented medium. In this culture system, production of the A12 form does not require activity or innervation.

Journal ArticleDOI
TL;DR: While the effects ofHTX on the synaptic channels are concentration, time, and pH dependent, the effects on the channels of the conducting membrane are, in addition, use dependent, suggesting interactions of HTX with the activated forms of these channels.
Abstract: 1. Histrionicotoxin (HTX) at low concentrations of 5–10µM blocks the postsynaptic potential of the electroplax ofElectrophorus electricus. 2. At 100-fold higher concentrations, HTX blocks the directly evoked action potentials of the conducting membrane. 3. The pH dependence of the blockade by HTX at synaptic channels is different from that at the conducting membrane. At the synapse HTX is more potent at acid pH, while at the conducting membrane it is more potent at basic pH. 4. HTX at high concentrations antagonizes the effects of batrachotoxin, indicative of an effect on the batrachotoxin-sensitive sodium channels involved in action potential generation. 5. While the effects of HTX on the synaptic channels are concentration, time, and pH dependent, the effects on the channels of the conducting membrane are, in addition, use dependent, suggesting interactions of HTX with the activated forms of these channels.

Journal ArticleDOI
TL;DR: Background discharges of pure infrared secondary neurons from the lateral descending nucleus were analyzed and there were no histograms with a normal distribution in these central neurons, which might indicate that the constant interspike intervals which appear in the primary afferent fibers are not utilized for information processing at this level but occur only as part of a receptor mechanism which is still unknown.
Abstract: 1. Background discharges (static responses) of warm fibers in the pit organs (infrared receptive organs) of two species of crotaline snakes were recorded at various temperatures (water, 18–33°C; air, 19–28°C). Mean interspike intervals\(\left( {\bar X} \right)\), standard deviations (SD), and coefficients of variation (CV) were calculated, and the goodness of fit of interspike interval histograms to a corresponding normal distribution (i.e., one having the same mean and SD) were tested.\(\bar X\), SD, and CV were smallest at a certain temperature, which might be the optimum receptor temperature for the species. More than half of the histograms (22/42 for water, 7/10 for air) showed a normal distribution at a significance level of 0.01. This suggests that the spike intervals generated at the spike initiation site are constant, with some random error. 2. Background discharges of three pure infrared secondary neurons from the lateral descending nucleus were analyzed in the same way and compared to the peripheral discharges. There were no histograms with a normal distribution in these central neurons, which might indicate that the constant interspike intervals which appear in the primary afferent fibers are not utilized for information processing at this level but occur only as part of a receptor mechanism which is still unknown. 3. The discharge patterns of primary afferent fibers are also discussed in relation to the known discharge patterns of cold fibers in other animals.

Journal ArticleDOI
TL;DR: A wrinkled pattern appeared on the surface of the axons while they were in hyperosmotic media containing excess NaC1 or KC1, and no such patterns appeared in media which were made hyper- and hypoosmotic by the addition of sucrose or sodium glutamate.
Abstract: 1. Volume and morphological changes of the squid giant axons in response to hyper- and hypoosmotic media were examined. 2. In hyperosmotic media, which were made by adding sucrose or sodium chloride to the artificial seawater, the axons behaved approximately as ideal osmometers. The fraction of the osmotically inactive volume was less than 0.05. 3. In hypoosmotic media down to half the osmolality of the artificial seawater, intact squid axons did not show significant volume increases. However, following a combined treatment with hyaluronidase and collagenase, the volume of the squid axons increased in these hypoosmotic media. 4. A wrinkled pattern appeared on the surface of the axons while they were in hyperosmotic media containing excess NaC1 or KC1. Trypsin treatment prevented appearance of this surface pattern. Furthermore, no such patterns appeared in media which were made hyperosmotic by the addition of sucrose or sodium glutamate.