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Cell Culture in Neurobiology

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Abstract

The sections in this article are:

1 Introduction
1.1 Primary Cell Cultures
1.2 Cell Lines
1.3 Muscles as Models
2 Techniques
2.1 Primary Cultures
2.2 Cell Lines
3 Cell Form
3.1 Distinctive Cell Shapes
3.2 Development of Cell Form
3.3 Growth Cones
3.4 Factors Affecting Process Formation
4 Resting Membrane Potential
5 Action Potential
5.1 Ionic Determinants
5.2 Developmental Aspects
6 Synaptic Transmission
6.1 Nerve‐Muscle Synapses
6.2 Synapses Between Neurons
6.3 Chemosensitivity of Neurons
6.4 Transmitter Metabolism
6.5 Factors Affecting Synapse Formation
7 Summary
Figure 1. Figure 1.

Rat spinal cord and attached spinal ganglion explant culture after 46 days in vitro. Arrows point to spinal ganglion cells. Tissue organization, including dorsal and ventral roots, is preserved to a remarkable degree. Note that fibers originating in the anterior horn or attached ganglion tend to follow a fairly straight course. Only myelinated fibers are seen in this bright‐field view

From Sobkowicz et al.
Figure 2. Figure 2.

Dissociation of a 7‐day embryonic chick spinal cord; at this age sensory ganglia are located within the cartilaginous spinal canal. A: sensory ganglia remain attached to the spinal cord when it is removed. B: same segment shown in A after “stripping’ the dorsal root ganglion by removing the meninges. C: minced fragments of the spinal cord. D: after incubating fragments in 0.1% trypsin in Pucks (divalent cation‐free) saline, they are resuspended in complete medium, triturated, and filtered. E: isolated, spherical cells. Note variation in cell diameter: vertical bar in B represents 1 mm and applies to A‐C; horizontal bar in E, 50 μm.

Figure 3. Figure 3.

Elimination of “background’ cells. Skeletal muscle fibers in a control (A) 12‐day culture and in a sister culture (α) treated with cytosine arabinoside (araC; 10−5 M) for 48 h beginning on the 3rd day. Inset in α is a typical hypolemmal muscle nucleus. Spinal cord neurons in a control (B) and in a sister culture (b) treated with araC. Sympathetic ganglion cells grown in media containing HCO3 and exposed to CO2 (C) and in media without HCO3 and exposed to air (c). Horizontal bars, 50 μm in each panel. Note the absence of background, fibroblast‐like cells in a‐c

A and α from Fischbach & Cohen ; B and b from Fischbach ; C and c from P. Patterson, unpublished observations
Figure 4. Figure 4.

Separation of dissociated 7‐day chick spinal cord cells by velocity sedimentation (see text). Interference contrast micrographs illustrate the wide range in cell size in the initial suspension (upper), small cells in trailing fractions (middle), and large cells in leading fractions (lower).

From D. K. Berg and G. D. Fischbach, unpublished observations
Figure 5. Figure 5.

Neuroblastoma cells clone N‐18. A: log phase. B: stationary phase — grown in the absence of serum for 4 days. C: aminopterin‐selected (10−6 M) cells grown in the presence of serum

From Peacock et al.
Figure 6. Figure 6.

Silver‐impregnated spinal cord cells. A and B illustrate the range in size and shape of neurons. Compare with the characteristically bipolar sensory ganglion cell (C) grown under identical conditions. Horizontal bar, 50 μm

From Fischbach & Dichter
Figure 7. Figure 7.

Isolated sympathetic ganglion cell: A, at time 0 (18 h after plating); B, at 60 min; C, at 120 min; D, at 240 min; E, at 360 min; F, at 500 min. A‐C same scale, horizontal bar 78 μm, D‐F same scale, horizontal bars 80 μm. Note that at the end of the sequence all growing tips are approximately equidistant from the cell body and also that branch points are apparently stable.

Figure 8. Figure 8.

Polarity of processes. A: sensory ganglion cell with one process issuing from the cell body after 4 days in culture.

From Nakai .] Note the “recurrent collateral’ near the cell body. B: spinal cord neuron in a 3‐wk culture with one prominent, long, relatively unbranched process. Cell body inked in. [From Fischbach . Copyright 1970 by the American Association for the Advancement of Science
Figure 9. Figure 9.

Growth of chick embryonic neurites in culture plotted from time‐lapse film records. A: control; small dots refer to a sensory ganglion cell and larger dots to a neuron isolated from the midbrain. B: neurites severed from their cell body at time 0

Adapted from Hughes
Figure 10. Figure 10.

Scanning electron micrographs of chick sensory ganglion cell growth cones 1 day after plating. Note the fine microspikes in A and the elaborate “undulating’ membrane in B. A, × 2,000; B, × 17,000.

Micrographs provided by J. Jabaily
Figure 11. Figure 11.

Ultrastructure of a chick sensory ganglion cell growth cone. A filamentous network (FN) which extends into the microspikes is evident beneath the surface membrane. Note that microtubules (MT) appear to end at the base of the growth cone and that neurofilaments (NF) are centrally located. Tubulovesicular profiles of the smooth endoplasmic reticulum (SER) are abundant and appear at the base of one microspike. V, D, and C are smooth, dense‐core, and coated vesicles, respectively; MC is a mitochondrion

From Yamada et al.
Figure 12. Figure 12.

Selective growth of sympathetic ganglion (sg) neurites toward nearby fragments of vas deferens (vd) rather than kidney (k); 5 days in vitro. Horizontal bar, 400 μm

From Chamley et al.
Figure 13. Figure 13.

Membrane potential (A) and the relation between membrane potential and [K+]0(B) of rat myotubes as a function of age. In A, • refers to multinucleated myotubes and Δ to mononucleated myoblasts. Vertical bars, ± SE. The number of cells at each point is indicated. In B, 4 myotubes of the indicated ages were examined. In each case the cultures were perfused with a continuous K+ gradient between 5.3 mM and 148 mM which was completed in 3 min. The relation between membrane potential and log [K+]0 predicted by the Nernst equation is indicated by the line labeled EK. Continuous lines through each set of points are predicted from the constant‐field equation (Eq. in text) assuming [Na]i = 13 mM and the PNa+‐to‐PK+ ratios are as indicated at each day

From Ritchie & Fambrough
Figure 14. Figure 14.

Action potentials evoked in spinal cord (A and C) and sensory ganglion (B and D) cells. Duration of current pulses indicated by bars above each trace; dorsal root ganglion (DRG) spikes are prolonged and are marked by a plateau on the falling phase. Many spinal cord neurons fire repetitively during a sustained depolarization (C), whereas most DRG cells rapidly adapt (D). The resting potential of cells in A‐C was 50–55 mV.

Figure 15. Figure 15.

Action potentials conducted along a process. All records obtained from a microelectrode located in the cell body shown in A. Spikes were evoked by extracellular stimulation (frames 2–4) as shown and by intracellular stimulation (frame 1). Stimulus artifacts appear as breaks in the records. Note the inflection on the rise of conducted spikes. In B‐4, superimposed traces show that hyperpolarization (inward current pulse not shown) accentuates the inflection and ultimately blocks the second component. Calibration pulse, 20 mV, 1 ms. Vertical bar in A, 50 μm

From Fischbach & Dichter
Figure 16. Figure 16.

Presumptive glial cells. A and B: two cells found to be electrically inexcitable. Current‐voltage relation of the cell shown in B is plotted in C. Sample traces are shown in inset. Note that the relation remains linear over a wide range of membrane potential.

Figure 17. Figure 17.

Ca2+ spikes in sensory neurons. A: relation between overshoot of the action potential evoked at the cell body and external Ca2+. Insets show representative traces recorded in the presence of TTX (10−7 g/ml) and 1.8 mM Ca2+ (trace 1) and 18 mM Ca2+ (trace 2). B: effect of Na+ on conducted spikes. Two cells (α and b) were bathed in Na+‐free media. In each case stimulation (arrows) of a process 300–400 μm away from the cell body failed to evoke a conducted spike (trace 1). An action potential was evoked via an intrasoma electrode in cell b. Immediately before trace 2, a 50 μm tip, NaCl‐filled electrode was lowered adjacent to the process. Identical extracellular stimuli now evoked conducted spikes in α and b. The axon spike in α invaded the cell body, whereas the one in b did not. Trace 3 in b was recorded after the NaCl electrode was removed. Note in b the decrease in duration of the soma spike in the Na+ atmosphere

From Dichter & Fischbach
Figure 18. Figure 18.

Action potential mechanisms in chick (A) and L‐6 (B) myotubes. A: in chick fibers, the sharp spike observed in normal saline (1) is abolished in Na+‐free (substituted with sucrose) media (2); a small approximately 50‐ms active response remains (wedge) which is abolished by Co2+ (10 mM) (3). The prolonged active response (arrows) which is not affected by removing Na+ or by addition of Co2+ is presumably due to an outward Cl current. The stimulus duration is indicated by a bar below each trace.

From Fuduka .] B: in normal saline the sharp spike of L‐6 myotubes (evoked by anode‐break excitation) is followed by a prolonged active response (1). The sharp spike is absent, but the late response is increased in size and duration in Na+‐free saline containing 10 mM Ca2+ (2). (Normal saline contains 1.8 mM Ca2+.) [From Kidokoro
Figure 19. Figure 19.

Changes in action potential generation in two cell lines as they “differentiate’ in vitro. A: L‐6 muscle cells. A small active response evoked in L‐6 mononucleated cell (myoblast) following a hyperpolarizing current pulse is contrasted with a large prolonged spike in a multinucleated (fused) myotube. In each pair, current is displayed in the upper trace and membrane potential in the lower trace.

From Kidokoro .] B: neuroblastoma cells (clone N‐18) in log phase (upper record), stationary phase (middle record), and selected in the presence of aminopterin (lower record). [From Peacock et al. .] The neuroblastoma spikes were recorded from the cells in Fig. which are indicated by the blurred image of a microelectrode
Figure 20. Figure 20.

Action potentials in neuroblastoma × L cell hybrids. Cells from two clones derived from the same hybridization are shown in A and B. A appears more neuronal and B more fibroblastic. The response of each cell type to depolarizing currents is shown in adjacent records. Cells in the neuronlike clone generate spikes, whereas those in the fibroblastlike clone do not. Both clones were grown in the presence of aminopterin for 10 days. Calibration pulse, 10 mV

From Peacock et al.
Figure 21. Figure 21.

A multipolar chick spinal cord neuron that has settled between muscle fibers on the collagen substratum. The neuron was dissociated from a 7‐day cord and plated about 48 h earlier. Note the microspike that extends from an expanded growth cone (gc) to contact a muscle fiber.

Figure 22. Figure 22.

Neuromuscular synaptic physiology. A: functional contact — a depolarizing synaptic potential (upper trace) follows an action potential in a nearby neuron evoked by intracellular stimulation (current pulse not shown). Bars, 3.5 and 50 mV for muscle and neuron records, respectively, and 10 ms for both. B: superimposed traces show the change in end‐plate potential (EPP) amplitude as the muscle membrane potential (Vm) was altered to the values shown at the left of each record by steady currents injected through a second intracellular microelectrode; calibration pulse, 5 mV, 5 ms. Each point in the graph represents the mean amplitude of 5–10 EPP's. C: ACh response (arrow) evoked by a 1‐ms pulse of ACh precisely mimics a spontaneous synaptic potential recorded on the same trace; calibration, 5 mV, 10 ms. D: reversible reduction in EPP size by d‐tubocurare (10−7 g/ml); record at extreme right obtained 30 min after removal of the drug; bars, 15 mV, 15 ms. E: miniature end‐plate potentials (MEPP's) recorded on contiguous segments of moving film; bars, 5 mV, 2 s. F: variation in amplitude (and one failure) of successive EPP's in a series evoked at a rate of 1/s, despite a constant stimulus (action potential) in the innervating neuron (upper traces); calibration pulse, 5 mV, 5 ms

A, B, D, and F from Fischbach ; C from Fischbach & Cohen
Figure 23. Figure 23.

A site of transmitter release identified by focal depolarization in the presence of TTX (see text). Interference contrast illumination of a chick muscle cell contacted by a neurite from a nearby spinal cord explant. Synaptic potentials were evoked by extracellular stimulation at α but not at b or elsewhere along the process which extends along the edge of the fiber

From Fischbach et al.
Figure 24. Figure 24.

Appearance of ACh receptors in muscle cells. A: increase in α‐bungarotoxin (α‐BGT) binding in control (▪) and in fusion‐arrested (•) chick muscle cultures (see text). ▴, fusion index (percent of nuclei incorporated into myotubes). Δ, fusion index in low‐Ca2+ media. Note that the increase in toxin binding lags behind the fusion index in control cultures and is even further delayed when fusion is blocked.

From Patterson & Prives .] B: elongated mononucleated muscles from rat muscle grown in standard media for 48 h. The oscilloscope record shows an ACh response (current on lower trace) recorded in cell 1. Horizontal bar, 100 μm. [From Fambrough & Rash .] C: autoradiographs of freshly dissociated (without the use of enzymes) mononucleated cells from chick muscle exposed in suspension to [125I]α‐BGT. Interference contrast and bright‐field views. Horizontal bar, 10 μm. [From Smilowitz et al.
Figure 25. Figure 25.

Synthesis and degradation of ACh receptors by cultured muscle fibers. A: following a saturating exposure to unlabeled α‐bungarotoxin (α‐BGT). The appearance of new receptors was determined by [125I]α‐BGT binding (•). ○, cultures treated as above but pretreated for 3 h with cycloheximide; Δ, cultures preincubated in dinitrophenol plus iodoacetate.

From Harzell & Fambrough .] B: release of radioactivity into the medium from chick or rat muscle fibers saturated with [125I]α‐BGT. Release, expressed as a fraction of that initially bound, is a first‐order process with a half‐time of about 20 h. [From Devreotes & Fambrough
Figure 26. Figure 26.

Histograms of ACh sensitivity of uninnervated (A) and innervated (B) muscle fibers in spinal cord muscle cell cultures.

From Fischbach and Cohen .] In (C) active and inactive uninnervated fibers are compared. No neurons were present. Active fibers stimulated intermittently for 2–3 days at 10/sec (\ \ \ \) were less sensitive than inactive fibers grown in the presence of TTX (/ / / /). [From Cohen & Fischbach . Copyright 1973 by the American Association for the Advancement of Science
Figure 27. Figure 27.

Relative peaks of ACh sensitivity on uninnervated (A) and innervated (B) muscle fiber; ordinate, mV/nC. In A, bars indicate position of hypolemmal muscle nuclei. [From Fischbach & Cohen .] B: a hot spot located exactly at a site of transmitter release. Inset, synaptic potentials evoked by focal depolarization at the termination of a nerve process. ACh was iontophoresed at the points indicated in the tracing (prepared from a photograph), and the sensitivities at each point are shown in register below. Bars, 5 mV, 5 ms.

From S. A. Cohen and G. D. Fischbach, unpublished observations
Figure 28. Figure 28.

Tubulovesicular network of cultured muscle fibers. In A note the openings to the exterior and also that some tubules form junctions with cisternae of the sarcoplasmic reticulum (arrow). Horizontal bar, 0.25 μm. B: unstained section of a muscle fiber exposed to horseradish peroxidase prior to fixation. The reaction product outlines areas continuous with the extracellular space. N, nucleus; G, Golgi; T, tubules; S, sarcoplasmic reticulum.

Micrographs provided by M. Henkart
Figure 29. Figure 29.

Spontaneously occurring synaptic potentials in dissociated chick spinal cord cells. A and B: superimposed traces show depolarizing potentials (EPSP's) in one cell (A), hyperpolarizing potentials (IPSP's) in another (B, upper record), and both in a third cell (B, lower record). Vertical bar, 10 mV for A, 12 mV for B. Horizontal bar, 5 ms for A, 10 ms for B, upper trace, 20 ms for B, lower trace. C: a continuous record shows regularly occurring large EPSP's that often trigger action potentials. Note the few small PSP's. D: Small PSP's recorded in the presence of TTX (10−7 g/ml). Vertical bar, 10 mV for C, 5 mV for D. Horizontal bar, 1 s for C, 5 s for D

From Fischbach & Dichter
Figure 30. Figure 30.

Stimulus‐evoked synaptic potentials in mouse spinal cord‐sensory ganglion cell cultures. A: monophasic EPSP (trace 3) evoked in one spinal cord neuron by stimulation (current in trace 1) of another spinal cord neuron (trace 2). B: action potential in a sensory neuron (evoked by a brief current pulse — not shown); trace 1 is followed by a small variable EPSP in a spinal cord cell (superimposed traces in 2). A larger less variable EPSP evoked by another sensory neuron in another spinal cord cell is shown in trace 3. C: complex responses in spinal cord neurons. An outward current plus (trace 3) evokes three spikes and is followed by a long‐lasting barrage of spikes and PSP's in the same (trace 2) and in an adjacent (trace 1) neuron.

Figure 31. Figure 31.

Electron micrographs of synaptic structures in chick spinal cord (A) and rat brain (B and C) cell cultures. In each, note the accumulations of small clear vesicles, the larger dense‐core vesicles, and increased density or thickening of the synaptic membranes. A, Several synaptic profiles appear along a single process. × 18,000. C: a synapse contacts a small process which may be a “spine.’ B and C, × 32,000.

A, from Fischbach & Dichter ; B and C provided by E. Neale
Figure 32. Figure 32.

Stimulation of a single presynaptic ending on a chick spinal cord neuron. Successive brief negative current pulses, delivered through a 2‐μm‐tipped NaCl‐filled pipette placed exactly over the bouton indicated by the arrow, evoked the synaptic potentials shown at right (traced from photographs). The response disappeared when the stimulating electrode was moved about 5 μm in any direction. Note that the incoming nerve processes pass out of the plane of focus. The postsynaptic nerve cell body overlies a muscle fiber. Calibration bars, 5 mV, 5 ms.

Figure 33. Figure 33.

Drug responses. A: ACh potentials in a sympathetic ganglion cell. The 20‐nA iontophoretic pulse is shown in the second trace; suprathreshold and subthreshold responses are shown in a and b. B: glutamate potential in a spinal cord neuron; iontophoretic pulse, 30 nA. This response is slower than EPSP's recorded in the same cell (trace 3). C: GABA potentials in a sympathetic ganglion cell. D: GABA potentials in a spinal cord neuron. A 25‐nA pulse (duration indicated by bar) was repeated as the membrane potential was shifted. The response reverses in polarity between −70 and −50 mV

A and C from Obata ; B and D from B. R. Ransom and P. G. Nelson, unpublished observations
Figure 34. Figure 34.

Nonuniform distribution of chemoreceptors on a mouse spinal cord neuron (A) and a neuroblastoma cell (B) about 4 wk after plating. A: penwriter records of glutamate responses at the sites indicated. Note the relatively large and rapid response at lower left. The same neuron impregnated with silver is shown in the inset.

From B. R. Ransom and P. G. Nelson, unpublished observations.) B: ACh produces a qualitatively different response at different sites — a depolarization over the soma, a hyperpolarization at relatively distant sites along processes, and a biphasic response at intermediate points. (From Peacock et al. 1973
Figure 35. Figure 35.

A: increase in synthesis of catecholamines (norepinephrine and dopamine) from [3H]tyrosine by rat sympathetic ganglion neurons with time in culture. Note the initial log of 7–8 days followed by a 50‐fold increase. This pattern is markedly different from the increase in total accumulation of radioactivity measured in the same cultures (B)

From Mains & Patterson
Figure 36. Figure 36.

Cholinergic synapses between sympathetic ganglion cells. A: simultaneous recordings from two sympathetic neurons in which each spike in one (the “driver,’ middle trace) is followed by a depolarizing synaptic potential in the other (the “follower,’ upper trace). Some PSP's in the follower are large enough to trigger spikes. The first spike in the driver cell is evoked by a brief pulse of outward current (lower trace). The other spikes arise spontaneously. B: the same cells in the presence of 50 μM hexamethonium, a nicotinic cholinergic antagonist. All spontaneous PSP's are abolished, and the evoked spike in the driver neuron is no longer followed by a PSP in the follower. C: the same cells after washing out the hexamethonium. Calibration bars: 40 mV, 40 ms.

From O. MacLeish, P. H. O'Lague, E. J. Furshpan, and D. D. Potter, unpublished observations


Figure 1.

Rat spinal cord and attached spinal ganglion explant culture after 46 days in vitro. Arrows point to spinal ganglion cells. Tissue organization, including dorsal and ventral roots, is preserved to a remarkable degree. Note that fibers originating in the anterior horn or attached ganglion tend to follow a fairly straight course. Only myelinated fibers are seen in this bright‐field view

From Sobkowicz et al.


Figure 2.

Dissociation of a 7‐day embryonic chick spinal cord; at this age sensory ganglia are located within the cartilaginous spinal canal. A: sensory ganglia remain attached to the spinal cord when it is removed. B: same segment shown in A after “stripping’ the dorsal root ganglion by removing the meninges. C: minced fragments of the spinal cord. D: after incubating fragments in 0.1% trypsin in Pucks (divalent cation‐free) saline, they are resuspended in complete medium, triturated, and filtered. E: isolated, spherical cells. Note variation in cell diameter: vertical bar in B represents 1 mm and applies to A‐C; horizontal bar in E, 50 μm.



Figure 3.

Elimination of “background’ cells. Skeletal muscle fibers in a control (A) 12‐day culture and in a sister culture (α) treated with cytosine arabinoside (araC; 10−5 M) for 48 h beginning on the 3rd day. Inset in α is a typical hypolemmal muscle nucleus. Spinal cord neurons in a control (B) and in a sister culture (b) treated with araC. Sympathetic ganglion cells grown in media containing HCO3 and exposed to CO2 (C) and in media without HCO3 and exposed to air (c). Horizontal bars, 50 μm in each panel. Note the absence of background, fibroblast‐like cells in a‐c

A and α from Fischbach & Cohen ; B and b from Fischbach ; C and c from P. Patterson, unpublished observations


Figure 4.

Separation of dissociated 7‐day chick spinal cord cells by velocity sedimentation (see text). Interference contrast micrographs illustrate the wide range in cell size in the initial suspension (upper), small cells in trailing fractions (middle), and large cells in leading fractions (lower).

From D. K. Berg and G. D. Fischbach, unpublished observations


Figure 5.

Neuroblastoma cells clone N‐18. A: log phase. B: stationary phase — grown in the absence of serum for 4 days. C: aminopterin‐selected (10−6 M) cells grown in the presence of serum

From Peacock et al.


Figure 6.

Silver‐impregnated spinal cord cells. A and B illustrate the range in size and shape of neurons. Compare with the characteristically bipolar sensory ganglion cell (C) grown under identical conditions. Horizontal bar, 50 μm

From Fischbach & Dichter


Figure 7.

Isolated sympathetic ganglion cell: A, at time 0 (18 h after plating); B, at 60 min; C, at 120 min; D, at 240 min; E, at 360 min; F, at 500 min. A‐C same scale, horizontal bar 78 μm, D‐F same scale, horizontal bars 80 μm. Note that at the end of the sequence all growing tips are approximately equidistant from the cell body and also that branch points are apparently stable.



Figure 8.

Polarity of processes. A: sensory ganglion cell with one process issuing from the cell body after 4 days in culture.

From Nakai .] Note the “recurrent collateral’ near the cell body. B: spinal cord neuron in a 3‐wk culture with one prominent, long, relatively unbranched process. Cell body inked in. [From Fischbach . Copyright 1970 by the American Association for the Advancement of Science


Figure 9.

Growth of chick embryonic neurites in culture plotted from time‐lapse film records. A: control; small dots refer to a sensory ganglion cell and larger dots to a neuron isolated from the midbrain. B: neurites severed from their cell body at time 0

Adapted from Hughes


Figure 10.

Scanning electron micrographs of chick sensory ganglion cell growth cones 1 day after plating. Note the fine microspikes in A and the elaborate “undulating’ membrane in B. A, × 2,000; B, × 17,000.

Micrographs provided by J. Jabaily


Figure 11.

Ultrastructure of a chick sensory ganglion cell growth cone. A filamentous network (FN) which extends into the microspikes is evident beneath the surface membrane. Note that microtubules (MT) appear to end at the base of the growth cone and that neurofilaments (NF) are centrally located. Tubulovesicular profiles of the smooth endoplasmic reticulum (SER) are abundant and appear at the base of one microspike. V, D, and C are smooth, dense‐core, and coated vesicles, respectively; MC is a mitochondrion

From Yamada et al.


Figure 12.

Selective growth of sympathetic ganglion (sg) neurites toward nearby fragments of vas deferens (vd) rather than kidney (k); 5 days in vitro. Horizontal bar, 400 μm

From Chamley et al.


Figure 13.

Membrane potential (A) and the relation between membrane potential and [K+]0(B) of rat myotubes as a function of age. In A, • refers to multinucleated myotubes and Δ to mononucleated myoblasts. Vertical bars, ± SE. The number of cells at each point is indicated. In B, 4 myotubes of the indicated ages were examined. In each case the cultures were perfused with a continuous K+ gradient between 5.3 mM and 148 mM which was completed in 3 min. The relation between membrane potential and log [K+]0 predicted by the Nernst equation is indicated by the line labeled EK. Continuous lines through each set of points are predicted from the constant‐field equation (Eq. in text) assuming [Na]i = 13 mM and the PNa+‐to‐PK+ ratios are as indicated at each day

From Ritchie & Fambrough


Figure 14.

Action potentials evoked in spinal cord (A and C) and sensory ganglion (B and D) cells. Duration of current pulses indicated by bars above each trace; dorsal root ganglion (DRG) spikes are prolonged and are marked by a plateau on the falling phase. Many spinal cord neurons fire repetitively during a sustained depolarization (C), whereas most DRG cells rapidly adapt (D). The resting potential of cells in A‐C was 50–55 mV.



Figure 15.

Action potentials conducted along a process. All records obtained from a microelectrode located in the cell body shown in A. Spikes were evoked by extracellular stimulation (frames 2–4) as shown and by intracellular stimulation (frame 1). Stimulus artifacts appear as breaks in the records. Note the inflection on the rise of conducted spikes. In B‐4, superimposed traces show that hyperpolarization (inward current pulse not shown) accentuates the inflection and ultimately blocks the second component. Calibration pulse, 20 mV, 1 ms. Vertical bar in A, 50 μm

From Fischbach & Dichter


Figure 16.

Presumptive glial cells. A and B: two cells found to be electrically inexcitable. Current‐voltage relation of the cell shown in B is plotted in C. Sample traces are shown in inset. Note that the relation remains linear over a wide range of membrane potential.



Figure 17.

Ca2+ spikes in sensory neurons. A: relation between overshoot of the action potential evoked at the cell body and external Ca2+. Insets show representative traces recorded in the presence of TTX (10−7 g/ml) and 1.8 mM Ca2+ (trace 1) and 18 mM Ca2+ (trace 2). B: effect of Na+ on conducted spikes. Two cells (α and b) were bathed in Na+‐free media. In each case stimulation (arrows) of a process 300–400 μm away from the cell body failed to evoke a conducted spike (trace 1). An action potential was evoked via an intrasoma electrode in cell b. Immediately before trace 2, a 50 μm tip, NaCl‐filled electrode was lowered adjacent to the process. Identical extracellular stimuli now evoked conducted spikes in α and b. The axon spike in α invaded the cell body, whereas the one in b did not. Trace 3 in b was recorded after the NaCl electrode was removed. Note in b the decrease in duration of the soma spike in the Na+ atmosphere

From Dichter & Fischbach


Figure 18.

Action potential mechanisms in chick (A) and L‐6 (B) myotubes. A: in chick fibers, the sharp spike observed in normal saline (1) is abolished in Na+‐free (substituted with sucrose) media (2); a small approximately 50‐ms active response remains (wedge) which is abolished by Co2+ (10 mM) (3). The prolonged active response (arrows) which is not affected by removing Na+ or by addition of Co2+ is presumably due to an outward Cl current. The stimulus duration is indicated by a bar below each trace.

From Fuduka .] B: in normal saline the sharp spike of L‐6 myotubes (evoked by anode‐break excitation) is followed by a prolonged active response (1). The sharp spike is absent, but the late response is increased in size and duration in Na+‐free saline containing 10 mM Ca2+ (2). (Normal saline contains 1.8 mM Ca2+.) [From Kidokoro


Figure 19.

Changes in action potential generation in two cell lines as they “differentiate’ in vitro. A: L‐6 muscle cells. A small active response evoked in L‐6 mononucleated cell (myoblast) following a hyperpolarizing current pulse is contrasted with a large prolonged spike in a multinucleated (fused) myotube. In each pair, current is displayed in the upper trace and membrane potential in the lower trace.

From Kidokoro .] B: neuroblastoma cells (clone N‐18) in log phase (upper record), stationary phase (middle record), and selected in the presence of aminopterin (lower record). [From Peacock et al. .] The neuroblastoma spikes were recorded from the cells in Fig. which are indicated by the blurred image of a microelectrode


Figure 20.

Action potentials in neuroblastoma × L cell hybrids. Cells from two clones derived from the same hybridization are shown in A and B. A appears more neuronal and B more fibroblastic. The response of each cell type to depolarizing currents is shown in adjacent records. Cells in the neuronlike clone generate spikes, whereas those in the fibroblastlike clone do not. Both clones were grown in the presence of aminopterin for 10 days. Calibration pulse, 10 mV

From Peacock et al.


Figure 21.

A multipolar chick spinal cord neuron that has settled between muscle fibers on the collagen substratum. The neuron was dissociated from a 7‐day cord and plated about 48 h earlier. Note the microspike that extends from an expanded growth cone (gc) to contact a muscle fiber.



Figure 22.

Neuromuscular synaptic physiology. A: functional contact — a depolarizing synaptic potential (upper trace) follows an action potential in a nearby neuron evoked by intracellular stimulation (current pulse not shown). Bars, 3.5 and 50 mV for muscle and neuron records, respectively, and 10 ms for both. B: superimposed traces show the change in end‐plate potential (EPP) amplitude as the muscle membrane potential (Vm) was altered to the values shown at the left of each record by steady currents injected through a second intracellular microelectrode; calibration pulse, 5 mV, 5 ms. Each point in the graph represents the mean amplitude of 5–10 EPP's. C: ACh response (arrow) evoked by a 1‐ms pulse of ACh precisely mimics a spontaneous synaptic potential recorded on the same trace; calibration, 5 mV, 10 ms. D: reversible reduction in EPP size by d‐tubocurare (10−7 g/ml); record at extreme right obtained 30 min after removal of the drug; bars, 15 mV, 15 ms. E: miniature end‐plate potentials (MEPP's) recorded on contiguous segments of moving film; bars, 5 mV, 2 s. F: variation in amplitude (and one failure) of successive EPP's in a series evoked at a rate of 1/s, despite a constant stimulus (action potential) in the innervating neuron (upper traces); calibration pulse, 5 mV, 5 ms

A, B, D, and F from Fischbach ; C from Fischbach & Cohen


Figure 23.

A site of transmitter release identified by focal depolarization in the presence of TTX (see text). Interference contrast illumination of a chick muscle cell contacted by a neurite from a nearby spinal cord explant. Synaptic potentials were evoked by extracellular stimulation at α but not at b or elsewhere along the process which extends along the edge of the fiber

From Fischbach et al.


Figure 24.

Appearance of ACh receptors in muscle cells. A: increase in α‐bungarotoxin (α‐BGT) binding in control (▪) and in fusion‐arrested (•) chick muscle cultures (see text). ▴, fusion index (percent of nuclei incorporated into myotubes). Δ, fusion index in low‐Ca2+ media. Note that the increase in toxin binding lags behind the fusion index in control cultures and is even further delayed when fusion is blocked.

From Patterson & Prives .] B: elongated mononucleated muscles from rat muscle grown in standard media for 48 h. The oscilloscope record shows an ACh response (current on lower trace) recorded in cell 1. Horizontal bar, 100 μm. [From Fambrough & Rash .] C: autoradiographs of freshly dissociated (without the use of enzymes) mononucleated cells from chick muscle exposed in suspension to [125I]α‐BGT. Interference contrast and bright‐field views. Horizontal bar, 10 μm. [From Smilowitz et al.


Figure 25.

Synthesis and degradation of ACh receptors by cultured muscle fibers. A: following a saturating exposure to unlabeled α‐bungarotoxin (α‐BGT). The appearance of new receptors was determined by [125I]α‐BGT binding (•). ○, cultures treated as above but pretreated for 3 h with cycloheximide; Δ, cultures preincubated in dinitrophenol plus iodoacetate.

From Harzell & Fambrough .] B: release of radioactivity into the medium from chick or rat muscle fibers saturated with [125I]α‐BGT. Release, expressed as a fraction of that initially bound, is a first‐order process with a half‐time of about 20 h. [From Devreotes & Fambrough


Figure 26.

Histograms of ACh sensitivity of uninnervated (A) and innervated (B) muscle fibers in spinal cord muscle cell cultures.

From Fischbach and Cohen .] In (C) active and inactive uninnervated fibers are compared. No neurons were present. Active fibers stimulated intermittently for 2–3 days at 10/sec (\ \ \ \) were less sensitive than inactive fibers grown in the presence of TTX (/ / / /). [From Cohen & Fischbach . Copyright 1973 by the American Association for the Advancement of Science


Figure 27.

Relative peaks of ACh sensitivity on uninnervated (A) and innervated (B) muscle fiber; ordinate, mV/nC. In A, bars indicate position of hypolemmal muscle nuclei. [From Fischbach & Cohen .] B: a hot spot located exactly at a site of transmitter release. Inset, synaptic potentials evoked by focal depolarization at the termination of a nerve process. ACh was iontophoresed at the points indicated in the tracing (prepared from a photograph), and the sensitivities at each point are shown in register below. Bars, 5 mV, 5 ms.

From S. A. Cohen and G. D. Fischbach, unpublished observations


Figure 28.

Tubulovesicular network of cultured muscle fibers. In A note the openings to the exterior and also that some tubules form junctions with cisternae of the sarcoplasmic reticulum (arrow). Horizontal bar, 0.25 μm. B: unstained section of a muscle fiber exposed to horseradish peroxidase prior to fixation. The reaction product outlines areas continuous with the extracellular space. N, nucleus; G, Golgi; T, tubules; S, sarcoplasmic reticulum.

Micrographs provided by M. Henkart


Figure 29.

Spontaneously occurring synaptic potentials in dissociated chick spinal cord cells. A and B: superimposed traces show depolarizing potentials (EPSP's) in one cell (A), hyperpolarizing potentials (IPSP's) in another (B, upper record), and both in a third cell (B, lower record). Vertical bar, 10 mV for A, 12 mV for B. Horizontal bar, 5 ms for A, 10 ms for B, upper trace, 20 ms for B, lower trace. C: a continuous record shows regularly occurring large EPSP's that often trigger action potentials. Note the few small PSP's. D: Small PSP's recorded in the presence of TTX (10−7 g/ml). Vertical bar, 10 mV for C, 5 mV for D. Horizontal bar, 1 s for C, 5 s for D

From Fischbach & Dichter


Figure 30.

Stimulus‐evoked synaptic potentials in mouse spinal cord‐sensory ganglion cell cultures. A: monophasic EPSP (trace 3) evoked in one spinal cord neuron by stimulation (current in trace 1) of another spinal cord neuron (trace 2). B: action potential in a sensory neuron (evoked by a brief current pulse — not shown); trace 1 is followed by a small variable EPSP in a spinal cord cell (superimposed traces in 2). A larger less variable EPSP evoked by another sensory neuron in another spinal cord cell is shown in trace 3. C: complex responses in spinal cord neurons. An outward current plus (trace 3) evokes three spikes and is followed by a long‐lasting barrage of spikes and PSP's in the same (trace 2) and in an adjacent (trace 1) neuron.



Figure 31.

Electron micrographs of synaptic structures in chick spinal cord (A) and rat brain (B and C) cell cultures. In each, note the accumulations of small clear vesicles, the larger dense‐core vesicles, and increased density or thickening of the synaptic membranes. A, Several synaptic profiles appear along a single process. × 18,000. C: a synapse contacts a small process which may be a “spine.’ B and C, × 32,000.

A, from Fischbach & Dichter ; B and C provided by E. Neale


Figure 32.

Stimulation of a single presynaptic ending on a chick spinal cord neuron. Successive brief negative current pulses, delivered through a 2‐μm‐tipped NaCl‐filled pipette placed exactly over the bouton indicated by the arrow, evoked the synaptic potentials shown at right (traced from photographs). The response disappeared when the stimulating electrode was moved about 5 μm in any direction. Note that the incoming nerve processes pass out of the plane of focus. The postsynaptic nerve cell body overlies a muscle fiber. Calibration bars, 5 mV, 5 ms.



Figure 33.

Drug responses. A: ACh potentials in a sympathetic ganglion cell. The 20‐nA iontophoretic pulse is shown in the second trace; suprathreshold and subthreshold responses are shown in a and b. B: glutamate potential in a spinal cord neuron; iontophoretic pulse, 30 nA. This response is slower than EPSP's recorded in the same cell (trace 3). C: GABA potentials in a sympathetic ganglion cell. D: GABA potentials in a spinal cord neuron. A 25‐nA pulse (duration indicated by bar) was repeated as the membrane potential was shifted. The response reverses in polarity between −70 and −50 mV

A and C from Obata ; B and D from B. R. Ransom and P. G. Nelson, unpublished observations


Figure 34.

Nonuniform distribution of chemoreceptors on a mouse spinal cord neuron (A) and a neuroblastoma cell (B) about 4 wk after plating. A: penwriter records of glutamate responses at the sites indicated. Note the relatively large and rapid response at lower left. The same neuron impregnated with silver is shown in the inset.

From B. R. Ransom and P. G. Nelson, unpublished observations.) B: ACh produces a qualitatively different response at different sites — a depolarization over the soma, a hyperpolarization at relatively distant sites along processes, and a biphasic response at intermediate points. (From Peacock et al. 1973


Figure 35.

A: increase in synthesis of catecholamines (norepinephrine and dopamine) from [3H]tyrosine by rat sympathetic ganglion neurons with time in culture. Note the initial log of 7–8 days followed by a 50‐fold increase. This pattern is markedly different from the increase in total accumulation of radioactivity measured in the same cultures (B)

From Mains & Patterson


Figure 36.

Cholinergic synapses between sympathetic ganglion cells. A: simultaneous recordings from two sympathetic neurons in which each spike in one (the “driver,’ middle trace) is followed by a depolarizing synaptic potential in the other (the “follower,’ upper trace). Some PSP's in the follower are large enough to trigger spikes. The first spike in the driver cell is evoked by a brief pulse of outward current (lower trace). The other spikes arise spontaneously. B: the same cells in the presence of 50 μM hexamethonium, a nicotinic cholinergic antagonist. All spontaneous PSP's are abolished, and the evoked spike in the driver neuron is no longer followed by a PSP in the follower. C: the same cells after washing out the hexamethonium. Calibration bars: 40 mV, 40 ms.

From O. MacLeish, P. H. O'Lague, E. J. Furshpan, and D. D. Potter, unpublished observations
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Gerald D. Fischbach, Phillip G. Nelson. Cell Culture in Neurobiology. Compr Physiol 2011, Supplement 1: Handbook of Physiology, The Nervous System, Cellular Biology of Neurons: 719-774. First published in print 1977. doi: 10.1002/cphy.cp010120