Werner T. Schlapfer

Tissue and Organ Culture

The term “tissue culture” has been used to denote the in vitro cultivation and maintenance of living biological materials from multicellular organisms. The techniques that have been developed for this purpose fall into four broad classes: (1) methods to dissociate cells, possibly isolating individual cell types, and to maintain and propagate the dispersed cells as monolayers or suspensions; (2) methods to reassociate previously dissociated cells into aggregates and to maintain these reaggregates in an in vitro environment; (3) methods to cultivate tissue fragments over prolonged periods of time under conditions that encourage cellular interactions as well as differentiation and maturation of the cellular elements in a way resembling the in vivo situation; and (4) methods for maintenance of organ fragments under conditions that discourage the migration of cells from the explant, and that maintain the differentiated cells as a group in a viable, organized, and functioning state. Short-term incubation methods (24 hr or less) of tissue slices or fragments, as are commonly used in biochemical experiments, short-term isolations, or in vitro procedures used in neurophysiology (e.g., isolated ganglia or nerve—muscle preparations), do not qualify as culture systems in the context of this discussion and are not included in this review.

Depression and frequency facilitation at a synapse inAplysia californica: Evidence for regulation by availability of transmitter

Summary Synaptic depression and frequency facilitation were observed in the monosynaptic and uritary excitatory postsynaptic potential (EPSP) obtained in the ‘parabolic burster cell’ (R15) by stimulating the right visceropleural connective in Aplysia californica . During trains of stimulation at 0.5–2 c/sec the size of the EPSP was initially depressed during the first few stimuli and subsequently slowly increased to reach a potentiated steady state after a few hundred stimuli. The relative amount of depression between the first two EPSPs depended on (a) the inter-stimulus interval, and (b) the size of the first EPSP. The larger the first EPSP the larger the percentage depression whether variations in the size of the first EPSP were due to natural interanimal variability or experimental manipulation of the concentrations of Ca 2+ , Mg 2+ and Co 2+ . The amount of frequency facilitation (expressed as the ratio of the 100th EPSP to the first EPSP of a train) increased with increasing frequency of stimulation and was inversely related to the log of the size of the first EPSP both for natural variations in EPSP size and manipulations of the EPSP size by changing the divalent cation concentrations. Neither the level of hyperpolarization of R 15 nor partial curarization affected the relative depression and potentiation. We suggest that the initial depression is largely due to depletion of a rather small pool of transmitter available for release, while the frequency facilitation is the result of a Ca 2+ and frequency dependent increase in the net rate of supply of transmitter into the available pool.

Increased membrane fluidity implicated in acceleration of decay of post-tetanic potentiation by alcohols.

AFTER repetitive stimulation of a synapse, the efficacy of synaptic transmission may be potentiated for a period. In systems that lend themselves to detailed analyses, this posttetanic potentiation (PTP) has been shown to be due to an increased amount of transmitter released by the presynaptic neurone rather than to postsynaptic mechanisms1. We have investigated the PTP of a monosynaptic, all-or-none, excitatory postsynaptic potential (e.p.s.p.) recorded in cell R15 of the abdominal ganglion of Aplysia californica with appropriate stimulation of the right visceropleural connective, and have presented evidence that the PTP of this e.p.s.p. is based on increased transmitter release2. We have shown3 that while PTP is slightly prolonged by lowering the temperature of the preparation from 20 to 12 °C, the duration of the PTP is increased tenfold by further lowering of the temperature to 10 °C. This demonstration of a transition temperature suggested that the fluidity of critical lipids in the presynaptic membrane regulates the decay of PTP, such that PTP decays more slowly in conditions of decreased membrane fluidity. If this inference is correct, agents, such as alcohols4, that increase membrane fluidity and thus act as ‘antifreezes’, should either prevent the occurrence of the temperature transition or lower the transition temperature. These agents should also, by themselves, accelerate the rate of PTP decay and their potency should be correlated with their lipophilicity. We present here experimental evidence in support of these predictions.

Dopamine, serotonin and related compounds: Presynaptic effects on synaptic depression, frequency facilitation, and post-tetanic potentiation at a synapse inAplysia californica

Dopamine, serotonin and related compounds (referred to collectively as biogenic amines) were found to modify transmission at the presumably cholinergic synapse made by an axon in the right visceropleural connective onto cell R15 of the abdominal ganglion of Aplysia californica. (1) With chronic application, dopamine hyperpolarizes R15, and serotonin depolarizes R15. Both actions upon the membrane potential desensitize in 10 min. All the actions described below were studied with chronic perfusion of the biogenic amines after desensitization of this postsynaptic action. (2) The biogenic amines drastically reduce the size of the EPSP evoked at the synapse under investigation; but they do not alter the ACh potential evoked in the soma of R15. (3) The biogenic amines reduce the amplitude of synaptic depression. The relationship between the effects of the amines on the size of an isolated EPSP and on synaptic depression differed from this relationship as affected by post-tetanic potentiation (PTP) or by changes in the Ca2+-Mg2+ balance. (4) The biogenic amines increase frequency facilitation, when the latter is defined as the ratio of the facilitated to the isolated EPSP. However, the absolute magnitude of the facilitated EPSP is always reduced at long times after introduction of the agent; shortly after introduction of the biogenic amines the absolute magnitude of the facilitated EPSP is unaffected in most preparations.

Synaptic depression at a synapse inAplysia californica: Analysis in terms of a material flow model of neurotransmitter

When a pair of stimuli separated by an appropriate interval is given to the right visceropleural connective of Aplysia californica the amplitude of the second EPSP elicited in cell R15 is usually smaller than the amplitude of the first EPSP. In the present paper we show that this phenomenon, synaptic depression, can be analyzed in terms of the material flow model of neurotransmitter economics developed in our preceding publications. We specifically show how changes in the 4 model parameters; A, the available pool of transmitter; F, the fraction of the available pool released by a presynaptic action potential; M, the rate of transmitter mobilization into the available pool; and D, the rate constant of demobilization of transmitter from the available pool, all effect synaptic depression. In addition, we show how transient changes in F and M, that are observed immediately and for seconds after a stimulus, influence the time course of synaptic depression. Using this analysis we then tested our previous inferences about changes in the model parameters produced either by pharmacological manipulations or repetitive stimulation, by comparing the observed effects of these manipulations on synaptic depression with the theoretical predictions. The theoretical and experimental findings agreed, thereby strengthening both our previous conclusions of the mode of action of these manipulations and the model itself.

Morphine and related compounds: evidence that they decrease available neurotransmitter inAplysia californica

Abstract Studies were made of the action of morphine on an identified monosynaptic, unitary excitatory postsynaptic potential (EPSP) in the abdominal ganglion of Aplysia californica . The synapse is presumed to be cholinergic. Upon repetitive stimulation of the right pleurovisceral connective the size of EPSPs recorded intracellularly in cell R15 undergo a progressive depression followed by facilitation to a frequency dependent plateau and a period of post-tetanic potentiation. Perfusion with10 −4 M morphine for 3 h reduced the size of every EPSP of the train to about 50% of its control value. The relative depression, facilitation and post-tetanic potentiation (measured as a fraction of the first EPSP of a train) were not affected by morphine. The minimal effective concentration of morphine was about10 −5 M . The morphine effect on EPSP amplitude does not appear to be due to blockade of the postsynaptic receptor since the potential obtained by iontophoretic application of acetylcholine onto the postsynaptic cell body was not decreased by morphine perfusion. The effect of morphine on the EPSPs appears to be due to reduction of neurotransmitter release from the presynaptic terminal. The reduction of transmitter release seems to result from a general reduction in the supply of transmitter to the pool available for release. This contrasts with previous studies on a group of drugs typified by trimethidinium, which appear to selectively block frequency dependent supply of neurotransmitter without affecting the frequency independent supply process. Naloxone, which is an antagonist of morphine in many other systems did not antagonize the morphine effect in this system. Instead, it too decreased the amplitude of all EPSPs of a train. Levorphanol and dextrorphan also produced these effects, indicating that the ‘receptor’ for this action of opiates is not stereospecific. The relatively high concentrations of morphine required to produce the effect, and the lack of stereospecificity distinguish these findings from some actions of morphine in vertebrate preparations. However, some morphine effects in vertebrate preparations are similar to those observed here. Whereas the relationship of these findings to the analgesic action of morphine is unclear, the characteristics of its action at this synapse assist in the pharmacological analysis of regulation of transmitter release in Aplysia .

Resting and stimulated values of model parameters governing transmitter release at a synapse inAplysia californica

Transmitter release (R) at a synpase in Aplysia californica can be analyzed in terms of a model with the following parameters: A, the available pool of transmitter; F, the fraction of available pool released by a presynaptic action potential; M, the rate of transmitter mobilization into the available pool; D, the rate constant of demobilization of transmitter from the available pool. In the present paper we show that: (1) beginning with an analysis of the recovery from depression of the second of a pair of disolated EPSPs separated by a series of intervals of about 10-60 sec, and assuming that the recovery is due to refilling of a depleted A, it is possible to estimate resting equilibrium values of these parameters; (2) changes in these parameters when a new equilbrium state is reached after prolonged stimulation (e.g., 300 stimuli at 1/sec) can then be quantitatively determined; (3) the increased rate of transmitter release observed during and after repetitive stimulation is the consequence of increases in F and M with changes in A passively following; and (4) there are significant correlations among certain resting parameters and between the values of certain resting parameters and these parameters upon stimulation. Preparations with a large resting F tend to have a relatively small resting A. Preparations with a large resting F or M tend to increase these less with stimulation than preparations with smaller resting values of these parameters. Preparations with large stimulus-dependent increases in F tend to have large stimulus-dependent increases in M.

Cholinergic agents affect two receptors that modulate transmitter release at a central synapse in Aplysia californica

A unitary, monosynaptic and presumably cholinergic EPSP recorded in cell R15 of the abdominal ganglion of Aplysia californica undergoes depression followed by facilitation when the presynaptic axon is repetitively stimulated at a rate of 1-3 pulses/sec. During trains of stimulation which produced this sequence of phenomena, the effects of a large number of agents known to affect cholinergic transmission in other systems were studied. The agents could be divided into 4 classes: (1) agents having no effect upon transmission at this cholinergic junction; (2) agents of a class typified by curare, which depressed all EPSPs of a train to the same extent, and which are believed to be acting in this system solely as competitive postsynaptic blockers; (3) agents typified by acetylcholine and carbachol (ACh class), which selectively depressed earlier EPSPs of a train more than later EPSPs and which appear to act by reducing the fractional release of transmitter; (4) agents typified by trimethidinium (trimethidinium class), which selectively depress later EPSPs of a train more than earlier EPSPs and which appear to act by reducing the rate of transmitter supply into the readily releasable pool. Neither the ACh class nor the trimethidinium class produced these selective effects on different pulses in the train by changes in the postsynaptic membrane potential or membrane resistance. Nor did they act by stimulating or inhibiting other recorded inputs onto R15. Iontophoretic application of acetylcholine onto R15 indicated that the effect of trimethidinium could not be explained by an alteration in desensitization of a postsynaptic acetylcholine receptor. The structural specificity of the presynaptic receptors mediating the action of the ACh and trimethidinium classes was demonstrated by the use of a larger number of structurally related compounds.

Heterosynaptic inhibition modifies the presynaptic plasticities of the transmission process at a synapse inAplysia californica

The monosynaptic and unitary excitatory postsynpatic potential (EPSP) observed in cell R15 of the abdominal ganglion of Aplysia californica upon minimal stimulation of the right visceropleural connective exhibits several presynaptic plasticities (synaptic depression, frequency facilitation, post-tetanic potentiation). We studiied effects of branchial nerve stimulation (heterosynaptic stimulation) on these plasticities of the homosynaptic (right connective) path. A burst of heterosynaptic stimulation (20 pulses at 4/sec) decreased the amplitude of an isolated homosynaptic EPSP. The rate of recovery from heterosynaptic inhibition (HSI) was a function of the rate of stimulation of the homosynaptic path so that at a stimulus frequency of 1 pulse/sec to the right connective (RC) the HSI lasted less than 20 sec while at a RC stimulus frequency of 1/10 sec the HSI persisted for more than 60 sec. While the frequency facilitated EPSP (during homosynaptic stimulation at 1/sec) was only transiently affected by heterosynaptic stimulation the effect on the subsequent post-tetanic potentiation was much more pronounced and longer lasting (more than 30 min). This suggests a specific effect of HSI on the rate constant of decay of elevated fractional release, as observed upon bath applications of biogenic amines. Heterosynaptic stimulation also reduces synaptic depression but the reduction in the depression is more than would be caused by comparable reduction of the first EPSP of a pair of high Mg2+, low Ca2+ or the addition of carbachol to the perfusion medium. The duration of the effect on synaptic depression was the same as the effect on EPSP1.

Marked prolongation of post-tetanic potentiation at a transition temperature and its adaptation

A UNITARY, monosynaptic excitatory postsynaptic potential (e.p.s.p.) can be recorded from cell R15 of the abdominal ganglion of Aplysia californica on appropriate stimulation of the right visceropleural connective. The amplitude of this e.p.s.p. undergoes a sequence of changes with trains of one or two stimuli per second. These changes include synaptic depression, frequency facilitation and post-tetanic potentiation. As described in detail elsewhere1,2, all these phenomena are apparently due to alterations in the amount of neurotransmitter released rather than to postsynaptic mechanisms. The frequency facilitation, a relatively transient phenomenon, is believed to be limited by the rate of neurotransmitter mobilisation into the pool available for release2. In contrast, the post-tetanic potentiation, a much more sustained phenomenon, is believed to be due to a change in the fraction of the available pool of transmitter released per stimulus2.