Harvey M. Fishman

Potassium-ion conduction noise in squid axon membrane.

SummarySpectral analysis (1–1000 Hz) of spontaneous fluctuations of potential and current in small areas of squid (Loligo pealei) axon shows two forms of noise:f−1 noise occurs in both excitable and inexcitable axons with an intensity which depends upon the driving force for potassium ions. The other noise has a spectral form corresponding to a relaxation process, i.e., its asymptotic behavior at low frequencies is constant, and at high frequencies it declines with a slope of −2. This latter noise occurs only in excitable axons and was identified in spectra by (1) its disappearance after reduction of K+ current by internal perfusion with solutions containing tetraethylammonium (TEA+), Cs+ or reduced [Ki+] and (2) its insensitivity to block of Na+ conduction and active transport. The transition frequency of relaxation spectra are also voltage and temperature dependent and relate to the kinetics of K+ conduction in the Hodgkin-Huxley formulation. These data strongly suggest that the relaxation noise component arises from the kinetic properties of K+ channels. Thef−1 noise is attributed to restricted diffusion in conducting K+ channels and/or leakage pathways. In addition, an induced K+-conduction noise associated with the binding of TEA+ and triethyldecylammonium ion to membrane sites is described. Measurement of the induced noise may provide an alternative means of characterizing the kinetics of interaction of these molecules with the membrane and also suggests that these and other pharmacological agents may not be useful in identifying noise components related to the sodium conduction mechanism which, in these experiments, appears to be much lower in intensity than either the normal K conduction or induced noise components.

Axolemmal repair requires proteins that mediate synaptic vesicle fusion.

A damaged cell membrane is repaired by a seal that restricts entry or exit of molecules and ions to that of the level passing through an undamaged membrane. Seal formation requires elevation of intracellular Ca(2+) and, very likely, protein-mediated fusion of membranes. Ca(2+) also regulates the interaction between synaptotagmin (Syt) and syntaxin (Syx), which is thought to mediate fusion of synaptic vesicles with the axolemma, allowing transmitter release at synapses. To determine whether synaptic proteins have a role in sealing axolemmal damage, we injected squid and crayfish giant axons with an antibody that inhibits squid Syt from binding Ca(2+), or with another antibody that inhibits the Ca(2+)-dependent interaction of squid Syx with the Ca(2+)-binding domain of Syt. Axons injected with antibody to Syt did not seal, as assessed at axonal cut ends by the exclusion of extracellular hydrophilic fluorescent dye using confocal microscopy, and by the decay of extracellular injury current compared to levels measured in uninjured axons using a vibrating probe technique. In contrast, axons injected with either denatured antibody to Syt or preimmune IgG did seal. Similarly, axons injected with antibody to Syx did not seal, but did seal when injected with either denatured antibody to Syx or preimmune IgG. These results indicate an essential involvement of Syt and Syx in the repair (sealing) of severed axons. We suggest that vesicles, which accumulate and interact at the injury site, re-establish axolemmal continuity by Ca(2+)-induced fusions mediated by proteins such as those involved in neurotransmitter release.

Plasmalemmal repair of severed neurites of PC12 cells requires Ca2+ and synaptotagmin

Ca2+ and synaptotagmin (a Ca2+‐binding protein that regulates axolemmal fusion of synaptic vesicles) play essential roles in the repair of axolemmal damage in invertebrate giant axons. We now report that neurites of a rat pheochromocytoma (PC12) cell line transected and maintained in a serum medium form a dye barrier (exclude an external hydrophilic fluorescent dye) and survive for 24‐hr posttransection (based on morphology and retention of another hydrophilic dye internally loaded at 6‐hr posttransection). Some (25%) transected neurites that form a dye barrier regrow. Most (83%) neurites transected in a saline solution containing divalent cations (PBS++) also exclude entry of an externally placed hydrophilic fluorescent dye at 15‐min posttransection. In contrast, only 14 or 17% of neurites maintained in a divalent cation‐free solution (PBS=) or in PBS= + Mg2+, respectively, form a dye barrier. Neurites that do not form a dye barrier do not survive for 24 hr. When PC12 neurites are loaded with an antibody to squid synaptotagmin, most (81%) antibody‐loaded neurites do not form a dye barrier, whereas most (≥81%) neurites loaded with heat‐inactivated antibody or preimmune IgG do form a barrier. These data show that: 1) transected neurites of PC12 cells have mechanism(s) for plasmalemmal repair (dye barrier formation and survival); 2) Ca2+ is necessary for dye barrier formation, which occurs minutes after transection and is necessary for survival and regrowth; and 3) synaptotagmin is an essential mediator of barrier formation. The similarity in the requirements for plasmalemmal repair in this mammalian cell preparation with those reported previously for invertebrate axons suggests that mechanisms necessary for plasmalemmal repair have been conserved phylogenetically. J. Neurosci. Res. 62:566–573, 2000.

Noise measurements in squid axon membrane

SummaryA small area (10−4 to 10−5 cm2 patch) of the external surface of a squid (Loligo pealei) axon was “isolated” electrically by means of a pair of concentric glass pipettes and sucrose solution to achieve a low extraneous noise measurement of spontaneous fluctuations in membrane potential and current. The measured “small-signal” impedance function of the isolated patch in seawater was constant at low frequencies and declined monotonically at frequencies beyond 100 Hz. It is shown that the power-density spectrum (PDS) of voltage noise, which generally reflects the current-noise spectrum filtered by the membrane impedance function, is equivalent to the power spectrum of current-noise up to frequencies where the impedance decline is significant (Fishman, 1973a, Proc. Nat. Acad. Sci. USA70:876). This result is in contrast to an impedance resonance measured under uniform constant-current (internal axial wire) conditions, for which the voltage-noise PDS reflects the impedance resonance. The overdamped resonance in the patch technique is a consequence of the relatively low resistance (1 MΩ) pathways through the sucrose solution in the interstitial Schwann cell space which surround and shunt the high resistance (10–100 MΩ) membrane patch. Current-noise measurements during patch voltage clamp extend observation of patch ionconductance fluctuations to 1 kHz. Various tests are presented to demonstrate the temporal and spatial adequacy of patch potential control during current-noise measurements.

Direct and Rapid Description of the Individual Ionic Currents of Squid Axon Membrane by Ramp Potential Control

Computations based upon the Hodgkin-Huxley equations and experimental data from squid axons show that ramp functions can be used as commands to a voltage clamp system to selectively observe either the fast (sodium) or slow (potassium) process in axon membranes without chemical separation techniques or computer assistance. Each process is characterized directly (on line) and rapidly (real time) by generating a current-potential curve on an oscilloscope for fast or slow rates of change of membrane potential (ramps). The speed and directness of this method of characterizing each of the essential axonal events permit quantitative measurement of the kinetics of rapid effects on these processes due to various pharmacological agents such as tetrodotoxin and tetraethylammonium ion or other experimental changes in the membrane environment.

Plasmalemmal sealing of transected mammalian neurites is a gradual process mediated by Ca2+‐regulated proteins

Cultured mammalian PC12 or B104 cells do not instantaneously restore a plasmalemmal barrier (seal) after neurite transection, as measured using fluorescent dye probes of various sizes and saline solutions with different [Ca2+]o. Rather, transected cells gradually (from 15 to 60 min postseverance) exclude probes (dye molecules) of progressively smaller size. Furthermore, an inhibitor (calpeptin) of a Ca2+‐activated cysteine protease (calpain) and antibodies or toxins to a Ca2+‐regulated protein (synaptotagmin) and other membrane fusion proteins (syntaxin and synaptobrevin) inhibit plasmalemmal sealing. These data obtained using molecular probes on mammalian cell lines are consistent with previous data on invertebrate giant axons indicating that Ca2+ plays many roles in the formation, accumulation, and fusion/interaction of vesicles gradually forming a seal at a site of plasmalemmal damage.

Barrier Permeability at Cut Axonal Ends Progressively Decreases until an Ionic Seal Is Formed

After axonal severance, a barrier forms at the cut ends to rapidly restrict bulk inflow and outflow. In severed crayfish axons we used the exclusion of hydrophilic, fluorescent dye molecules of different sizes (0.6-70 kDa) and the temporal decline of ionic injury current to levels in intact axons to determine the time course (0-120 min posttransection) of barrier formation and the posttransection time at which an axolemmal ionic seal had formed, as confirmed by the recovery of resting and action potentials. Confocal images showed that the posttransection time of dye exclusion was inversely related to dye molecular size. A barrier to the smallest dye molecule formed more rapidly (<60 min) than did the barrier to ionic entry (>60 min). These data show that axolemmal sealing lacks abrupt, large changes in barrier permeability that would be expected if a seal were to form suddenly, as previously assumed. Rather, these data suggest that a barrier forms gradually and slowly by restricting the movement of molecules of progressively smaller size amid injury-induced vesicles that accumulate, interact, and form junctional complexes with each other and the axolemma at the cut end. This process eventually culminates in an axolemmal ionic seal, and is not complete until ionic injury current returns to baseline levels measured in an undamaged axon.

Patch voltage clamp of squid axon membrane

SummaryA small area (patch) of the external surface of a squid axon can be “isolated” electrically from the surrounding bath by means of a pair of concentric glass pipettes. The seawater-filled inner pipette makes contact with the axon and constitutes the external access to the patch. The outer pipette is used to direct flowing sucrose solution over the area surrounding the patch of membrane underlying the inner pipette. Typically, sucrose isolated patches remain in good condition (spike amplitude >90 mV) for periods of approximately one half hour. Patches of axon membrane which had previously been exposed to sucrose solution were often excitable. Membrane survival of sucrose treatment apparently arises from an outflow of ions from the axon and perhaps satellite cells into the interstitial cell space surrounding the axolemma. Estimate of the total access resistance (electrode plus series resistance) to the patch is about 100 kΩ (7 Ω cm2). Patch capacitance ranges from 10–100 pF, which suggests areas of 10−4 to 10−5 cm2 and resting patch resistances of 10–100 MΩ. Shunt resistance through the interstitial space exposed to sucrose solution, which isolates the patch, is typically 1–2 MΩ. These parameters indicate that good potential control and response times can be achieved on a patch. Furthermore, spatial uniformity is demonstrated by measurement of an axoplasmic isopotential during voltage clamp of an axon patch. The method may be useful for other preparations in which limited membrane area is available or in special instances such as in the measurement of membrane conduction noise.

DELAMINATING MYELIN MEMBRANES HELP SEAL THE CUT ENDS OF SEVERED EARTHWORM GIANT AXONS

Transected axons are often assumed to seal by collapse and fusion of the axolemmal leaflets at their cut ends. Using photomicroscopy and electronmicroscopy of fixed tissues and differential interference contrast and confocal fluorescence imaging of living tissues, we examined the proximal and distal cut ends of the pseudomyelinated medial giant axon of the earthworm, Lumbricus terrestris, at 5-60 min post-transection in physiological salines and Ca2+-free salines. In physiological salines, the axolemmal leaflets at the cut ends do not completely collapse, much less fuse, for at least 60 min post-transection. In fact, the axolemma is disrupted for 20-100 microm from the cut end at 5-60 min post-transection. However, a barrier to dye diffusion is observed when hydrophilic or styryl dyes are placed in the bath at 15-30 min post-transection. At 30-60 min post-transection, this barrier to dye diffusion near the cut end is formed amid an accumulation of some single-layered and many multilayered vesicles and other membranous material, much of which resembles delaminated pseudomyelin of the glial sheath. In Ca2+-free salines, this single and multilayered membranous material does not accumulate, and a dye diffusion barrier is not observed. These and other data are consistent with the hypothesis that plasmalemmal damage in eukaryotic cells is repaired by Ca2+-induced vesicles arising from invaginations or evaginations of membranes of various origin which form junctional contacts or fuse with each other and/or the plasmalemma.

Interaction of apical and basal membrane ion channels underlies electroreception in ampullary epithelia of skates.

The exquisite sensitivity of elasmobranch fishes to electric fields is thought to reside in electroreceptive organs called ampullae of Lorenzini. We measured the stimulus-response behavior of ampullary organs excised from skates. Under open-circuit conditions, the ampullary organ showed three distinct response states: spontaneous repetitive spikes, evoked spikes, and small, damped oscillatory responses. Under short-circuit conditions, the amplitude range for a linear current response to a sinusoidal (0.5 Hz) voltage clamp of an organ (assessed by spectral analysis of the harmonics generated) was 7-200 microV rms. Changes in the spike firing rate of the afferent nerve innervating the organ were evident for voltage clamps of the ampullary epithelium of 3 microV and the spike rate saturated for clamp steps exceeding 100 microV. Thus, the linear response range of the ampullary epithelium exceeded the range in spike firing rate of the afferent nerve. The steady-state transorgan electrical properties under voltage clamp conditions were obtained by analysis of complex admittance determinations in the frequency range 0.05-20 Hz for perturbations (< 100 microV rms) in the linear range. Admittance functions were distinctly related to the preparation states observed under open-circuit conditions. A negative real part in the organ admittance (i.e., a steady-state negative conductance generated by the preparation) was a common characteristic of the two (open-circuit) excitable states. The negative conductance was also confirmed by the direction of current flow through the ampullary epithelium in response to step voltage clamps. We conclude that the steady state-negative conductance is an essential property of the ampullary epithelium,and we suggest that the interplay of negative and positive conductances generated by ion channels in apical and basal membranes of receptor cells results in signal amplification that may contribute significantly to the electric field sensitivity of ampullary organs.