Brain Research, 185 (1980) 125-137 125 © Elsevier/North-Holland Biomedical Press

A C T I V A T I O N OF T H E E F F E R E N T SYSTEM I N T H E ISOLATED F R O G L A B Y R I N T H : EFFECTS ON T H E A F F E R E N T EPSPs A N D SPIKE D I S C H A R G E R E C O R D E D F R O M SINGLE FIBRES OF T H E POSTERIOR NERVE

MARIA LISA ROSSI, IVO PRIGIONI, PAOLO VALLI and CESARE CASELLA

Institute of General Physiology, University of Pavia, Pavia (Italy)

(Accepted July 19th, 1979)

Key words: frog labyrinth - - efferent system - - receptor inhibition - - inhibitory transmitter - - receptor facilitation - - peripheral control

SUMMARY

Intra-axonal recordings were obtained from single afferent fibres of the posterior nerve in the isolated labyrinth of the frog (Rana esculenta). EPSPs and spike discharge were recorded both at rest and during rotatory stimulation of the canal. Electrical stimulation of either the distal end of the cut posterior nerve or of the central stumps of the anterior-horizontal nerves elicited a frequency-dependent inhibitory effect on the afferent discharge arising from the posterior canal. Denervation experiments revealed that inhibition is mediated by efferent fibres exhibiting a high degree of branching in the proximal part of the eighth nerve. The inhibitory effect was selectively cancelled by (1)D-tubocurarine 10 -6 M; (2) atropine 5 × l0 -5 M; (3) acetylcholine or carbachol 10 -4 M; (4) eserine l0 -5 M. Inhibition is thus most likely to be sustained by the release of acetylcholine from the efferent nerve terminals. Experiments in which the ionic composition of the external medium was modified suggest that the transmitter acts mainly by opening the chloride ion channels of the hair cell membrane. In some units the same stimulation pattern evoked a consistent increase in both EPSP and spike discharge, instead of inhibition. Such facilitation was unaffected by drugs or ionic modifications which block the efferent synapse, but disappeared after denerva- tion. Inhibition and facilitation, therefore, act as two control mechanisms which are able to modify substantially, at the first stage of processing, the sensory information which is sent to the vestibular second order neurones.

INTRODUCTION

Frog vestibular receptors receive an efferent innervation originating in the brain.

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After leaving the central nervous system (CNS), the efferent fibres enter the eighth nerve and on reaching the Scarpa ganglion they split off, running to the individual vestibular end-organs. Efferent nerve terminals form axo-somatic synapses with type 1I hair cells, so that sensory activity can be controlled peripherally at the receptor levell4,17,19,22-26. The presence of an efferent innervation has been clearly demon- strated in the vestibular and auditory system of several animals 17,2a,35, as well as in the lateral line organsS, 17. It is generally accepted that these efferent systems are inhibitory in nature and, at least in the cochlea and lateral line organs, their inhibitory action is sustained by the release of acetylcholine (ACh) from the efferent nerve terminals 2,7,9, 13,17,21,32,33. Though it has been reported that in the frog labyrinth the efferent system has an inhibitory effect on the afferent discharge 19,2a,24, its actual importance in controlling the receptor activity needs to be clarified further. In fact the actual role of the efferent system has recently been challenged by the findings of Gribenski and Caston; according to these authors efferent neurones seem to play a double role: their activity can in fact result either in peripheral inhibition or excitation if driven by a sensory input from the contralateral or homolateral sense organs respectively lz. Moreover the homolateral inhibitory control of the peripheral receptor activity is believed to be sustained by inhibitory receptor-receptor fibres 3,4,11. Therefore it was felt to be of interest to investigate more closely the effect of the efferent nerve fibres on the afferent EPSPs and propagated spike discharge by recording them intracellularly both at rest and during rotatory stimulation from single fibres of the posterior canal in the frog labyrinth. A preliminary report on the subject of this paper has already been presented 29.

METHODS

Experiments were carried out on frogs (Rana esculenta) weighing 25-30 g, at room temperature (20-22 °C). The right half head of the frog was mounted on a small turn-table and positioned so that the posterior canal lay approximately in the plane of rotation. This canal could thus be stimulated by sinusoidal velocity changes of constant amplitude (10-110 deg/sec) at different frequencies (0.1-0.5 Hz). Intracellular recordings were performed both at rest and during rotation by inserting a glass microelectrode of 30-40 Mf~ resistance, filled with 2 M potassium citrate in the posterior nerve close to the cytoneural junction. Electrical stimulation at different frequencies (10-200/see) and increasing times (200 msec-10 sec) was applied by means of fluid electrodes either to the distal end of the cut posterior nerve or to the central stumps of the anterior-horizontal nerves. To reveal the presence of fibres commonly distributed to the three canals of the same labyrinth, the latter stimulation pattern was also performed while extracellular potentials were recorded from the central stump of the posterior nerve. In some frogs, previously anaesthetized by immersion in 1%o solution of tricaine methane sulphonate, the roof of the mouth was opened and the right eighth nerve was exposed and cut between the Scarpa ganglion and the CNS. After two weeks the animals were killed and the effect of denervation was thus tested by repeating the same stimulation experiments. Details of experimental procedures have

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been extensively reported in previous papers 3°,a4. The composition of the Ringer solution was (mM): NaCI 117, KC1 2.5, NaHCO3 1.2, NaH2PO4 0.17, CaC12 1.8. In experiments in which K + or Ca2+-Mg 2+ concentrations were altered, osmolarity was kept constant by appropriate modifications in the NaCI concentration. Finally, when the external C1- level had to be reduced, NaC1 was replaced by an equivalent amount of sodium isethionate. Control and test solutions were applied to the preparation during the intervals between the rotation periods without affecting the fibre impale- ment. The following drugs were used: acetylcholine chloride, acetyl-fl-methylcholine chloride, atropine sulphate, D-tubocurarine chloride, physostigmine salicylate, car- bacholamine chloride, norepinephrine bitartrate, epinephrine chloride, a-dibenamine and fl-D-INPEA.

RESULTS

At rest, intracellular recordings from afferent nerve fibres of the posterior canal reveal a typical discharge pattern consisting of both EPSPs and propagated spikes (Fig. 1A). Stimulation at 10/sec of the distal end of the cut posterior nerve eliminated the orthodromic spikes and slightly reduced the frequency and amplitude of the EPSPs (Fig. 1B), whereas any resting afferent discharge disappeared when the stimulation frequency was raised to 50/sec. This effect is illustrated in Fig. 1 C, in which only antidromic spikes are exhibited in the tracing. This pattern of events was commonly observed in the majority of the impaled fibres.

To avoid the presence of antidromic spike activity, which could mask the inhibitory effect on the receptor discharge, we looked for an experimental condition which might allow a selective activation of the efferent system in the isolated labyrinth. This condition could occur if some of the efferent fibres branch in the eighth nerve providing collaterals to the different organs in the same labyrinth. In an attempt to test this hypothesis, we stimulated the central stumps of the anterior-horizontal nerves while extracellular potentials were recorded from the posterior nerve (Fig. 1D). Stimulation of this part of the anterior branch does in fact evoke a compound action potential in the posterior nerve (Fig. 1E). Conversely, when the same experiment was repeated in denervated frogs, no propagated spikes were recorded from this nerve (Fig. 1F). These results clearly show that the connections among the canals are sustained by common fibres belonging to the efferent system. When this stimulation pattern was applied while recording the potentials intracellularly from the posterior nerve an evident inhibitory effect on the resting discharge was observed, similar in its time course and frequency dependence to that produced by direct stimulation of the posterior nerve. Moreover antidromic spikes were actually recorded in hardly any of the impaled fibres. Fig. 2B shows the effect of stimulation at 50/sec on the resting discharge of an afferent fibre. Inhibition was complete (Fig. 2D) and could be maintained as long as stimulation was applied (up to 5-10 see). The inhibitory effect was followed by a marked increase in the resting discharge of both EPSPs and propagated spikes, the frequency of which was in some cases up to 3-4 times its initial level (Fig. 2B, E).

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.4 B

I C

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Fig. 1. A-C: effect of electrical stimulation of the posterior nerve on the resting activity recorded from an afferent fibre of the same nerve. A: afferent activity at rest; B and C: inhibition of the resting dis- charge evoked at 10/sec and 50/sec, respectively. Arrows in B and C show the antidromic spikes evoked by stimulation. D is a diagram of the frog labyrinth and experimental arrangement used to test the presence of fibres commonly distributed among the three canals: RE, recording electrode; STE, stimulating electrode; VIII, eighth nerve; AV, anterior vertical canal; PV, posterior vertical canal; H, horizontal canal; S, sacculus. E shows the compound action potential recorded from the posterior nerve when stimulating the anterior-horizontal nerves; F is the same experiment repeated after degener- ation of the efferent fibres.

Fig. 3A shows the time course o f the response evoked by sinusoidal stimulation at

0.1 Hz in an afferent nerve fibre, which displayed full inhibition o f the resting discharge at 50/sec. I t m ay be noted that activation o f the efferent fibres at 20/sec reduced the response evoked by one Cycle o f sinusoidal st imulation (Fig. 3A2), and when the frequency was raised up to 50/sec a complete block of the afferent discharge

could be achieved (Fig. 3A3). A complete block similarly occurred when the frequency of the sinusoid was raised f rom 0.1 Hz to 0.5 Hz, thus increasing the acceleratory peak f rom 39.4 to 985 deg/sec 2, The inhibitory effect persisted during successive cycles as

long as the efferent system was maintained in activity (Fig. 3B). Al though the inhibition o f the afferent discharge was the most c o m m o n result o f

stimulation, bo th o f the posterior nerve and of the anterior-horizontal nerves, the same stimulation pat tern in some units led to a substantial increase in bo th EPSPs and

propagated spikes during the stimulation period. Dissection proved to be a crucial step in determining the effect o f stimulation since the fibres sustaining inhibition appear to be extremely sensitive to damage; in some preparations in fact, inhibition could never

definitely be evoked. Under the most favourable conditions, inhibit ion is present in

A

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129

B E I

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Fig. 2. Effect of activation of the efferent inhibitory system of the posterior canal by electrical stimula- tion at 50/sec of the anterior-horizontal nerves; recordings are taken from an afferent fibre arising from the posterior canal. The diagram of the experimental arrangement (A) shows: ME, recording micro- electrode; STE, stimulating electrode; VIII, eighth nerve; AV, anterior vertical canal; PV, posterior vertical canal; H, horizontal canal; S, sacculus. The resting discharge, the effect of stimulation (solid bar) and the post-inhibitory discharge are shown in a sequence in B and in detail at a faster sweep speed in C, D, E respectively.

some 60 ~ of the impaled fibres, while 20 ~ of them, in the same preparation, are facilitated. Fig. 4 illustrates an example of such a facilitation of both resting (A) and evoked (B) receptor discharge. Like inhibition, facilitation is a striking effect that can be maintained for a relatively long time (5-10 sec), especially at 50/sec (Fig. 4A). The facilitatory effect, when present, proved to be strong enough to overcome the disfacilitation brought about by the deceleratory phase of the sinusoid (Fig. 4B, short bar). The degree of receptor inhibition or facilitation differs widely in different units at the same electrical stimulation frequency and is moreover independent of the resting discharge level. The inhibitory or facilitatory pattern was stable in a single unit when tested at different times; in no instance did inhibition shift to facilitation or vice versa. Complete inhibition or facilitation can also be effectively produced by a short stimulus train at high frequency (200/sec for 200 msec, Fig. 5A and B). Full inhibition ensues within a few milliseconds (about 10 msec in Fig. 5A2) and outlasts the stimulus train

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A B

IillIIlUl .... ulllml ~100deglsec L 1 0 s e e z

Fig. 3. Effect of activation of the efferent inhibitory system of the posterior canal during rotatory stimu- lation. All recordings are taken from the same unit. Column A shows the effect of activation at increasing frequencies of the inhibitory system, while rotatory stimulation was maintained constant at 0.1 I-Iz: 1, control recording; 2 and 3, effect of stimulation at 20/sec and 50/sec (solid bars), respectively. Column B shows the effect of 50/sec stimulation on the afferent response evoked by sinusoidal stimulation at increasing frequencies: 1,0.2 Hz; 2, 0.3 Hz; 3, 0.5 Hz. The turn-table angular velocity (10-110 deg/sec) is indicated under the corresponding tracings. In all recordings the activation of the efferent system was achieved via the axon-reflex described in the text.

for some 50-100 msec (Fig. 5A1). Similarly, the onset o f facilitation was very early (about 10-15 msec, Fig. 5B2) and persisted in the fibre for 100-200 msec after the end of the train (Fig. 5B1). The period during which the receptors remained alternatively inhibited or facilitated after the end of the stimulation was positively related to the durat ion o f the stimulus train applied to the nerve. In parallel experiments on

denervated frogs, electrical stimulation o f the ampullary nerves resulted neither in inhibition nor in facilitation o f the resting and evoked receptor activity, which proved to be virtually normal ~7.

To test the hypothesis that A C h may be the transmitter at the efferent synapse in frog labyrinth a series o f pharmacological agents were applied to the preparation. Drugs were simply added to the bathing medium since the experimental conditions did

not allow more direct application at the synaptic sites. All the pharmacological experiments were performed on units which exhibited a complete inhibition o f the afferent discharge. A C h 10 -4 M did not by itself produce a direct inhibition o f the

A

131

B

20mY _

~ IlOOdeglsec Fig. 4. Facilitation, instead of inhibition, evoked in a unit by electrical stimulation at 50/sec of the anterior-horizontal nerves. A shows the effect of stimulation (solid bar) on the resting discharge of a fibre from the posterior canal. B illustrates the results of stimulation (solid bars) on the afferent dis- charge during rotatory stimulation at 0.1 Hz. Note in B that the facilitatory effect prevails over the dis- facilitation brought about by the deceleratory phase of the sinusoid.

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B

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Fig. 5. Inhibition (A) and facilitation (B) elicited in two different fibres arising from the posterior canal by short stimulus trains (200/see, 200 msec; solid bars) applied to the anterior-horizontal nerves. The whole time course of the effects is illustrated in A1 and B1 and in detail, at a faster sweep speed, in A2 and B~ to show the full development of inhibition and facilitation within a few milliseconds from the onset of stimulation.

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afferent discharge; however, stimulation o f the efferent fibres failed to inhibit the

receptor discharge in the presence o f ACh. The same results were obtained after a few

minutes application o fca rbacho l 10 -4 M or eserine 10 -5 M, whereas acetyl-fl-methyl-

choline 5 × 10 -4 M was found to be ineffective. The block of the efferent system

observed in these experiments was easily reversible on washing and is most likely to be related to desensitization o f the cholinergic receptor16, 20. The application o f higher

concentrations of ACh (10-3-10 -2 M) actually resulted in an enduring decrease o f the

afferent discharge which was not followed by a spontaneous recovery, as might be expected f rom receptor desensitization; this effect could thus be due to an aspecific

depressant effect o f the drug. On the other hand, D-tubocurarine chloride 10 -6 M

caused a selective block of the electrically evoked inhibition within 3-5 min. Atropine was effective only when its concentrat ion was raised up to 5 × 10 -5 M; moreover its

effect occurred slowly and was less marked than that o f curare. The effect resulting f rom the blockade of the efferent system, typically through the action o f curare, is

illustrated in Fig. 6. It will be noted that the mechanically evoked response was

unaffected by the drugs tested and that the effect o f the drugs was the same when inhibition o f the afferent activity was elicited either by stimulation o f the posterior nerve or o f the anterior-horizontal nerves, as might be expected if the inhibitory effect

2

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B C

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i i i i i l l A l ~ l t JJ I 1 ~ 1 1 1 I

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50ms

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Fig. 6. Blockade of the inhibitory effect in the presence of D-tubocurarine 10 -8 M. Colunm A shows recordings from three different afferent fibres of the posterior canal at rest (A1 and A~) and during rotato- ry stimulation (A3). Column B shows the inhibition evoked in the same units by electrical stimulation at 50/sec of the posterior nerve (B1) and anterior-horizontal nerves (B2 and Bs). Column C shows the dis- appearance of inhibition brought about by curare. The arrows in B1 and C1 indicate the antidromic spikes evoked by stimulation of the fibre from which the recording is being obtained.

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is sustained by the same cholinergic pathway and the same synapse. Fig. 6Ca shows that the postinhibitory discharge also disappears when the efferent synapse is blocked. When all such pharmacological agents were applied to those fibres which exhibited facilitation instead of inhibition, no effects were detectable. Moreover, facilitation was apparently unaffected either by catecholamines (up to 10 -3 M) or by similar concentrations of a-dibenamine and fl-D-INPEA, thus ruling out the possibility that the facilitatory effect might be due to activation of the sympathetic innervation of the labyrinthS,13,17.

When K + concentration in the bath was progressively raised from 2.5 to 10 mM the resting receptor discharge increased, whereas the inhibition evoked by stimulation at 50/sec decreased and eventually disappeared. However in the presence of a 10 mM K + solution, though the occurrence of the orthodromic spike potentials in the afferent fibres was apparently normal, the compound efferent potential (Fig. 1 E) was greatly reduced, thus indicating that conduction in the efferent fibres was almost completely blocked. On the other hand, the progressive substitution of sodium isethionate for NaC1 reduced the efferent inhibition when more than 50 ~ of the normal chloride was removed from the bath; when 80 ~o of the chloride was replaced, inhibition completely disappeared. Modification in chloride concentration did not by itself affect either the normal receptor discharge or the compound efferent potential. At variance with inhibition, changes in chloride concentration did not affect electrically evoked facilitation, which on the other hand was greatly reduced by the increase in K + concentration (up to 10 mM).

An attempt to block the efferent inhibitory synapse selectively by appropiate modifications in Ca 2+ and Mg 2+ concentrations, as obtained in other systems is,a2, proved to be unsuccessful. In fact, when Ca 2+ was lowered to 0.9 mM and Mg 2+ was raised up to 6-10 mM both the resting discharge and the mechanically evoked response decreased, but full inhibition could invariably be produced. In the presence of 10 mM Mg 2+ a further decrease in Ca 2+ concentration beyond 0.9 mM resulted in a drastic block of the receptor afferent discharge 30, which prevented inhibition from being tested. In a similar experiment, in which Ca 2+ and Mg 2+ concentrations were adjusted to reduce the resting discharge significantly in a unit exhibiting facilitation, electrical stimulation was still effective in producing a clear-cut increase in both EPSPs and spike frequency.

DISCUSSION

Our results clearly show that in the frog inner ear inhibition of the afferent discharge is sustained by activation of the efferent system, whose fibres provide an inhibitory pathway among the different organs in the same labyrinth. Moreover, the denervation experiments definitely rule out the possibility that the observed effects might be sustained by axons belonging to neurones located peripherally to the CNS and thus different from the classically described efferent neurones.

Inhibition is presumably sustained, both in the cochlea and lateral line organs, by the release of ACh from the efferent nerve terminals2,7,9,13,17,3~, 83. The present

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results have similarly shown that inhibition of the frog labyrinthine receptors is mediated by a chemical cholinergic synapse. The inhibitory effect has in fact been prevented by treatments with ACh antagonists, such as curare and atropine2,13,17,3L However, at variance with the lateral line organs 32, the postsynaptic receptor appears to be nicotinic instead of muscarinic, since curare proved to be much more effective than atropine in blocking the efferent synapse. The failure of ACh and cholinomime- tics to evoke inhibition, as has been observed in the cochlea and lateral line organs 2,7,13,17,21,32, does not disprove the cholinergic nature of the efferent synapse, since, even though these drugs do not mimic inhibition, they cause inhibition to disappear most effectively. This may be due to the desensitization of the postsynaptic receptorZ,16, zo. The onset and time course of desensitization induced by ACh treatment is known to be closely dependent on the mode of administration and the peculiarities of individual synapsesl6, 20. Thus it may be that desensitization induced in our preparation by ACh proceeds at the same rate as activation, thus leading to a synaptic block without any appreciable inhibition of the receptor discharge. This mechanism should also hold for carbachol and eserine, which can by itself act as a weak agonist 15 and is likely to produce ACh accumulation in the tissue.

Potassium and/or chloride ions are usually involved in the development of inhibition at a chemical synapse. Although the effect of the inhibitory transmitter could not be directly tested in our experiments at the postsynaptic level, evidence has been produced that, as in the cat cochlea 6, chloride ions are mainly involved at the inhibitory synapse in the frog labyrinth. The increase in external K + concentration actually resulted in the disappearance of the efferent inhibition, but the true postsynaptic nature of this effect is at least questionable since blockade in conduction occurs at K + concentrations which interfere with the inhibitory system. A more clear- cut effect was produced by modifications in C1- concentration, which selectively affects the ionic synaptic mechanism without modifying the resting potential of the nerve fibres and hair cells, as indirectly shown by recording a normal pattern of afferent discharge and a normal compound efferent potential.

Although the inhibition of the receptor discharge is the most frequent effect produced by stimulation of the efferent fibres, some units were unaffected, whereas an evident facilitation was produced in others. Whatever may be the mechanism sustaining facilitation, it can hardly be interpreted as a stimulation artefact. Facilita- tion, as well as inhibition, is in fact cancelled by degeneration of the efferent fibres, thus proving its true efferent nature. As suggested by Gribenski and Caston ~2, the efferent fibres in the frog labylinth could form excitatory synapses with the hair cells. Our experiments however failed to demonstrate that facilitation is mediated by a chemical synapse, either adrenergic or cholinergic in nature. Finally, facilitation can also be evoked when the afferent synapse is greatly depressed by modifications in calcium and magnesium concentration. As regards the basic mechanism of afferent facilitation, Goldberg and Fernandez 10 have suggested that in the squirrel monkey inner ear receptor facilitation might be due to an accumulation of potassium extracellularly. Our experiments have shown that the resting afferent discharge actually increases when potassium concentration increases in the bath. However, when

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the inhibitory control of an afferent fibre was blocked, for example by curare, inhibition did not reverse to facilitation when the mass nerve stimulation was applied, as would be expected if facilitation were due to an accumulation of potassium ions in the extracellular space near the hair cells.

Though it has been reported that under natural stimulation the effect of the efferent system on the afferent discharge is rather weak13,17,19,23-25, al, it seems of

interest to point out the potential effectiveness of this system, which, when fully activated, can lead not only to a complete and long-lasting suppression of the resting discharge, but also to the blockade of any response to very intense acceleratory stimuli. This supports the view that efferent control in the semicircular canal can extend the dynamics of this sensory organ to a wide range of stimulus intensities. Our results, however, have clearly shown that stimulation of the efferent fibres is able to produce not only an inhibitory, but also a facilitatory widespread effect on the afferent discharge. I f both actions actually cooperate in the control of the sensory information in frog vestibular organs, inhibition and facilitatio n interact not only in the midbrain centers 23, but also peripherally at the receptor level. The response of vestibular receptors thus depends on the balance between two opposite control channels which are able to modify profoundly the sensory information which is sent to the vestibular second order neurons. Moreover, the physiological meaning of the extensive multi- labyrinthine convergence on a given efferent neuron, which has been clearly demon- strated in the frog1,22, 24, can be more fully understood if we consider that this efferent

neuron can by itself control the information arising from different organs in a labyrinth and also in its contralateral labyrinth, irrespective of their directional sensitivity, as in the lateral line system 3x.

ACKNOWLEDGEMENTS

We thank Dr. N. Dieringer for his critical reading of the manuscript. This work was supported by Grant 78.02034.04 f rom the Consiglio Nazionale

delle Ricerche and by a grant from the Ministero della Pubblica Istruzione, Rome, Italy.

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