Aplysia Ganglion Research Articles

Biomechanical micromotion at the neural interface modulates intracellular membrane potentials in vivo

Objective. Respiration and vascular pulsation cause relative micromotion of brain tissue against stationary implants resulting in repetitive displacements of 2–4 µm (due to vascular pulsation) and 10–30 µm (due to breathing) in rats. However, the direct functional impact of such tissue micromotion on the cells at the neural interface remains unknown. This study aims to test the hypothesis that micromotion in brain tissue causes changes in membrane potentials (MPs) through the activation of mechanosensitive ion channels. Approach. Intracellular MPs were recorded from Aplysia ganglion cells (n = 8) and cortical cells (n = 15) in vivo in n = 7 adult rats. Cyclic stresses between 0.2 and 4 kPa repeated at 1 Hz were tested in Aplysia ganglion cells. For the in vivo experiments, 30 μM of gadolinium chloride (Gd3+), a non-selective blocker of mechanosensitive ion channels, was used to assess the role of such ion channels. Main results. In Aplysia ganglion cells, there were no MP changes for <1.5 kPa, and action potentials were observed at >3.1 kPa. Drug studies utilizing 5-HT showed an 80% reduction in firing frequency from controls. In in vivo experiments, periodic pulsations (1–10 mV) were observed in the MPs of cells that corresponded to breathing and heart-rate. In response to the addition of 30 µM Gd3+, we observed a significant reduction (0.5–3 mV) in the periodic pulsations in MP in all cortical cells across four different rats, suggesting the role of mechanosensitive ion channels in mediating MP fluctuations due to tissue micromotion at the neural interface. Significance. Under chronic conditions, the tissue at the interface stiffens due to scar tissue formation, which is expected to increase the likelihood of recruiting stretch-receptors due to tissue micromotion. It is speculated that such chronic sub-threshold pulsations in MPs might trigger the immune response at the neural interface.

Somatic inhibition by microscopic magnetic stimulation

Electric currents can produce quick, reversible control of neural activity. Externally applied electric currents have been used in inhibiting certain ganglion cells in clinical practices. Via electromagnetic induction, a miniature-sized magnetic coil could provide focal stimulation to the ganglion neurons. Here we report that high-frequency stimulation with the miniature coil could reversibly block ganglion cell activity in marine mollusk Aplysia californica, regardless the firing frequency of the neurons, or concentration of potassium ions around the ganglion neurons. Presence of the ganglion sheath has minimal impact on the inhibitory effects of the coil. The inhibitory effect was local to the soma, and was sufficient in blocking the neuron’s functional output. Biophysical modeling confirmed that the miniature coil induced a sufficient electric field in the vicinity of the targeted soma. Using a multi-compartment model of Aplysia ganglion neuron, we found that the high-frequency magnetic stimuli altered the ion channel dynamics that were essential for the sustained firing of action potentials in the soma. Results from this study produces several critical insights to further developing the miniature coil technology for neural control by targeting ganglion cells. The miniature coil provides an interesting neural modulation strategy in clinical applications and laboratory research.

Specific Expression of Aplysia Phosphodiesterase 4 in Bag Cells Revealed by in situ Hybridization Analysis.

Phosphodiesterases (PDEs) play a key role in the regulation of cyclic adenosine monophosphate (cAMP), which in turn mediates various cellular functions including learning and memory. We previously cloned and characterized three PDE4 isoforms (ApPDE4) from Aplysia kurodai. Using reverse transcription polymerase chain reaction (RT-PCR), we found that ApPDE4 isoforms are primarily expressed in the central nervous system. However, the detailed distribution of ApPDE4 mRNA in Aplysia individual ganglions was not evident. In this study, to determine the distribution of ApPDE4 mRNAs in Aplysia ganglions, we performed in situ hybridization (ISH) using a probe targeting ApPDE4, including the PDE catalytic domain. Interestingly, we found the strongest ISH-positive signals in the symmetrical bag cell clusters of the abdominal ganglion. The R2, R14, L7, L2 and L11 neurons in the abdominal ganglion, LP1 neuron in pleural ganglion, and metacerebral (MCC) neurons were ISH-positive. Mechanosensory neurons of the sensory cluster were also stained on the ventral aspect of the right and left pleural ganglia. Taken together, we found the detailed distribution of ApPDE4 mRNA in Aplysia ganglion and support their roles in serotonin (5-HT)-induced synaptic facilitation of Aplysia mechanosensory neurons.

Blockade of ionotropic receptor responses by progesterone in the ganglion cells of Aplysia.

To compare nongenomic effects of progesterone on various receptor responses of neurons, Aplysia ganglion cells were pretreated with 30 microM progesterone for 5 min and various receptor responses were tested using a conventional voltage-clamp method. Progesterone reduced nicotinic receptor-activated Na(+)-currents, nicotinic receptor-activated Cl(-)-currents, gamma-aminobutyric acid receptor-activated Cl(-)-currents, and dopamine receptor-activated Na(+)-currents. These depressant effects are similar at two different agonist concentrations. On the other hand, progesterone affected neither muscarinic receptor-activated K(+)-currents nor dopamine receptor-activated K(+)-currents. The former four types of receptors are known to be ionotropic while the latter two types of receptors are known to be metabotropic. Therefore, progesterone selectively inhibited all the types of ionotropic receptor responses, presumably in a noncompetitive manner.

Physiological and pharmacological characteristics of quisqualic acid-induced K(+)-current response in the ganglion cells of Aplysia.

The extracellular application of either quisqualic acid (QA) or Phe-Met-Arg-Phe-NH2 (FMRFamide) induces an outward current in identified neurons of Aplysia ganglion under voltage clamp. The time course of the QA-induced response is significantly slower than that induced by FMRFamide. The reversal potential for both responses was -92 mV and was shifted 17 mV in a positive direction for a twofold increase in the extracellular K(+) concentration. The QA-induced response was markedly depressed in the presence of Ba(2+), a blocker of inward rectifier K(+)-channel, whereas TEA, a Ca(2+)-activated K(+)-channel (BK(Ca)) blocker, or 4-AP, a transient K(+) (A)-channel blocker, had no effect on the response. The QA-induced K(+)-current was significantly suppressed by CNQX and GYKI52466, antagonists of non-NMDA receptors. However, the application of either kainate or AMPA, agonists for non-NMDA receptors, produced no type of response in the same neurons. The QA-induced K(+)-current response was not depressed at all by an intracellular injection of either guanosine 5'-O-(2-thiodiphosphate) (GDP-betaS) or guanosine 5'-O-(3-thiotriphosphate) (GTP-gammaS), but the FMRFamide-induced response was markedly blocked by both GDP-betaS and GTP-gammaS in the same cell. Furthermore, the QA- and FMRFamide-induced K(+)-current responses were both decreased markedly when the temperature was lowered to 15 degrees C, from 23 degrees C. These results suggested that the QA-induced K(+)-current response is produced by an activation of a novel type of QA-receptor and that this response is not produced by an activation of the G protein.

Quisqualic acid produces a novel K +-current response in identified neurons of aplysia ganglion

Quisqualic acid produces a novel K +-current response in identified neurons of aplysia ganglion

320 Depressing effect of budilast on the K + -current response induced by dopamine in APLYSIA ganglion cells

320 Depressing effect of budilast on the K + -current response induced by dopamine in APLYSIA ganglion cells

Intracellular injection of cyclic AMP inhibits the dopamine-induced K(+)-dependent responses of Aplysia ganglion cells.

Intracellular injection of cyclic AMP inhibits the dopamine-induced K(+)-dependent responses of Aplysia ganglion cells.

A role for the interchain disulfide or its participating thiols in the internalization of botulinum neurotoxin A revealed by a toxin derivative that binds to ecto-acceptors and inhibits transmitter release intracellularly.

Botulinum neurotoxin type A consists of a disulfide-linked light and heavy chain, with an intradisulfide present within the C-terminal half of the latter. The functional consequences of reducing these bonds and alkylating the thiols were investigated. Modification of free cysteine residues had no effect on the toxicity in mouse bioassays or on acetylcholine release in the mouse nerve-diaphragm and the buccal ganglion of Aplysia californica. However, reduction of the toxin prior to alkylation drastically decreased neuroparalytic potency; yet, this derivative inhibited transmitter release if injected directly into a presynaptic neuron in the Aplysia ganglion or added to bovine permeabilized adrenal chromaffin cells. Its antagonism of the action of botulinum neurotoxin A at mammalian motor nerve endings and Aplysia neurons indicates retention of the ability to bind to the toxin's productive ecto-acceptors. Thus, the abolition of the toxicity of extracellularly applied botulinum neurotoxin A by the cleavage of both disulfides, and the alkylation of the half-cystines involved, results from ineffective uptake. Modified forms of the isolated chains of botulinum neurotoxin A were utilized to determine which of the disulfides were necessary for internalization. Alkylation of the cysteines in the light and heavy chains, including those involved in the interchain bond but excluding those of the intact disulfide in the heavy chain, revealed that the intermolecular bond must be present, or the thiols concerned unmodified, for botulinum neurotoxin A to undergo membrane translocation into Aplysia neurons.

Multi-Channel Optical Monitoring of Neural Signals from Aplysia Ganglion

Multi-Channel Optical Monitoring of Neural Signals from Aplysia Ganglion

Temperature effects on dopamine-induced inhibitory and excitatory responses recorded from Aplysia ganglion cells.

Effects of temperature change on different dopamine-induced responses were studied in Aplysia ganglion cells. When the temperature was raised from 11 to 35 degrees C, the K+-dependent inhibitory response to dopamine increased to reach a maximum at about 23 degrees C, then decreased at higher temperature. The Na+-dependent slow excitatory one kept increasing without showing the maximum. On the contrary, the Na+-dependent fast excitatory one rather decreased to reach the minimum near 30 degrees C.

Effects of furosemide on the resting membrane potentials and the transmitter-induced responses of the Aplysia ganglion cells.

Effects of Furosemide (FM) on the ionic channels in the ganglion cells of Aplysia were investigated using a conventional electrophysiological method. Application of 1 mM FM for 30 min did not alter the resting membrane potential or conductance. Na+-dependent responses to acetylcholine (ACh) were slightly depressed but Cl--dependent responses to ACh and gamma-aminobutyric acid (GABA) were markedly depressed by FM. On the other hand, K+-dependent responses to ACh and dopamine (DA), and cyclic adenosine-3', 5' monophosphate (cAMP)-dependent response to DA were not appreciably altered by 1 mM FM. The depressing effects of FM on both Na+- and Cl--dependent responses were due to the non-competitive inhibition of their receptor activities. It was concluded that 1 mM FM can change neither Na+-K+-pump nor Cl--pump mechanisms in the ganglion cells of Aplysia, but it depresses the receptor-operated Na+-and Cl--channel activities, the greater effect on the open Cl--channels. In addition, it was postulated that FM effect may be highly specific to the open state of Cl--channels, regardless of whether the membrane is excitable or not.

Temperature-sensitive activation of G-protein regulating the resting membrane conductance of aplysia ganglion cells

Temperature-sensitive activation of G-protein regulating the resting membrane conductance of aplysia ganglion cells

Desensitization of dopamine receptors observed in Aplysia ganglion cells.

The time course of the current response produced by a prolonged application of dopamine (DA) was studied in Aplysia ganglion cells with different types of DA receptors. The Na+-dependent fast excitatory receptors were found to be more readily desensitized than K+-dependent inhibitory ones, whereas Na+-dependent slow excitatory ones showed little sign of desensitization.

Selective blocking action of tetraethylammonium ion on three types of acetylcholine receptors in Aplysia ganglion cells.

The ganglion cells of Aplysia have three types of acetylcholine (ACh)-receptors in their membranes. The activation of one type produces Na+-dependent depolarization (DNa-type). The other receptors are HK- and HC1-types, the activation of which produces either K+- and Cl- dependent hyperpolarization, respectively. ACh-induced membrane current (IACh) was measured under the voltage clamp at different holding potential levels (V) of the membrane. The effects of tetraethylammonium (TEA) were analyzed quantitatively on each type of receptor activity by observing the changes in dose-inhibition curves and IACh-V relations obtained under voltage clamp. TEA exhibited a dose dependent block on both DNa- and HK-type of receptor activities but no effect on HCl-type. The mode of blocking action was found to be competitive in HK-type but noncompetitive in DNa-type. In the DNa-type receptor, 3 mM TEA shifted the reversal potential of ACh-induced current response from +5 to +40 mV, and caused a decrease in the slope of the IACh-V curve. In HK-type, TEA simply decreased the slope of IACh-V curve without changing the reversal potential of the ACh-induced response. It was concluded that TEA competed that with ACh for common binding sites at the receptor of the HK-type, but not of the DNa-type, and that in DNa-type, TEA made a binding with an allosteric site of the ACh-receptor and depressed the increase in membrane permeability toward Na+ as well as K+.

Blocking effect of serotonin on inhibitory dopamine receptor activity of Aplysia ganglion cells.

The abdominal ganglion of Aplysia includes neurons with a characteristic dopamine (DA) receptor, the activation of which induces a marked hyperpolarization with a specific increase in the permeability of the membrane to K+. The DA receptor of this type is called the "HK-type." A 2-min exposure to 1 microM serotonin (5-HT) had little effect on the resting membranes with the receptor of HK-type, but significantly depressed the responses to 10 microM DA. The depressing effect of 5-HT on this type of response was completely reversible after a 15-min washing with normal artificial Aplysia blood. Lineweaver-Burke type plotting of the DA-induced responses showed a systematic shift of the straight lines when the concentration of 5-HT was increased; the slope of the line became steeper but the intercept on the ordinate remained unchanged. The dose-inhibition curves, in which relative responses to a given [DA] were plotted against log [5-HT], showed a parallel shift toward the right when the concentration of DA increased. These findings suggest that 5-HT competes with DA for common binding sites at the DA receptor of HK-type, and that the blockade is not due to the interaction of 5-HT with K+-channels in the receptor membrane. The effect of other indole derivatives suggests that the DA receptor of HK-type includes anionic and cationic sites to which the NH2 group and 5-HO group of 5-HT could specifically bind, thus exhibiting competitive blockade.

Different effects of cAMP and cGMP derivatives on the activity of an identified neuron: Biochemical and electrophysiological analysis

Eight-position substituted cAMP and cGMP derivatives, and phosphodiesterase inhibitors, modify endogenous ‘bursting’ activity in Aplysia neuron R15. Several different patterns of activity were elicited depending on the agent used. 8-Benzylthio-cAMP or 8-parachlorophenylthio-cAMP, at concentrations between 5 μM and 0.3 mM, markedly enhanced the depth and duration of the interburst hyperpolarization, and in some cells bursting was inhibited completely. In contrast, 8-parachlorophenylthio-cGMP treatment led to some depolarization and to the appearance of long slow bursts, with little effect on the interburst phase. When the parachlorophenylthio-derivatives of cAMP and cGMP were added together at equal concentrations, a pattern consisting of long bursts interrupted by long and deep interburst hyperpolarizations was observed. This pattern could also be elicited by the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX). IBMX inhibited cAMP and cGMP phosphodiesterases and caused both cAMP and cGMP to accumulate in intact ganglia and in individual identified neuronal cell bodies including that of R15. Another phosphodiesterase inhibitor, Ro 7-2956, was a more potent inhibitor of cAMP than of cGMP phosphodiesterase; Ro 7-2956 also modified bursting activity, and seemed to enhance preferentially the interburst hyperpolarization. At high concentrations the 8-substituted cAMP and cGMP derivatives also inhibited cAMP and cGMP phosphodiesterases. The 8-parachlorophenylthio-derivatives of cAMP and cGMP were indistinguishable from each other in this assay, and thus phosphodiesterase inhibition cannot be responsible for their differential effects on bursting activity. The derivatives stimulated protein kinase activity in Aplysia ganglion homogenates, as measured by the incorporation of 32P from ATP into histone. IBMX and Ro 7-2956 had no detectable effect on protein kinase activity. The concentrations of cAMP and cGMP derivatives required for protein kinase activation (10 −8M–10 −6M) were much lower than those required for phosphodiesterase inhibition (10 −5M–10 −3M). Thus, differential protein phosphorylation is more likely to be responsible for the effects of cAMP and cGMP derivatives on neuron R15 bursting activity than is differential phosphodiesterase inhibition.

Rapid adaptation to neuronal membrane effects of ethanol and low temperature: Some speculations on mechanism

There is increasing evidence that ethanol exerts its primary effect at neuronal membranes by influencing specific lipid--protein or lipid--lipid interactions that control the state of organization of a specific membrane component; for example, a specific lipid--protein complex that controls a particular physiological property. This implies that tolerance to ethanol is the result of a change in the composition and/or state of organization of this critical membrane component. This altered state confers ethanol resistance. It may or may not have an effect on function in the absence of ethanol. One basis for these speculations comes from experiments using a sensitive and specific neurophysiological assay -- the rate of decay of posttetanic potentiation (PTP) at an identified synapse in an isolated, perfused Aplysia ganglion. We review evidence that PTP decay rate is strikingly accelerated by ethanol (a membrane-fluidizing agent) and strikingly decelerated below a transition temperature, presumably reflecting a transition in the structure of a membrane component. The ethanol and low-temperature effects are antagonistic. The system develops adaptation (tolerance) to either ethanol or low temperature within hours of its exposure. Tolerance persists for at least 12 hours, the longest interval tested thus far. In the absence of ethanol and at normal temperature the system behaves normally, that is it shows no "physical dependence". The system also has the remarkable property that when it becomes tolerant to either of these treatments, it shows tolerance to the other treatment that normally has the opposite effect. Therefore, the adaptation to either treatment cannot be a simple change in membrane composition governing overall membrane fluidity. A hypothesis which could explain the bidirectional cross-tolerance is considered in which adaptation to both treatments involves a shift from a homogeneous to a more heterogeneous composition of the critical membrane component, for example increasing heterogeneity in boundary lipid surrounding a critical membrane protein. It is becoming increasingly clear that ethanol exerts its primary effect by altering cell membrane structure -- by "fluidizing" or expanding neuronal and other membranes [1 - 6]. This effect results when ethanol, a somewhat hydrophobic molecule, intercalates between some fatty acid chains of membranes, reducing the degree of order of their alignment and increasing the lateral mobility of some membrane components. Decrease in the order of the fatty acid chains results in a measurable expansion of the membrane; and the change in fluidity is reflected in the change in mobility of appropriate probes that can be dissolved in the membrane [6, 7]. It is presumed that the physiological effects of ethanol are consequences of its fluidization of some critical membrane components; and that tolerance to ethanol is based on some form of resistance to this fluidization...

Selective blocking action of LSD on inhibitory dopamine receptors

Dopamine-induced postsynaptic responses of both excitory and inhibitory types were recorded from identified neurones of Aplysia ganglion. A 30 sec exposure to 0.1 μm d-lysergic acid diethylamide (LSD) significantly depressed the inhibitory responses to 1 mM dopamine, while a 60 sec exposure to 10 μm LSD did not alter the excitatory responses to 0.1 mm dopamine. Dose-response curves obtained from cells with inhibitory receptors indicate that the mode of LSD interaction was non-competitive.

The cholinergic excitatory and inhibitory postsynaptic responses of Aplysia ganglion cells during and after lithium-Ringer perfusion.

AbstractThe excitatory and inhibitory postsynaptic responses of Aplysia ganglion cells were elicited by either stimulating cholinergic afferent fibers or by topically applying acetylcholine (ACh) to the cell under study. The responses were evaluated as an increase in membrane conductance and compared before, during, and after perfusion with Li+‐Ringer. Excitatory postsynaptic responses were significantly depressed during 30–60 min of Li+‐Ringer perfusion. Upon return to normal Ringer, they increased sharply beyond the control, showing post‐Li+ augmentation.Inhibitory postsynaptic responses were augmented during 30–60 min of Li+‐Ringer perfusion when the cells were examined before treatment with anticholinesterase. After treatment with neostigmine, however, they were found to be also depressed in Li+‐Ringer. This finding suggested a suppressing effect of Li+‐Ringer on the acetylcholinesterase (AChE) activity.The reversal potential of ACh‐induced depolarizing response was altered from the normal value of –25 mV to –5 mV during 1–2 hr of Li+‐Ringer perfusion and gradually returned to normal value in 2–3 hr after return to normal Ringer. The reversal potential of ACh‐induced hyperpolarizing response remained practically unchanged (–62 mV) during 1–2 hr of Li+‐Ringer perfusion.Post‐Li+ augmentation of ACh‐induced depolarizing responses was explained by an increase in driving force for Na+ movement resulting from a depletion of intracellular Na+. The systematic shift of the dose‐response curves during Li+‐Ringer perfusion suggested that the affinities of both excitatory and inhibitory receptors to ACh are depressed and the effect is greater on the excitatory receptors.