In paired recordings, the proportion of sIPSCs that were elicited by the recorded presynaptic neurone was rather small, indicating that on average 4·25 interneurones contributed to the sIPSCs (Kondo & Marty, 1998). But in one case (cell pair 15) the proportion of sIPSCs that were associated with presynaptic action potentials was as high as 0·79. Shortly after starting a whole-cell recording in the presynaptic cell, we observed in this experiment the gradual disappearance not only of eIPSCs (presynaptic 'washout', see Kondo & Marty, 1998), but also of sIPSCs that were not linked to presynaptic action potentials, indicating that all sIPSCs observed before washout originated from the recorded presynaptic cell. We took advantage of the special situation of this recording to obtain the rate of mIPSCs simply by subtracting eIPSCs from all recorded events. The results indicated that NA increased the frequency of mIPSCs, with a ratio to the control value that was similar to that previously observed in the presence of TTX (Kondo & Marty, 1997). Meanwhile, the success rate of eIPSCs was not modified (Fig. 7B). This result shows that NA increases the frequency of action potential-independent IPSCs even in the absence of TTX while in the same synapse, it fails to increase evoked transmitter release.
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Figure 7. NA increases action potential-independent IPSCs recorded without TTX
In this paired recording it appeared that all IPSCs, whether they were evoked by a presynaptic action potential or not, originated from a synapse made by the recorded presynaptic interneurone. A, pre- and postsynaptic traces on a slow time scale, both in the control (Aa) and in NA (10 µM; Ab). Spike-evoked IPSCs are marked ( ). B, NA did not increase the proportion of successful IPSCs but it increased significantly the rate of action potential-independent IPSCs (labelled 'mIPSCs').
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DISCUSSION |
The principal result that emerges from the present work is that at synapses among stellate cells sIPSCs, eIPSCs and mIPSCs are regulated separately from each other following NA application. Thus while the frequencies of mIPSCs and sIPSCs are both increased, the underlying mechanisms are different, and the mean amplitude of eIPSCs is actually slightly decreased.
Excitatory action of NA on action potential generation
NA markedly increased the action potential frequency in cerebellar stellate cells. Our results show that this increase arises from a direct effect of NA on action potential generation rather than from a change in the efficacy of excitatory or inhibitory inputs. The NA-induced excitation was mimicked by a specific 
-adrenoceptor agonist and persisted in the presence of an 
-adrenoceptor antagonist, suggesting that -adrenoceptors are mainly responsible for this action. The slow recovery of the effects of NA or Iso on spike frequency upon washing are also consistent with the involvement of -receptors. -Receptor-mediated excitatory effects on action potentials have been reported in several central nervous system preparations, including hippocampal pyramidal cells and thalamic neurones. In hippocampal pyramidal cells, blockade of slow afterhyperpolarization (AHP) seems to be the major cause for excitation (Madison & Nicoll, 1986) whereas in thalamic neurones, a slow depolarization resulting from a decrease in hyperpolarization-activated current (Ih) predominates (McCormick & Pape, 1990).
Effects of NA on eIPSCs
In paired recordings, NA applications did not lead to the expected increase in mean eIPSC, but to a significant decrease (Table 1). The reduction in mean eIPSC amplitude resulted from decreases in both the success rate and mean amplitude excluding failures, and was accompanied by a small but significant increase in mean latency (Table 1). This pattern of changes is strikingly similar to that observed in control saline during periods of higher presynaptic firing frequency (Kondo & Marty, 1998; Fig. 6B), raising the possibility that the changes in eIPSCs observed in NA are a mere consequence of the increase in presynaptic firing rate.
The increased latency and failure rate indicate that the release probability is reduced in NA. This could happen by either of two mechanisms. First, the increased rate of GABA release associated with the increase in the frequency of sIPSCs could activate presynaptic GABAB receptors and thus reduce the probability of evoked release. Alternatively, the high exocytosis rate could induce a depletion of the pool of vesicles available for evoked release.
The amplitude reduction of eIPSCs excluding failures in NA is observed both for 'multiple' and for 'simple' synapses (see Figs 5 and 6 for the latter). Due to the combination of multivesicular release and of a high degree of receptor occupancy, a reduction in release probability at one release site results in a reduction in the quantal size (C. Auger, S. Kondo & A. Marty, unpublished observations). The reduced probability of evoked release could therefore be the cause of the observed reduction in mean IPSC amplitude. Alternatively, the amplitude decrease could result from cumulative desensitization. Previous results on -latrotoxin-induced bursts indicate that at single-site synapses, high release rates induce a gradual decrease in peak mIPSC amplitudes, presumably reflecting cumulative desensitization (Auger & Marty, 1997). However, this effect is negligible for mIPSC frequencies below 3 Hz; since the maximum eIPSC frequency observed in the present experiments was 3·3 Hz, cumulative desensitization is unlikely to have played an important part in the reduction in mean eIPSC amplitude.
In summary, the most parsimonious explanation for the NA-induced changes in eIPSCs is that the increased rate of presynaptic firing leads to a decrease in the probability of evoked transmitter release, which then leads to both an increase in latency and to a decrease in mean amplitude of eIPSCs.
Action potential-dependent and -independent transmitter release are differentially modulated by NA
We conclude from this work and from previous publications (Llano & Gerschenfeld, 1993b; Kondo & Marty, 1997) that stimulation of -adrenergic receptors increases the frequency of both mIPSCs and sIPSCs to a similar extent while eIPSCs are slightly reduced. The effects on mIPSCs and sIPSCs are superficially similar but the underlying mechanisms are different. The increase in mIPSC frequency is not linked to any membrane permeability change and is due to the direct enhancement of the vesicular release probability (Llano & Gerschenfeld, 1993b; Kondo & Marty, 1997). Here we show on the other hand that the effects on sIPSCs are mostly accounted for by an increase in the rate of firing of presynaptic interneurones, apparently due to a change in presynaptic membrane conductance. Thus the location of the NA action is different in the two cases: the enhancement of mIPSC frequency takes place at the nerve terminals whereas the acceleration of sIPSC discharge is elicited in the somato-dendritic domain or near the site of generation of the spikes, at the axon hillock.
The experiment of Fig. 7 suggests that action potential-independent IPSCs occurring in normal saline solution are regulated by NA in the same manner as mIPSCs in TTX. If this is so it should be possible to predict the change in the rate of sIPSCs produced by NA on the basis of the other measurements made in this study and in our preceding work (Kondo & Marty, 1997). To this end we first note that: frequency of sIPSCs = frequency of mIPSCs + frequency of eIPSCs.
To estimate the frequency of mIPSCs, we assume that this frequency remains the same with and without TTX. Therefore, we measured the proportion of action potential-independent IPSCs among the IPSCs occurring in control conditions simply by adding TTX (200 nM) to block the action potentials. The mean proportion of TTX-insensitive IPSCs estimated with this method was 19 % (n = 11; range 10 to 29 %). This number is the same as that previously reported by Llano & Gerschenfeld (1993a).
The above equation is valid both in control saline solution and in the presence of NA:
sctl = mctl + ectl, (1)
sNA = mNA + eNA, (2)
where sctl, mctl and ectl are the frequencies of sIPSCs, mIPSCs and eIPSCs, respectively, in control conditions, and the same symbols with NA indices are the corresponding quantities in NA. We are assuming here that the frequency increase of action potential-independent release in normal saline solution is the same as in TTX. According to our previous measurements (Kondo & Marty, 1997) the mIPSCs frequency was 2·24 times higher in NA than in the control:
mNA/mctl = 2·24. (3)
The eIPSC frequency was 1·48 larger in NA than in the control (see Table 1):
eNA/ectl = 1·48. (4)
Finally, the proportion of mIPSCs in total IPSCs was 0·19:
mctl/sctl = 0·19. (5)
Equations (1)-(5) are a set of five equations with six unknowns. Therefore these unknowns are linked to each other. One obtains:
sNA = 1·62 sctl.
This calculation predicts a frequency increase of sIPSCs by NA of 1·62 over the control. This number is quite close to the experimental value (1·70, see Table 1). This match provides evidence for the above hypothesis that sIPSCs are the sum of two components, mIPSCs and eIPSCs, which are regulated by NA independently of each other.
Other examples of differential regulation of evoked and miniature synaptic currents
There are many examples where neuromodulatory substances modify in parallel evoked and miniature synaptic currents, as if a regulatory step downstream of Ca2+ entry was common to spontaneous and triggered exocytosis (see the review by Thompson, Capogna & Scanziani, 1993, for examples in the hippocampus). However, recent measurements of intraterminal Ca2+ concentration suggest that at least in two of these cases (inhibition by adenosine and by GABAB receptor activators of the CA3-CA1 synapse), the regulation of evoked synaptic currents is actually due to modulation of voltage-dependent Ca2+ currents (Wu & Saggau, 1994, 1995). Such an effect is unlikely to modify miniature synaptic currents (Thompson et al. 1993). Therefore, even though adenosine and GABA similarly affect miniature and evoked release, they appear to do so by separate mechanisms.
Many examples have been previously reported where a neuromodulatory agent changes evoked transmitter release without altering the rate of miniature currents. This is the case for the inhibitory effects of GABAB stimulation at inhibitory synapses in hippocampal CA1 neurones (Scanziani, Capogna, Gähwiler & Thompson, 1992), for those of -adrenergic stimulation at excitatory synapses in hippocampal CA3 pyramidal cells (Scanziani, Gähwiler & Thompson, 1993), or for those of the metabotropic glutamate agonist trans-ACPD in cerebellar interneurone- interneurone synapses (Llano & Marty, 1995). Such cases can easily be explained by a reduction of Ca2+ influx following the regulation of presynaptic excitability. Thus in all the above examples an inhibition of Ca2+ channels is likely, even though this possibility has not been tested directly.
To our knowledge there has been no report until the present study of a neuromodulatory action where miniature events are modified without an accompanying change in evoked currents. In this case the discrepancy cannot be explained easily on the basis of an alteration in membrane conductances of the presynaptic terminals.
It is finally worth mentioning that in Drosophila, mutations of the synaptotagmin gene can lead to a situation where the frequency of miniature synaptic currents is enhanced while the amplitude of evoked synaptic currents is reduced (DiAntonio & Schwartz, 1994). One possible interpretation of this finding is that the primary defect in mutated animals is an enhancement in the rate of fusion of synaptic vesicles, leading to a decrease in the number of vesicles available for evoked release.
Functional consequences of enhanced mIPSCs and sIPSCs and reduced eIPSCs in the presence of NA
Since in this preparation there is little difference between the mean amplitudes of mIPSCs and of sIPSCs (Llano & Gerschenfeld, 1993a), action potential-dependent and -independent currents contribute in proportion to their relative frequencies (80 and 20 %, respectively) to the inhibition exerted by stellate cells. In slices, excitatory mossy-fibre and climbing-fibre inputs are not active, so that interneurone IPSCs reflect mIPSCs and the random spontaneous firing of presynaptic interneurones. It is this background inhibition which is enhanced by NA. Because of the powerful inhibition resulting from one IPSC it may seem paradoxical that NA manages to enhance significantly the rate of firing of interneurones. Recent results suggest that one IPSC prevents spiking for a period of about 7 ms (Häusser & Clark, 1997). Since the mean frequency of sIPSCs across experiments was 4·7 Hz in the control and 8·7 Hz in NA, the deficit of spiking due to interneurone- interneurone synapses can be estimated at 3 % in the former and at 6 % in the latter case. Thus, due to the low frequency of sIPSCs in interneurones, their rate of firing is determined by their intrinsic action potential generating conductances and not by the rate of synaptic currents (see, however, Häusser & Clark, 1997 for a different point of view). This is confirmed by the lack of net effects of bicuculline on the rate of action potentials in our experiments, and by the finding that the potentiation of the action potential frequency by NA was as strong in control saline solution as in bicuculline.
In vivo , phasic stimulation of the excitatory afferent fibres generates a powerful synchronized inhibition among interneurones and in postsynaptic Purkinje cells (Eccles, Llinas & Sasaki, 1966a,b). The present results indicate that, at interneurone-interneurone synapses, this phasic response is not enhanced, but is somewhat reduced by NA. Even though only interneurone-interneurone synapses have been investigated in the present study it can be speculated that the same situation holds at interneurone-Purkinje cell synapses. If such is the case, NA increases desynchronized inhibitory background activity but decreases the inhibition produced by synchronized parallel fibre stimulation. In Purkinje cells, the increased background inhibition would reduce the spontaneous activity in the absence of stimulation, while the inhibition of eIPSCs would enhance the response to parallel fibre stimulation. Thus noradrenergic modulation appears to tune the contrast between the rate of discharge observed under low excitatory input conditions and the phasic responses to excitatory input stimulation.
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REFERENCES |
Auger, C. & Marty, A. (1997). Heterogeneity of functional synaptic parameters among single release site. Neuron 19, 139-150.
[Medline]
Bergles, D. E., Doze, V. A., Madison, D. V. & Smith, S. J. (1996). Excitatory actions of norepinephrine on multiple classes of hippocampal CA1 interneurones. Journal of Neuroscience 16, 572-585.
[Abstract]
Chavez-Noriega, L. E. & Stevens, C. F. (1994). Increased transmitter release at excitatory synapses produced by direct activation of adenylate cyclase in rat hippocampal slices. Journal of Neuroscience 14, 310-317.
[Abstract]
DiAntonio, A. & Schwartz, T. L. (1994). The effect on synaptic physiology of synaptotagmin mutations in Drosophila. Neuron 12, 909-920.
[Medline]
Eccles, J. C., Llinas, R. & Sasaki, K. (1966a). The inhibitory interneurones within the cerebellar cortex. Experimental Brain Research 1, 1-16.
[Medline]
Eccles, J. C., Llinas, R. & Sasaki, K. (1966b). Parallel fibre stimulation and the responses induced thereby in the Purkinje cells of the cerebellum. Experimental Brain Research 1, 17-39.
[Medline]
Gereau, R. W. IV & Conn, J. (1994). Presynaptic enhancement of excitatory synaptic transmission by -adrenergic receptor activation. Journal of Neurophysiology 72, 1438-1442.
[Medline]
Häusser, M. & Clark, B. (1997). Tonic synaptic inhibition modulates neuronal output pattern and spatiotemporal synaptic integration. Neuron 19, 665-678.
[Medline]
Hoffer, B. J., Siggins, G. R., Oliver, A. P. & Bloom, F. E. (1972). Cyclic AMP-mediated adrenergic synapses to cerebellar Purkinje cells. Advances in Cyclic Nucleotides Research1, 411-423.
Kondo, S. & Marty, A. (1997). Protein kinase A-mediated enhancement of miniature IPSC frequency by noradrenaline in rat cerebellar stellate cells. The Journal of Physiology 498, 165-176.
[Abstract]
Kondo, S. & Marty, A. (1998). Synaptic currents at individual connections among stellate cells in rat cerebellar slices. The Journal of Physiology 509, 221-232. &uid=9547395&dopt=r">
[Abstract/Full Text]
Lipscombe, D., Kongsamut, S. & Tsien, R. W. (1989). -Adrenergic inhibition of sympathetic neurotransmitter release mediated by modulation of N-type calcium-channel gating. Nature 340, 639-642.
[Medline]
Llano, I. & Gerschenfeld, H. M. (1993a). Inhibitory synaptic currents in stellate cells of rat cerebellar slices. The Journal of Physiology 468, 177-200.
[Abstract]
Llano, I. & Gerschenfeld, H. M. (1993b). -Adrenergic enhancement of inhibitory synaptic activity in rat cerebellar stellate and Purkinje cells. The Journal of Physiology 468, 201-224.
[Abstract]
Llano, I. & Marty, A. (1995). Presynaptic metabotropic glutamatergic regulation of inhibitory synapses in rat cerebellar slices. The Journal of Physiology 486, 163-176.
[Abstract]
McCormick, D. A. & Pape, H.-C. (1990). Noradrenergic and serotonergic modulation of a hyperpolarization-activated cation current in thalamic relay neurones. The Journal of Physiology 431, 319-342.
[Abstract]
Madison, D. V. & Nicoll, R. A. (1986). Actions of noradrenaline recorded intracellulary in rat hippocampal CA1 pyramidal neurones, in vitro.Journal of Physiology 372, 221-244.
Midtgaard, J. (1992). Membrane properties and synaptic responses of Golgi cells and stellate cells in the turtle cerebellum. The Journal of Physiology 457, 329-354.
[Abstract]
Mitoma, H. & Konishi, S. (1996). Long-lasting facilitation of inhibitory transmission by monoaminergic and cAMP-dependent mechanisms in rat cerebellar GABAergic synapses. Neuroscience Letters 217, 141-144.
[Medline]
Scanziani, M., Capogna, M., Gähwiler, B. H. & Thompson, S. M. (1992). Presynaptic inhibition of miniature excitatory synaptic currents by baclofen and adenosine in the hippocampus. Neuron 9, 919-927.
[Medline]
Scanziani, M., Gähwiler, B. H. & Thompson, S. M. (1993). Presynaptic inhibition of excitatory synaptic transmission mediated by alpha adrenergic receptors in area CA3 of the rat hippocampus in vitro.Journal of Neuroscience 13, 5393-5401.
Thompson, S. M., Capogna, M. & Scanziani, M. (1993). Presynaptic inhibition in the hippocampus. Trends in Neurosciences 16, 222-227.
[Medline]
Ungerstedt, U. (1971). Stereotaxic mapping of the monoamine pathways in the rat brain. Acta Physiologica Scandinavica, suppl. 367, 1-48.
Wu, L. G. & Saggau, P. (1994). Adenosine inhibits evoked synaptic transmission primarily by reducing calcium influx in area CA1 of hippocampus. Neuron 12, 1139-1148.
[Medline]
Wu, L. G. & Saggau, P. (1995). GABAB receptor-mediated presynaptic inhibition in guinea-pig hippocampus is caused by reduction of presynaptic Ca2+ influx. Journal of Physiology 485, 649-657.
[Abstract]
Acknowledgements
We thank C. Pouzat for sharing his analysis routines, and C. Auger for comments on the manuscript. This work was supported by the Human Frontier Science Program (fellowship to S. K.) and by the Deutsche Forschungsgemeinschaft (program S. F. B. 406).
Corresponding author's present address
S. Kondo: Laboratory for Neural Circuits, Bio-mimetic Control Research Center, RIKEN, 2271-130 Anagahora, Shimoshidami, Moriyama-ku, Nagoya, 463 Aichi, Japan.
Email: skondo{at}nagoya.riken.go.jp
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Introduction
