Contribution of an electrogenic pump to the modulation of synaptic efficacy

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CONTRIBUTION OF AN ELECTROGENIC PUMP TO THE MODULATION OF SYNAPTIC EFFICACY BEHRUS JAHAN-PARWAR and STEVEN M. FREDMAN Worcester Foundation for Experimental Biology, Shrewsbury. MA 01545, U.S.A. (Rrcriwd

8 October 1978)

A new postsynaptic mechanism for modulating synaptic efficacy is described. 2. The A cluster neurons in the cerebral ganglion of Aplysia have a resting membrane potential (RMP) which increases steadily from - 30 mV when initially impaled to a final level of - 50 to - 55 mV. This is close to the Cl- equilibrium potential (Ec,-). 3. This increase in RMP is due lo an electrogenic pump, and is blocked by ouabain, cooling and K’ free seawater. 4. When initially impaled, both spontaneous and sensory evoked synaptic input to the A neurons consists primarily of IPSPs. With increasing RMP, these first decrease in amplitude and may invert to become depolarizing. 5. Sensory evoked IPSPs have two components. The first is fast and Cl- mediated. The second is slow and K+ mediated. 6. During sensory stimulation. the slow component can pull the membrane potential below Ec,causing the fast component to invert (Fig. 3). These inverted IPSPs can summate with other excitatory inputs to produce A neuron firing. 7. Due to the interaction of the electrogenic pump and the two component inhibition, the efficacy of sensory and other synaptic input can be modulated by relatively small changes in membrane potential. Abstract-l.

INTRODUCTION Several different mechanisms are known which alter or modulate the efficacy of synaptic transmission.

These can generally be divided into two classes based on whether they exert their influence presynapticly or postsynapticly. Some examples of presynaptic effects are post-tetanic potentiation (Eccles, 1964; Hubbard, 1963; Larrabee, 1947; Lloyd 1949), presynaptic inhibition (Dude1 & Kuffler, 1961; Eccles, 1964) and heterosynaptic facilitation (von Baumgarten & Jahan-Parwar, 1967; Castellucci & Kandel, 1976; Jahan-Parwar & von Baumgarten, 1967; Kande1 & Taut, 1965; Kandel, et al., 1976). The means by which these work, all appear to involve alterations in the amount of transmitter released by the presynaptic nerve terminals. Postsynaptic mechanisms include changes in membrane resistance which can either shunt or facilitate the synaptic potential (Carew & Kandel, 1977). Other postsynaptic mechanisms involve changes in receptor sensitivity, particularly at multiple component synapses such that one component is facilitated and the other depressed (Gardner & Kandel, 1977; Wachtel & Kandel, 1971). We now report a different postsynaptic mechanism for modulating synaptic transmission. This utilizes alterations in the membrane potential of the postsynaptic neuron, resulting from the interaction between an electrogenic sodium pump and a two-component synaptic inhibition such that previously inhibitory postsynaptic potentials (IPSPs) are inverted and summated with excitatory postsynaptic potentials (EPSPs) to give rise to spikes. While the contribution of an electrogenic pump to the resting potential of some neurons (Carpenter & Alving, 1968; Thomas, 503

1969) and multiple component synapses (Gardner & Kandel, 1972; Kehoe, 1972; Wachtel & Kandel, 1971) have been described, the combined influence of these elements has not been considered as a mechanism for modulating synaptic transmission. As we shall demonstrate below, alterations in the sign of the IPSPs can occur both spontaneously and due to natural sensory stimulation alone.

MATERIALS

AND

METHODS

Adult Aplysiu calijornica were dissected and the CNS was removed with the nerves to the tentacles left intact. Each anterior tentacle was placed in its own compartment which was then sealed. The margins of the tentacular groove were pinned so as to expose the sensory mucosa. The central ganglia were pinned to the floor of the central compartment of the experimental chamber and the connective tissue sheath over the cerebral ganglion was surgically removed. Typically 3 cerebral A neurons were impaled with micropipettes filled with 2.5 M potassium citrate. Since they receive input directly from tentacular receptors. a fourth electrode was placed in a cerebral B neuron to monitor the effectiveness of sensory stimulations (Fredman & Jahan-Parwar, 1975; Jahan-Parwar & Fredman, 1976). Reproducible tactile stimuli were presented to the tentacles by a glass rod attached to a square-wave driven solenoid. For food chemosensory stimulation, I ml of seaweed extract (SWE) (Jahan-Parwar, 1972) was delivered 1 cm from the tentacle.

RESULTS

The caudal half of the cerebral ganglion of Aplysia contains two conspicuous pairs of symmetrical neuron clusters, which have been described in detail

BIHKC’SJAHAN-PARWAK md Sit.w% M. Ftwr)MA~

K+

C7

FREE SW

h

Fig 1. Evidence for an electrogenic pump in cerebral A neurons. (a) Penetrating an A neuron whtle passing constant current hyperpolarizing pulses (approximately I nA, 10 msec) shows that the membrane resistance quickly increased. then stabilized as the membrane potential increased. There were no further increases in membrane resistance even after several hours of recording. (Note: the bridge was slightly unbalanced. Actual current was monitored on a separate channel.) Gain: 20mV. (b) Impaling an A neuron in normal SW. There was a steady increase in membrane potential. Initial gain: 20 mV; after the downward deflection, 10 mV. (c) Penetrating an A neuron in K + free SW. The steep, steady increase in membrane potential was absent. Gain: 20mV. (d) When an A neuron was impaled at 7°C. there was no steady initial increase in membrane potential. The downward drift seen later in the record was due to warming. Initial gain: 20 mV; after the downward deflection, 10 mV. (e) Cooling the bathing SW from 17.5”C to 7S”C caused A neurons to depolarize approx ISmV. The small superimposed depolarizing potentials may have been axon-spikes in the distal part of the neuron. The subsequent slight repolarization was due to warming. Gain: 10 mV. Time: a. 2 mitt; b-d. 24 sec.

previously (Fredman & Jahan-Parwar. 1975; JahanParwar & Fredman, 1976). The more lateral and caudal of these clusters, the A neurons have several distinguishing characteristics. Upon penetration, they fire briefly and then typically are silent. Spontaneous synaptic input following penetration consists of IPSPs. Tactile stimulation of the tentacles at this time evokes an inhibitory response in the A neurons (Jahan-Parwar & Fredman, 1976). The neurons are also characterized by a continuous, often rapid, increase in resting membrane potential (RMP). This is of sufficient magnitude that after a period of 5-60 min. the spontaneous and evoked IPSPs first decrease in amplitude, reach a null point and often invert. As a result, synaptic inputs which initially were hyperpolarizing become depolarizing. When initially impaled, A neurons had a RMP of -30 to -45 mV (Fig. 1). Except during the brief period when they fired, the membrane resistance, as monitored by constant current hyperpolarizing pulses

remained constant for several hours despite the steady increase in membrane potential. This tends to eliminate recovery from damage during penetration as a significant factor in the changes in RMP. The final resting potential of the A neurons typically were - 50 to -60 mV, although some o~asionally reached -65 mV. We believe that this increase in RMP is due to an electrogenic sodium pump (Carpenter & Alving, 1968; Thomas, 1969) which is strongly activated by the large Na* influx caused by penetration with a microelectrode. This is based on several observations. As seen in Fig. I, the RMP of A neurons impaled in K’ free seawater (SW) increased only a few mV over the initial resting level. The IPSPs of these neurons did not invert spontaneously. When the bathing medium was replaced with normal SW, the membrane potential increased and the IPSPs inverted. Injecting A neurons with TEA (tetraethylammonium) sufficient to cause significant (greater than 50%) broadening of the falling phase of the spike.

505

Electrogenic pump synaptic efficacy

a

b

C

d

A,

Fig. 2. Spontaneous inversion of the A neuron tactile response. (a) Electromechanical stimulation of one of the anterior tentacles (arrows) soon after penetration produced inhibition in two simultaneously recorded A neurons. (b) After approx 20min the resting potential of both neurons had increased. The same stimulus produced very little change in membrane potential in either neuron. This was due to their resting potential being near the equilibrium potential of the fast synaptic component. These neurons lacked a prominent slow component. (c) After the membrane potential of both neurons had further increased. A, was depolarized to a level near its initial resting potential. The stimulus again caused IPSPs. The response in A2 whose membrane potential was not manipulated was a depolarization. (d) With A, held hyperpolarized the stimulation caused a depolarization. The depolarizing response in A, as in (c) was not due to manipulation of its membrane potentials. These results show that the inversion of the response in the A neurons is due to changes in their membrane potential rather than changes in the stimulus. Note: The relative position of the traces in a-d do not represent the actual changes in resting potential. The traces were repositioned between stimulations. Gain: IO mV; time: 400 msec.

had no effect on either the increase in RMP or the inversion of the IPSPs. This tends to eliminate a steady K+ leakage as a mechanism for the RMP increase. Cooling from 175°C to 75°C and applying 5 x 10e4 M ouabain both caused A neurons to depolarize (Fig. 1). A neurons impaled at 7°C or in the presence of ouabain also failed to show increases in membrane potential. These results are consistant with an electrogenic Na+-K+ pump. The increase in RMP produced by this pump is capable of exerting a significant influence on the effects of sensory inputs to the A neurons. A neuron responses to sensory stimulation were both inhibitory and excitatory depending on their membrane potential. Fig 2 shows the responses of two A neurons to tactile stimulation of one of the tentacles. The initial response in both was a hyperpolarization. After their membrane potentials had increased, the same stimulus produced depolarization in one while in the other, which was artificially depolarized, the stimulus produced a hyperpolarization. Thus the sign of the response evoked by tactile stimulation was dependent upon the membrane potential and was not due to a change in the stimulus itself. In another A neuron after the RMP had increased due to the action of the electrogenic pump, an inversion of IPSPs could be seen during the course of a single sensory stimulation. This was achieved by running SW rapidly over the surface of the tentacle, providing a’ strong mechanosensory stimulus. As seen in Fig. 3, the initial spontaneous synaptic input was IPSPs. The stimulation produced a hyperpolarizing response, and an inversion of the IPSPs. Superimposed on the hyperpolarization were axon spikes (A spikes). It is not

clear at this time whether the A-spikes were produced by the inverted IPSPs or facilitated EPSPs, or the combined effect of the two. The latter is the most likely. A similar alteration in the sign of the synaptic inputs was seen during food chemosensory stimulation. SWE applied to one of the tentacles also produced an initial hyperpolarization which was sufficient to cause the inversion of the IPSPs. Since SWE also activates polysynaptic pathways which excite the A neurons (Fredman & Jahan-Parwar, 197713) considerable firing resulted. This spiking activity sometimes caused a shift in the RMP back to a more depolarized level. In A neurons which had inverted IPSPs, this was sufficient for spontaneous inputs to become true IPSPs again. The inhibition in A neurons produced by sensory stimulation has two components. These could be distinguished in some, but not all preparations. The first component consists of fast IPSPs. These reversed at RMP of -50 to -55 mV, and were chloride mediated. Bathing the ganglia with Cll free SW resulted in a rapid inversion of all the spontaneous IPSPs. Responses evoked by tactile stimulation were also depolarizing and gave rise to spikes. This was due to a shifting of the Cl- equilibrium potential (Ec,-) in a positive direction such that increases in the A neurons permeability to Cll resulted in an outward, depolarizing current (Blankenship, et al.. 1971: Kehoe, 1972). Similar results have been obtained with iontophoretic applications of neurotransmitters in Cl- free solutions (Blankenship, et al., 1971; Carpenter, et al., 1977). It is this fast component that was reversed by increasing the RMP. The second component is slower and longer lasting. It appears

506

B~HR~ISJAHAN-PAHWARand

STLVEN M. FRHIMAN

a

b

SW

!iwE

C

Fig. 3. Inversion of IPSPs during sensory stimulation. (a) Washing SW rapidly over a tentacle caused spontaneous A neuron IPSPs to invert due to increase in membrane potential resulting from the slow inhibitory component. The stimulation was sufficient to trigger two A-spikes. (b) Control stimulation of an anterior tentacle with 1 ml of SW (arrow). There was no response. (c) Stimulating the same tentacle with I ml of SWE (arrow) caused a long duration discharge of A-spikes. In this record

individual IPSPs were difficult to see. Gain: IO mV: time: IOsec.

to be mediated by an increased K’ conductance. No increase in membrane resistance was seen during this component. This makes it unlikely that it was produced by a decreased Na+ conductance. In addition this component reversed at RMP’s in excess of - 80 mV and was blocked by injecting TEA. The separation of these components is shown in Fig. 4. In this cell the spontaneous fast IPSPs had already inverted. When a tentacle was touched it was possible to see these inverted IPSPs superimposed on a hyperpolarization. This clearly demonstrates that two separate ionic mechanisms are operating simultaneously. At the present time we do not know if the same presynaptic neuron(s) mediate both the fast and slow components. However, it appears that the presynaptic neurons responsible for the spontaneous fast IPSPs are the same as those providing the fast inhibitory component of the sensory response. Further analysis of these synaptic inputs must await the identification of the presynaptic neuron or neurons. DISCL’SSION

We are proposing a mechanism for modulating synaptic activity that is fundamentally different from others previously described. It consists of an electro-

genie pump which can bring the membrane potential of the postsynaptic neuron near the reversal potential of one component of a two-component synaptic inhibition. The inversion of Cl- mediated IPSPs by artificially hyperpolarizing a neuron is well known (Eccles. 1964). This can also happen spontaneously due to leakage of Cl- from the tip of the electrode when KC1 is used as the electrolyte (Eccles, 1964; Kandel & Spencer, 1961). This is not the case here since spontaneous inversion of IPSPs occurred using potassium citrate filled micropipettes and as the result of sensory stimulation, not by artificially manipulating either the membrane potential or ionic concentrations. Citrate ions are not known to substitute for Cl- in chloride mediated synaptic potential (Eccles. 1964; Ito. er al.. 1962). Due to their relatively large hydrated radius (Ito. et al.. 1962). citrate ions would be expected to diffuse more slowly than K’. Any leakage from the tip of the electrode would be expected to result in a steady depolarization. Furthermore, we have not seen equivalent hyperpolarizations of other neurons (such as the cerebral ganglion B neurons) when the! were impaled with the same electrodes. This makes it unlikely that either the steady hyperpolarization of the RMP of the A neurons or the inversion of spontaneous and evoked IPSPs is due to loading the ceils

RA

Fig. 4. Two components in the A neuron response lo tactile stimulation. A ractlle stimulus to the left anterior tentacle (presented by hand) caused a small initial depolarization due to inverted fast IPSPs. This was followed by a longer slow hyperpolarization on which inverted IPSPs were superimposed. This clearly illustrates that the tactile response has two distinct com$onents. Gain: 10mV: time: 400 msec.

Electrogenic pump synaptic efficacy with anions from the micropipette. As indicated by our results, the overall hyperpolarization of the A neurons to a RMP near or even below EC,- is consistent with an electrogenic potassium dependent sodium pump. The steady hyperpolarization observed following penetration was blocked by K* free SW, cooling and ouabain, all of which are known to block electrogenic pumps (Carpenter & Alving 1968; Thomas, 1969). Multiple component synapses have also been described previously (Blankenship, et al., 1971; Fiore & Meunier, 1975; Gardner & Kandel, 1972; Gardner & Kandel. 1977; Kehoe, 1972; Shimahara & Taut, 1975; Wachtet & Kandel, 1971). These have included various combinations of excitation and inhibition mediated by single presynaptic neurons. Unfortunately because the inhibitory response seen in the A neurons is due to stimulating polysynaptic sensory pathways, it has not been possible to analyze the synaptic input to these neurons in detail, since repeated stimulations depress the entire pathway. However, one hypothetical mechanism for the inversion of the IPSPs can be largely eliminated. This would consist of an IPSP with a hidden fast excitatory component. Blockage of the inhibitory component and facilitation of the excitatory component might produce an “inversion” of IPSPs similar to that seen in our results. Applying hyperpolarizing and depolarizing steps to the A neurons failed to disclose any excitatory phases hidden in the fast IPSP. All of the results are consistent with the fast IPSPs being purely Cl- mediated. What is more likely is that the presynaptic neurons produce a two-component inhibition similar to that described by Kehoe (1972). One of the questions raised by the spontaneous inversion of IPSPs in A neurons is what the normal resting state (and sign of the IPSPs) really is. Obviously the neurons are disturbed when penetrated by an electrode. Presumably the initial strong hyperpolarization is due to activating the Na+-pump by the large Na+ influx caused by the micropipette puncturing the neuronal membrane. It may not be possible to determine whether the PSPs in the A neurons are normally excitatory or inhibitory by any direct means, when the means of observing the system alters it. One possibility is that the electrogenic pump keeps the RMP of the A neurons very close to the equilibrium potential of the Cl- IPSPs (Ec,-). This is suggested by experiments where the A neurons were recorded for several hours. Often the spontaneous IPSPs were all but absent. Small manipulations of the membrane potential disclosed that the presynaptic neurons were still active, but that the IPSPs were very close to their equilibrium potential. We believe that this is the normal resting state of the A neurons. By keeping the RMP near Ec,- small changes in membrane potential could effectively modulate the efficacy of synaptic inputs. IPSP’s are generally considered to produce inhibition through two mechanisms. One is by moving the RMP away from threshold. The second is by shunting excitatory input, reducing its effectiveness. In the absence of obvious resistance changes during the IPSPs, the former mechanism becomes the most significant in this system. The sign of the synaptic potential thus becomes of critical significance. Small depolarizations such as

507

those seen following a train of spikes would shift the RMP away from Ec,- sufficiently that subsequent inputs would be inhibitory. Further hyperpolarizations would result in the same inputs being in effect, excitatory. The latter permit additional synergistic effects. True EPSPs would have greater amplitudes and thus be more effective. Inverted IPSPs could summate with these EPSPs (Dude1 & Kuffler, 1961) to trigger A neuron firing. As has been shown by Hagiwara et al. (1960) under some conditions inverted IPSPs can be of sufficient magnitude to trigger spikes. The two component inhibition seen in the A neuron responses is particularly important in this regard, since the slow component is capable of pulling the fast component past its equilibrium potential and converting inhibition to excitation. Similar results have been obtained with neurons artificially hyperpolarized near Ec,- or by altering the internal and external Cl- concentrations. The difference here is that the A neurons do this as the result of an electrogenic pump and without their membrane potential being artificially manipulated. Since the slow inhibitory component results from an increased K+ conductance, the change in synaptic efficacy is produced by quite a different means than that of other postsynaptic mechanisms which utilize a conductance decrease EPSP (Carew & Kandel, 1977). In addition, since the summated slow IPSP can be very long lasting, the possibility for long duration modulation exists. This type of modulation could be of behavioral significance. We have found evidence that food chemosensory stimuli activate long latency excitatory inputs to the A neurons (Fredman & Jahan-Parwar, 1977b). The efficacy of these pathways could thus be influenced by changes in the IPSPs evoked by shorter latency pathways such as those from tentacular mechanoreceptors. The A neurons may collectively serve as a command system for food mediated behaviors. This hypothesis is based on the observation that the A neurons not only appear to be motor neurons for the foot, parapodia (Jahan-Parwar & Fredman. 1978) and other parts of the body (unpublished data). but also make extensive excitatory synaptic connections both within the cerebral ganglion (Fredman & Jahan-Parwar, 1975) and other ganglia (unpublished). Thus whether the A neurons fire as a result of a given stimulus could have widespread behavioral effects. The experimental results show that the A neurons of the cerebral ganglion of Aplysia give both excitatory and inhibitory responses to the same sensory stimuli. Which response is obtained is a function of the membrane potential of the A neurons. An electrogenie pump brings the resting potential near the equilibrium potential of chloride IPSPs. Mechanosensory and food chemosensory stimuli activate a two component synaptic response. The first consists of fast Cl- mediated IPSPs. The second component is a slow hyperpolarization which can drive the fast IPSPs past their equilibrium potential. The result is the inversion of IPSPs and excitation rather than inhibition. This mechanism by combining the effects of an electrogenie pump and this dual component inhibition, can modulate the effectiveness of synaptic inputs to the A neurons and as a result, the behavior of the animal mediated by these neurons (Jahan-Parwar & Fredman, 1978).

50x

BI 11~11sJAHAN-PAHWAK and ST~WN M. .‘RI I)MAK SI,MMARY

A new postsynaptic mechanism for modulating synaptic transmission is described. The A cluster neurons in the cerebral ganglion of ~pl~.siu utilize an electrogcnic sodium pump and a two-component synaptic inhibition to alter the eficacq of synaptic input. When first impaled with potassium citrate filled electrodes. both the spontaneous and sensory evoked synaptic input to the A neurons is hyperpolarizing. As the membrane potential of the A neurons increases due to an clectrogenic pump. these inputs become depolarizing. A neurons have an initial resting potential of - 30 mV. This increases steadily to a final level of about -55 mV. The increase is blocked by K’ free seawater. cooling and ouabain. It is TE,A insensitive. The pump is capable of bringing the membrane potential near or even beiow the A neuron Cl- equilibrium potential (EC-, ). The synaptic input evoked by both mechanosensory and food chemosensory stimulation has two components. The first is a fast Cl mediated IPSP. This component becomes depolarizing in Cl- free seawater. The second is ;1 slow K‘ mediated inhibition which is blocked bv TEA injection. At membrane potentials near EC,. the slow component can pull the membrane potential below EC-, cdtsing the fast IPSPs to invert. These can summate with other excitatory inputs (which are more effective due to the increased membrane potential) to produce A neuron firing. Shifts in the membrane potential in 2, more depolarized direction cause the same stimuli to be inhibitory. Due to the interaction of the electrogenic pump and the two component inhibition. the efficacy of scnsort imd other synaptic inputs can be modulated h> relatively small changes m membrane potential. Since the A neurons have been shown to exert a widespread motor influence on the foot :tnd parapodia. this modulation m:ly be of behavioral significance.

A~litlv,vlrdyrmrnt-This work was supported NS 12483 and BNS 77-24174 to BJ-P

by grants

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