INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The cerebellum receives its input via two different afferent systems, the climbing and the mossy fibers. While the sole source of climbing fibers is the inferior olive, the vast majority of mossy fibers in mammals arise from the pontine nuclei (PN). It is well documented that the membrane properties of inferior olivary neurons are massively influenced by serotonin [5-hydroxytryptamine (5-HT)] in vivo and in vitro (Goldstein et al. 1969; Headley et al. 1976; Placantonakis et al. 2000; Sugihara et al. 1995). In particular, the rhythmic firing and subthreshold oscillations are under control of serotonergic neurons located within medullary raphe and reticular nuclei (Placantonakis et al. 2000). Thus the "working state" of the climbing fiber system is subject to serotonergic modulation. Such detailed information is lacking for the mossy fiber system. Our present knowledge about a possible serotonergic regulation of information transfer and processing within the PN is limited. More than a decade ago, Mihailoff et al. (1989) demonstrated projections from well-established serotonergic regions like the pontine and medullary raphe nuclei by retrograde tracing from the PN. Interestingly, parts of these structures, namely the medullary raphe nuclei, also provide serotonergic input to the inferior olive (Bishop and Ho 1986; Compoint and Buisseret-Delmas 1988). Since then, the presence of 5-HT receptors within the PN has been implicated by in situ hybridization studies and immunocytochemistry (Cornea-Hérbert et al. 1999; Hamada et al. 1998; Pompeiano et al. 1994; Wright et al. 1995). However, the functional consequences of these serotonergic afferents and receptors have as yet not been explored.

5-HT is one of the best-known neuromodulators, affecting many neurons throughout the whole CNS (Jacobs and Azmitia 1992). The diversity of 5-HT receptor types (Hoyer and Martin 1997) is in line with the diversity of actions 5-HT exerts on its target cells (for review, see Anwyl 1990). Most receptor types act on second messenger systems increasing or decreasing membrane conductances as well as synaptic transmission. Using a brain slice preparation (Möck et al. 1997; Schwarz et al. 1997), we have recently shown that the PN possess many of the putative pre- and postsynaptic targets known to be modulated by 5-HT in other preparations. On this basis, it is plausible to speculate that also the second cerebellar input system, arising from the PN, is under serotonergic control. In this study we tested whether 5-HT affects the membrane properties and synaptic transmission in PN neurons in vitro. Our results clearly demonstrate distinct effects on both the excitability and the synaptic transmission via two different receptor types allowing the PN to function as frequency filter, which can be adjusted according to functional needs.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Slice preparation and maintenance, the basic in vitro electrophysiology techniques, data sampling, and analysis methods employed in this study resemble those described elsewhere (Möck et al. 1997; Schwarz et al. 1997). Briefly, Sprague-Dawley rats (18-25 days old) were deeply anesthetized with ketamine and decapitated. During the dissection of the brain, care was taken not to impose mechanical stress onto the brain stem. To cool the brain as fast as possible, the dissection was performed under the superfusion of cold (4°C) artificial cerebrospinal fluid (ACSF; see following text). Parasagittal slices were cut to a thickness of 400 µm using a vibrating microtome (Leica, Wetzlar, Germany). They were stored in ACSF containing (in mM) 123 NaCl, 5 KCl, 1.25 NaH₂PO₄, 2 MgCl₂, 26 NaHCO₃, 2 CaCl₂, and 10 D-glucose, oxygenated with 95% O₂-5% CO₂ at room temperature. For recording, the slices were transferred to a submerged recording chamber and superfused with ACSF at 35°C.

Standard intracellular current-clamp recordings were performed with glass microelectrodes filled with 3 M potassium acetate (50-100 M) using an Axoclamp 2A amplifier (Axon Instruments) in the bridge mode. The microelectrodes had a linear I-V relationship within 1.0-1.0 nA. The voltage records were low-pass filtered (cutoff frequency 10 kHz) and digitized at a sample rate of 5 kHz or 20 kHz using a PC with a 1401plus interface and Spike2 software (Cambridge Electronic Design, Cambridge, UK).

Postsynaptic potentials (PSPs) were evoked by applying negative current pulses of 0.1-ms duration via lacquer-coated tungsten electrodes (impedance, 2.5-7 M, at 1 kHz). The stimulation electrode was placed into the cerebral peduncle. Square current pulses were generated by a constant current bipolar stimulus isolator (A365R; WPI) and controlled by the 1401plus interface. If not explicitly noted otherwise, current pulses were delivered at frequencies of 0.075 or 0.15 Hz during data recording. The minimal stimulus intensity required to elicit PSPs was determined for every individual neuron. To study the intensity dependence of PSPs, the intensity was increased from these thresholds in 5- or 10-µA steps until spikes were generated, or to 250 µA maximal. PSPs were recorded at a sampling rate of 10 kHz. For every stimulus intensity or every test membrane potential, 10 trials were recorded subsequently. To monitor changes in apparent membrane resistance, small negative current pulses (0.1 nA, 200-ms duration) were applied via the recording electrode 300 ms before afferent fibers were stimulated. Paired-pulse stimulations were performed with interstimulus intervals of 10, 20, 50, 100, 200, 500, and 1,000 ms. The sequence of different interpulse intervals was pseudorandom. Ten trials were recorded for every interpulse interval. The interval between individual trials was 14 s.

Drugs were diluted in ACSF to final concentrations from stock solutions and saturated with 95% O₂-5% CO₂ before use. We applied 5-HT at a concentration of 5 µM, cinanserin hydrochloride (20 µM), and cyanopindolol hemifumarate (10 µM), all obtained from Tocris (Bristol, UK). The slices were superfused with these drugs for several minutes before recording their steady-state effects. To allow proper comparison between control and test recordings, intracellular current application was used to readjust the somatic membrane potential to the value measured during control recordings. Recovery was recorded in all experiments as long as the recording was stable enough. Every neuron served as its own control.

For statistical comparison of control and test recordings, we employed the Wilcoxon signed-rank test for paired samples (Systat; SPSS). A first-order error probability of P < 0.05 was considered as significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Intracellular recordings obtained from 79 PN neurons were accepted for analysis according to the criteria described in Schwarz et al. (1997). Briefly, neurons were included in our sample if they developed a stable resting potential within a few minutes after impalement and if the apparent membrane resistance was 15 M. Furthermore, only neurons with spike amplitudes of more than 50 mV (measured from the resting potential to the peak) and a spike width no greater than 1 ms (measured at half-amplitude between resting potential and peak of the spike) were accepted. Under control conditions, none of these neurons was spontaneously active. They displayed a resting membrane potential of 67.4 ± 4 (SD) mV. Most likely the neurons used in the present study were impaled at the soma, and therefore the membrane potential measurements may not be directly valid for distal parts of the cells. To take this into account, we will use the term somatic membrane potential (SMP). The apparent membrane resistance (R_in) was 49.5 ± 14.6 M, and the apparent membrane time constant () was 6.6 ± 2.0 ms. R_in and  were determined from voltage responses to small negative current pulses (0.1 nA) to minimize contamination by activation of voltage-dependent conductances. As even small negative current pulses activate a fast and sustained inward rectification in PN neurons (Schwarz et al. 1997), the true passive membrane properties may be slightly different.

5-HT effects on membrane potential responses to subthreshold stimuli

In all cells tested (n = 79), application of 5 µM 5-HT led to a significant (P < 0.001) and persistent depolarization of the SMP (Fig. 1, A, B, D, and F). In 22% of the neurons, the depolarization was strong enough to drive the SMP above the firing threshold, causing previously silent cells to fire spontaneously (Fig. 1, B and D). In these cases, measuring the steady-state level of 5-HT-induced depolarization was not possible, and therefore we determined the maximal depolarization before firing occurred. On average the neurons were depolarized by 6.5 ± 3.5 mV. Removal of 5-HT resulted in a complete recovery of the initial SMP within a few minutes (Fig. 1, A, B, and D). Population data are given in Fig. 1F.

View larger version (31K): [in this window] [in a new window]   Fig. 1. Serotonergic modulation of the effects of 5-hydroxytrptamine (5-HT) receptor antagonists on the somatic membrane potential (SMP) and the apparent input resistance (Rin). A: continuous intracellular recording of a representative pontine nuclei (PN) neuron during 5-HT application and recovery. Small negative current pulses (0.1 nA, 200 ms) were applied every 6 s to monitor changes in Rin. 5-HT (5 µM) induced a depolarization of the SMP by 13.8 mV, paralleled by an increase in Rin. After removal of 5-HT from the bath, both SMP and Rin returned to their initial value within a few minutes. B: similar recording to A. In this example, however, the 5-HT-induced depolarization reversibly drove the SMP across the firing threshold. Note that the variability in spike amplitude throughout this figure results from the low sampling rate used in these experiments. As in A, 5-HT induced a reversible increase in Rin. C: coapplication of 5 µM 5-HT and 10 µM cyanopindolol to the same neuron in B. Cyanopindolol did not block depolarization and increase in Rin. D: another PN neuron responding to 5-HT application with a reversible suprathreshold depolarization and increase in Rin. E: coapplication of 5 µM 5-HT and 20 µM cinanserin to the same neuron in D. Cinanserin largely prevented any change in SMP and Rin. F and G: scatter plots showing changes in SMP (F) and Rin (G), for the entire population. 5-HT alone and with coapplication of cyanopindolol induced variable depolarization and an increase in Rin (open circles, filled squares). None of them were hyperpolarized. Effects were blocked almost completely by treatment with 5-HT plus cinanserin (gray triangles). See Table 1 for statistics.

The 5-HT-induced depolarization of the SMP was accompanied by a significant (P < 0.001) and reversible increase of the R_in from 49.5 ± 14.6 to 62.7 ± 21.1 M (n = 79). No such change was observed for : 6.6 ± 2.0 ms under control conditions versus 6.8 ± 2.3 ms under 5-HT (P > 0.2). Population data for R_in are given in Fig. 1G. It is important to take the increase of R_in into account for the analysis of membrane potential responses to current stimuli. Because changes in R_in per se alter membrane potential responses, analysis of 5-HT-induced alterations independent of changes in R_in required a correction of the quantified data according to these changes in R_in, i.e., by a factor derived from the ratio between R_in values measured under both conditions. In the following, data corrected for changes in R_in are indicated by a ().

Characteristically, the I-V relationship of PN neurons shows a fast inward rectification in response to negative and subthreshold positive current pulses of 200-ms duration (Fig. 2, A and C, left). The strength of inward rectification depends on the extracellular potassium concentration (Schwarz et al. 1997). Moreover, inward rectification is altered to outward rectification in response to positive current pulses when sodium channels are blocked with tetrodotoxin (Schwarz et al. 1997). Application of 5 µM 5-HT reversibly weakened the inward rectification in response to negative current pulses (Fig. 2, B and C, left). The summary plot in Fig. 2C (right) shows the deviation of the I-V curves after 5-HT application from the ones obtained from control recordings fitted by a linear regression (r = 0.454). In contrast to the membrane potential responses to hyperpolarizing current pulses, 5-HT reversibly enhanced inward rectification in depolarizing direction (Fig. 2, B and C). Since only small positive current pulses were subthreshold, and therefore available for analysis, this enhancement is not very conspicuous. Statistical analysis, however, revealed a significant (P < 0.05) difference to the control values for current pulses between 0.1 and 0.3 nA.

View larger version (31K): [in this window] [in a new window]   Fig. 2. 5-HT effects on I-V relationship. A: typical membrane potential responses (top) of a PN neuron to subthreshold intracellular current applications (bottom; 1.0-0.4 nA in this case). Here, as in other figures, the stimulus artifacts were truncated. B: same neuron and stimulus protocol as in A after application of 5 µM 5-HT. The 5-HT-induced depolarization was compensated by intracellular current injection. Stimulation with 0.4 nA elicits a suprathreshold response (data not shown). C: I-V relationships of the neuron shown in A and B, calculated from the membrane potential responses measured 10 ms before stimulus offset (left) and difference between the I-V relationships under control conditions and serotonin for the entire population (right). I-V relationships are characterized by inward rectification in hyperpolarizing and depolarizing direction under both conditions. During 5-HT application (open circles) inward rectification in hyperpolarizing direction is reduced, but it is enhanced when depolarizing current pulses were applied. Summary data plot shows a polynomial regression of 1st order (solid line) plus 95% confidence interval (dotted line) fitted to the differences between control and test values (Vt  Vc) for each current amplitude used. 5-HT clearly diminishs inward rectification in response to negative current pulses and increases inward rectification in response to positive current pulses. D: summary data plots (similar procedure as in C) for all neurons tested with 5-HT alone (black lines) and together with receptor antagonists (gray lines). Cyanopindolol did not block the serotonergic modulation of inward rectification (left). Differences to control values were almost identical during 5-HT application (black lines) and coapplication of 5-HT and cyanopindolol. In contrast, cinanserin largely prevented the 5-HT-effect changes in inward rectification (right).

5-HT effects on membrane potential responses to suprathreshold stimuli

As previously described by Schwarz et al. (1997), depolarizing current pulses driving the SMP of PN neurons just above the firing threshold usually elicit one single action potential that is followed by a pronounced afterhyperpolarization (AHP; Figs. 3A and 5A). In response to higher current amplitudes, PN neurons fire a train of action potentials with a marked firing rate adaptation (Fig. 4, A and B). In the present sample, the firing threshold was 45.4 ± 3 mV and the rheobase (i.e., the minimal current necessary to reach firing threshold) was 0.32 ± 0.14 nA. After superfusion with 5 µM 5-HT, we observed no change in firing threshold (45.4 ± 3 vs. 45.6 ± 3.5 mV after 5-HT). In contrast, there was a decrease of the rheobase to 0.24 ± 0.14 nA (0.28 ± 0.16 nA; the mean contribution of changes in R_in to the decrease in rheobase was 53%). This decrease was significant (P < 0.001) for both the uncorrected and the corrected values. Population data are shown in Fig. 3, B and C.

View larger version (15K): [in this window] [in a new window]   Fig. 3. 5-HT effects on spike generation. A: superposition of membrane potential responses (top) to just suprathreshold current injections under control conditions (black) and serotonin (gray). Membrane potential was readjusted to the control value during 5-HT application by current injection. Amplitude of intracellular current application (bottom) needed to drive the membrane potential across the firing threshold (i.e., rheobase) was smaller under 5-HT (0.49 vs. 0.30 nA in this case). The firing threshold, defined as the begin of the steep rise of the spike, was similar in both conditions. Note that the negative peak of the afterhyperpolarization is reduced under 5-HT (for details see Fig. 5). B: scatter plot showing summary data for serotonergic modulation of the rheobase and the effects of 5-HT receptor antagonists. In most neurons, the rheobase was higher under control conditions compared with those measured during 5-HT application (white circles). In some cases, however, we observed no change or even an increase in rheobase by 5-HT. Similar observations were made during coapplication of 5-HT and cyanopindolol (filled squares), whereas coapplication of 5-HT and cinanserin (gray triangles) resulted in a attenuation of the 5-HT effect. The cinanserin effect was, however, not significant. C: scatter plot showing summary data for serotonergic modulation of the firing threshold. Values are randomly distributed across the diagonal. No significant difference was observed between control and test values.

View larger version (37K): [in this window] [in a new window]   Fig. 4. -HT effects on firing rate and adaptation. A: Membrane potential trajectories in response to various positive current pulses (bottom traces; 0.3-1.0 nA) under control conditions (left) and 5-HT (right). 5-HT-induced depolarization was compensated by current injection. During 5-HT application, initial (i.e., under control conditions) subthreshold stimulus amplitudes (0.3 and 0.4 nA) were sufficient to elicit spike. At initial suprathreshold stimulus amplitudes, the neuron responded with a higher number of spikes during 5-HT application compared with control conditions. Note that interspike intervals (ISI) within individual spike trains increased under both conditions. B: Duration of ISIs (from the neuron shown in A) plotted against their number of occurrence. Positive slope of the resulting curves indicate firing rate adaptation. Under both conditions (control, left; 5-HT, right) the neuron displayed marked adaptation especially at lower stimulus amplitudes. At higher stimulus amplitudes, marked firing rate adaptation was only observed during early parts of the spike train. This observation, however, holds true for both conditions. Thus 5-HT induced no change in firing pattern. C: instantaneous firing rate of the neuron shown in A plotted vs. the stimulus current amplitude (control, left; 5-HT, right). Current-frequency relationships were approximately linear for every ISI under both conditions; however, the instantaneous firing frequency was almost twice as high for the first ISIs during 5-HT application. D: normalized current-frequency plots for the entire population under control conditions and 5-HT (top left) and control conditions and 5-HT/cinanserin (top right). The highest instantaneous firing rate in each neuron under control conditions was set to 1. Polynomial regressions of the 1st order were fitted to the mean of the normalized values for any current amplitude (control, black lines; 5-HT or 5-HT/cinanserin, gray lines). To test whether factors other than changes in Rin contributed to the increased firing rate, the normalized instantaneous firing rats were plotted vs. current amplitudes corrected for the corresponding change in Rin. For clarity, only the 1st to 5th ISIs are plotted. The corresponding percent changes obtained for the 1st and 5th ISI are shown separately at the bottom. The regression lines for control and 5-HT differed significantly only for the 1st ISI. No such difference was observed during coapplication of 5-HT and cinanserin.

The typical firing behavior of PN cells is shown in Fig. 4. Once the depolarizing current application is strong enough to drive the SMP clearly above firing threshold, the cell responded with a train of action potentials, which displayed a substantial firing rate adaptation (Fig. 4A, left). To quantify the adaptation, we plotted the duration of the interspike interval (ISI) versus the spike number in the train for each trial (Fig. 4B). Typically, the slope of the resulting curves was positive at all stimulus amplitudes, indicating firing rate adaptation. However, they differed in steepness for different current amplitudes. The steepest curves were obtained with lower current amplitudes, and firing rate adaptation decreased when the amplitude current was increased. Furthermore, the firing rate of PN neurons typically increased with higher current amplitudes. The current-frequency relationship was fairly linear in the range of current amplitudes tested (Fig. 4C, left). According to the marked firing rate adaptation, the instantaneous firing rate was highest for the first ISI and reached almost constant levels at the end of the stimuli. Application of 5 µM 5-HT had clear effects on the firing behavior of PN neurons (Fig. 4A, right). First, previously subthreshold current amplitudes (0.3 and 0.4 nA in the example shown) now caused the neuron to fire spikes. Second, similar suprathreshold current amplitudes evoked higher numbers of spikes, i.e., a higher firing frequency under 5-HT. The increase in instantaneous firing frequency was typically highest during the first ISIs (Fig. 4C, right). However, 5-HT did not cause a qualitative change of the firing behavior. All neurons tested showed strong adaptation under both conditions. An increase in firing rate without change in adaptation raised the question of whether this increase was based solely on the increase in R_in. To test whether other factors also contribute to the firing rate increment by 5-HT, we compared the current amplitude needed to evoke five spikes under control conditions with the corrected current amplitude (i.e., corrected for the increase in R_in), eliciting the same number of spikes after 5-HT application. The mean current amplitude was 0.63 ± 0.15 nA under control conditions and 0.58 ± 0.20 nA (uncorrected, 0.49 ± 0.17 nA) under 5-HT. The Wilcoxon signed-ranks test yielded a significant difference at a probability level of P < 0.01 (n = 64). Therefore we conclude that the 5-HT-induced increase in firing rate cannot be explained purely by an increase in R_in (in this case the changes in R_in accounted for 42% of the 5-HT effect). This notion is further supported by the current-frequency plot for the entire population in Fig. 4D (top left). As stated above, the current-frequency curves for individual neurons were fairly linear (see example in Fig. 4C); we therefore used polynomial fits of first order for the population plot. These polynomial fits (fitted to the mean of the normalized values for any current amplitude) show the mean instantaneous firing rate for each ISI plotted versus the current amplitude under control conditions and the corrected current amplitude under 5-HT. The amount of change in instantaneous firing rate was estimated by subtraction of the corresponding fit curves. As shown in Fig. 4D (bottom left), the change in normalized firing rate was about 5% at any current amplitude for the first ISI (whereas it was about 43% if changes in R_in were neglected). These differences, as well as those obtained for the second ISI (about 3%, data not shown), were significant (P < 0.05). In contrast, as exemplified by the differences for the fifth ISI, the normalized firing rates of the following ISIs were not significantly different between both conditions. Thus in particular for the first two ISIs, the increase in R_in is not entirely sufficient to account for the increase in instantaneous firing rate.

PN neurons possess membrane conductances responsible for the generation of at least two kinds of AHPs: one of medium duration (~50 ms; mAHP) immediately following each spike, and one (sAHP) lasting several hundreds of milliseconds, which requires the generation of several spikes to be initiated (Schwarz et al. 1997). While the influence on the firing rate exerted by the sAHP is thought to be restricted to later parts of a spike train due to its late onset, the mAHP supposedly controls each ISI. The mAHP is therefore a likely candidate for the remaining 5-HT action on the firing rate of PN neurons not accounted for by changes in R_in. To test this possibility, we measured the amplitudes of mAHPs (defined as the difference between firing threshold and the negative peak of the mAHP) before and after 5-HT application. These measurements were done using depolarizing intracellular stimuli just strong enough to elicit a single spike and thus a single mAHP (Fig. 3A). The superposition of corresponding membrane potential trajectories recorded before (black trace) and after 5-HT application (gray trace) as shown in Fig. 5A clearly indicate a substantial reduction of the mAHP amplitude. The summary plots in Fig. 5, B and C, demonstrate that similar reductions were found in almost every cell tested. The statistical analysis revealed a significant difference at a probability level of P < 0.001 (for both uncorrected and corrected values). In the present sample, the mean mAHP amplitude was 14.9 ± 2.0 mV under control conditions and 12.3 ± 2.4 mV (10.4 ± 2.6 mV) under 5-HT. From these results, it may be concluded that a joint increase in R_in and decrease in mAHP amplitude accounts for the 5-HT-induced enhancement of firing rate observed in PN neurons.

View larger version (18K): [in this window] [in a new window]   Fig. 5. 5-HT reduces medium-duration afterhyperpolarization (mAHP) amplitude. A: superposition of an action potential and the subsequent mAHP before (black trace) and during 5-HT application (gray trace). Note that the action potentials have been truncated as indicated by the dotted line. Recordings were made with stimulus amplitudes just high enough to elicit 1 spike. 5-HT reduced the negative peak potential of the mAHP by 2.6 mV in this case. B: scatter plot showing summary data for serotonergic modulation of the mAHP amplitude and the effectiveness of 5-HT receptor antagonists. In the vast majority of neurons, 5-HT reduced the mAHP amplitude by several millivolts. A few neurons display no change or a small increase in mAHP amplitude. In 1 example, the increase in amplitude was as large as 6.6 mV. Coapplication of 5-HT and cyanopindolol (filled triangles) revealed values similar to those obtained during 5-HT application. The mAHP amplitudes measured during coapplication of 5-Ht and cinanserin were evenly distributed across the diagonal and not significantly different from the control values. C: scatter plot showing the same data as in B but corrected for 5-HT-induced change in Rin. In all but 3 cases there was a true reduction in the conductance underlying the mAHP.

Pharmacology of 5-HT-induced modulation of the membrane properties

To fully understand the serotonergic modulation of PN neurons, it is necessary to identify the receptor types involved. In this study, we used cinanserin and cyanopindolol, two broad antagonists of 5-HT₂ and 5-HT₁ receptors, respectively, to block 5-HT action on PN neurons. Application of 5 µM 5-HT in the presence of 10 µM cyanopindolol still substantially depolarized the SMP. As shown in Fig. 1C, the depolarization was strong enough to evoke spontaneous firing in several cases. In contrast, coapplication of 10 µM cinanserin largely prevented a depolarization of the SMP (Fig. 1E). Summary data for the entire population are given in Fig. 1F. As noted above, the 5-HT-induced depolarization of the SMP was accompanied by an increase in R_in (Fig. 1, A, B, and D). A similar increase occurred during application of 5-HT plus cyanopindolol (Fig. 1, C and G), whereas no such change in R_in was detectable during coapplication of 5-HT and cinanserin (Fig. 1, E and G). Mean values and statistical analysis are given in Table 1. Similar results were obtained for the effects of 5-HT receptor antagonists on the I-V relationships of PN neurons. As shown in Fig. 2D (left), cyanopindolol was not able to prevent 5-HT-induced weakening of inward rectification in hyperpolarizing direction and enhancement of inward rectification in depolarizing direction. The plot shows the deviations of the I-V curves obtained from recordings under 5-HT and 5-HT plus cyanopindolol from those under control conditions. No significant (P > 0.5) difference between the two test populations (n = 6) was observed. In contrast, cinanserin (n = 6) largely reduced the 5-HT effects on the I-V curves of PN neurons (Fig. 2D, right). The remaining difference between control condition and 5-HT/cinanserin coapplication was not significant (P > 0.1). Next, we tested the effects of cyanopindolol and cinanserin on the serotonergic modulation of the mAHP amplitude (Fig. 5, B and C, Table 1). Cyanopindolol was not able to significantly prevent 5-HT-induced reduction of mAHPs. In contrast, blocking 5-HT₂ receptors with cinanserin was sufficient to obtain mAHP amplitudes, which were not significantly different from the control values. Finally, probing the pharmacology of 5-HT action on the rheobase (Fig. 3B and Table 1) revealed a tendency in favor of a 5-HT₂ receptor-mediated decrease in rheobase. This possibility was, however, not substantiated by statistical significance. In summary, 5-HT-induced changes in SMP, R_in, mAHP amplitude, inward rectification, and rheobase were all blocked by cinanserin but not by cyanopindolol. Therefore the serotonergic modulation of these intrinsic membrane properties can be explained by activation of 5-HT₂ receptors.

5-HT effects on PSPs

5-HT receptors are located at the soma, along dendrites, along axons, and at the presynaptic terminals in different brain regions (Cornea-Hébert et al. 1999; DeFilipe et al. 2001; Hamada et al. 1998; Sari et al. 1999). Thus 5-HT may also modulate the synaptic transmission on the neurons within these regions. Consequently, we asked whether this is true for PN neurons. In the rat, PN neurons receive massive synaptic input from the entire cerebral cortex (Legg et al. 1989) and numerous subcortical structures (Mihailoff et al. 1989). We have recently described the characteristics of the PSPs in rat PN neurons in vitro (Möck et al. 1997).

In this study we investigated how 5-HT acts on PSPs in PN neurons that were elicited by applying small current pulses (5-250 µA, 0.1 ms) at sites within the cerebral peduncle, which include the cerebropontine fibers. Typically, these stimuli evoked excitatory PSPs with short latencies (0.8-5.0 ms), a fast rise, and a slower decay (Fig. 6, A and B). To quantitatively assess 5-HT effects, we determined several characteristic parameters of the PSPs. The magnitude is well characterized by the peak amplitude, their time course by the maximal slope (for the rising phase), and the time required to decay from the peak to half-maximal amplitude (t_{1/2 PSP}). Finally, the time integral was calculated as measure for magnitude and total length of the PSPs.

View larger version (39K): [in this window] [in a new window]   Fig. 6. -HT effects on synaptically evoked postsynaptic potentials. A: electrical stimulation (15-30 µA in 5-µA steps, 0.1-ms duration) of afferent fibers within the cerebral peduncle evoked postsynaptic potentials (PSPs) in pontine neurons, which increased in amplitude with increased stimulus intensities. The presented PSPs are, as in other figures, averages of 10 individual PSPs subsequently recorded at each stimulus intensity. In all recordings the membrane potential was, if necessary, readjusted to the control value. During application of 5 µM 5-HT, the magnitude of these PSPs was substantially reduced at any stimulus intensity. After removing 5-HT from the bath, the PSPs recovered within 30 min. In this example, 30-µA stimulation was strong enough to elicit a spike. Application of 5-HT plus cyanopindolol after the recovery from 5-HT did not result in a similar reduction of the PSPs. Their magnitude was similar to the corresponding control and recovery recordings. B: average PSPs recorded in a different PN neuron but following a similar protocol as in A (except for maximal stimulus intensity). As in A, 5-HT strongly and reversibly reduced the magnitude of the PSPs at any stimulus intensity. A similar reduction in magnitude was observed during coapplication of 5-HT and cinanserin. C: quantified data showing amplitude, maximal slope, and time integral of the PSPs in A as means ± SD of 10 individual trials plotted as function of the stimulus intensity. All PSP parameters were strongly reduced by 5-HT (open squares) to <30% of the control at any stimulus intensity. During coapplication of 5-HT and cyanopindolol (light gray triangles), the PSP parameters parallel those recorded after recovery (dark gray triangles). D: quantified data calculated from the data in B plotted as in C. The values obtained during coapplication of 5-HT and cinanserin (light gray triangles) almost perfectly parallel those measured under 5-HT (open squares).

Our routine paradigm was to determine the minimal stimulus intensity, i.e., the smallest current amplitude that evoked detectable PSPs (>3 times the SD of the noise), for each neuron under control conditions, followed by application of successively larger stimulus amplitudes (until spikes were generated). The same stimulus intensities were then used to test synaptic transmission during 5-HT application. To allow comparison between control and test experiments, the 5-HT-induced depolarization of the SMP was compensated by constant current injection. Figure 6 shows two representative examples of PSP in PN neurons and their modulation by 5-HT. Under control conditions, the minimal stimulus intensities evoked PSPs with mean amplitudes of 1.71 ± 1.66 mV, mean maximal slope of 1.25 ± 1.16 mV/ms, mean t_{1/2 PSP} of 6.15 ± 3.69 ms, and mean integral of 6.12 ± 3.57 V · s. Elevation of stimulus intensities resulted in a nonuniform increase of all PSP parameters (Fig. 6, C and D). We have proposed that this reflects a successive recruitment of additional fibers or fiber bundles (Möck et al. 1997). Application of 5 µM 5-HT substantially reduced the magnitude of the PSPs at any given stimulus intensity (Fig. 6). At threshold stimulus intensities there was no PSP detectable in 41% of the cases. The mean PSP amplitude during 5-HT application, averaged over all cells tested (n = 44) and all stimulus intensities, was 46.2 ± 23.4% (41.9 ± 25.9%) of the corresponding control values. Similar reductions were found for maximal slope and time integral: 45.7 ± 23.7% (42.3 ± 26.4%) and 61.4 ± 28.4% (55.6 ± 28.1%), respectively. On the other hand, 5-HT had virtually no effect on the decaying phase of the PSPs (mean t_{1/2 PSP}: 97.1 ± 41.9% and 87.9 ± 41.3%). In the following, the analysis will therefore be limited to amplitude, maximal slope, and time integral. After removal of 5-HT from the medium, the PSPs recovered usually within 30-60 min (Fig. 6). Because, as shown in a previous paragraph, 5-HT depolarizes PN neurons, it has to be ruled out that the reduction of PSP amplitude is based on depolarization at the synaptic sites in dendrites with a consecutive reduction of driving force. This might be the case if the compensatory current did not reach the synaptic site due to a possible space-clamp error. Three arguments will be presented in detail in the following paragraphs that indicate that this is not the case. First, voltage-sensitive changes of the form of PSPs could be readily modulated under control and 5-HT conditions using somatic current injections. This fact demonstrated that current injected at the soma indeed reaches postsynaptic sites. Second, the 5-HT effect on PSPs was observed under selective pharmacological blockade of the 5-HT-induced depolarization. Third, 5-HT showed frequency-dependent effects on paired-pulse facilitation, supporting the notion of a presynaptic 5-HT effect.

We have recently shown in detail that the amplitude of excitatory PSPs is independent of the SMP over a wide range of SMPs (Möck et al. 1997). To test whether this independence is preserved during 5-HT application, we recorded medium-sized PSPs (5-10 mV at resting SMP under control conditions) at various SMPs. The representative example shown in Fig. 7A clearly shows the typical results found in PN neurons under control conditions: the amplitude remained fairly constant across the entire range of SMPs tested. Note however, that the PSP decay is strongly influenced by depolarizing current injection at the soma. As described elsewhere (Möck et al. 1997), this current injection removes the Mg²⁺ block from N-methyl-D-aspartate (NMDA) receptor-gated channels and possibly actives the persistent sodium conductance. Application of 5 µM 5-HT reduced the PSP amplitude as expected (Fig. 7B) but did not alter its independence from the SMP (Fig. 7C).

View larger version (29K): [in this window] [in a new window]   Fig. 7. 5-HT effects voltage dependence of postsynaptic potentials. A: average PSP (n = 10) recorded in a PN neuron at different SMPs under control conditions. They were evoked by electrical stimulation (120 µA, 0.1-ms duration) within the cerebral peduncle. PSP amplitude was almost independent of SMP. B: same neuron, stimulation, and recording protocol as in A, tested for the voltage dependence of the PSP during 5-HT application. Expectedly, the magnitude of the PSP was largely reduced by 5-HT, but it displayed an unchanged voltage dependence. C: quantified data showing amplitude, maximal slope, and time integral of the PSPs in A and B (conventions as in Fig. 6). 5-HT (white squares) reduced amplitude and maximal slope to about 30% of the control value (black circles) at any given SMP. The integral was reduced to a lesser extent at more negative SMPs. Its stronger reduction at less negative potentials is explained by the lack of activation of voltage-dependent boosting mechanisms due to the small PSP amplitude.

Does this reduction of PSPs mean that synaptic transmission on PN neurons is less effective in the presence of 5-HT, resulting in a decreased cerebrocerebellar information transfer? To answer this question, it has to be taken into account that 5-HT, while diminishing the PSP amplitude, also depolarized the SMP by several millivolts. Thus the crucial factor for pontine signal transmission under 5-HT is the effective peak depolarization reached at the somatic zone of spike generation under both conditions. We, therefore determined the "total" depolarization as the sum of PSP amplitude and the net steady-state depolarization under 5-HT (this calculation was done because the steady-state potential was compensated before recording PSPs) and compared it with the control PSP. The mean difference in peak depolarization was found to vary from case to case, but on average was negligible (0.05 ± 5.5 mV, n = 38). Since 5-HT did not alter the firing threshold in PN neurons, the conclusion would be that, on average, single PSPs are equally effective in driving PN neurons under both conditions. In line with this conclusion, we found in four cases in which our stimulus protocol was applied without compensating the 5-HT-induced depolarization of the SMP, spike generation under control and test conditions were at similar stimulus strengths.

In many neurons, synaptic transmission is not invariant during repetitive stimulation, but rather is influenced by previous events. The synaptic transmission on PN neurons is known to show robust paired-pulse facilitation (PPF) at interstimulus intervals between 10-200 ms (Möck et al. 1997). Two questions motivated us to study whether 5-HT also modulates PPF in the PN. First, alteration of PPF would indicate a presynaptic site of 5-HT action on synaptic transmission, whereas an unaltered PPF would argue for a postsynaptic site. Second, modulation of PPF in either direction would have strong functional implications, since, as shown above, the net effectiveness of a single PSP was unchanged by 5-HT. To this end, we applied pairs of electrical stimuli with delays between 10 and 1,000 ms at sites in the cerebral peduncle under control conditions and during 5-HT application. The amount of facilitation was determined by comparing the amplitude of the two PSPs evoked by the two stimuli. At the smallest interstimulus intervals (10 ms, or in some, 20 ms) the resulting PSPs overlapped in time. To obtain the isolated second PSP in these cases, we subtracted an average control PSP. Typical results for PPF in PN neurons are presented in Fig. 8. In this example, we observed weak PPF for long interstimulus intervals. Typically, PPF increased with shorter intervals between the pulse-pair length (Fig. 8, A and B) and was seen in all cases examined. In the example shown in Fig. 8B, the strongest enhancement of synaptic transmission occurred at a delay of 50 ms. Application of 5-HT expectedly reduced the amplitudes of both PSPs elicited by the paired stimuli (Fig. 8, C and D). As in the control recording, we observed a clear facilitation of the second PSP for shorter interstimulus intervals, whereas it was weak with longer delays. Quantification of the amount of facilitation (as percentage of the control amplitude) revealed that in the example shown, the PPF under 5-HT was about the same for longer interstimulus intervals as the one observed under control conditions (Fig. 8E). However, 5-HT enhanced the facilitation of the second PSP if the interval between pulses was short (10 and 20 ms). The summary plot in Fig. 8F demonstrates that 5-HT exerted similar effects on all cells tested (n = 6). Finally, we statistically analyzed the differences in the amount of facilitation for each class of interstimulus intervals over the entire population. It turned out that 5-HT significantly enhanced PPF at the smallest intervals (10 and 20 ms; P < 0.05), while the difference at larger intervals did not reach significance (P > 0.1). What is the reason for this differential, frequency-dependent modulation of PPF? Before attributing a presynaptic action to the observed 5-HT effect, a possible saturation of PSP amplitudes at shorter intervals under control conditions (e.g., due to glutamate receptor saturation) has to be ruled out. If the enhanced PPF under 5-HT for smaller intervals were due to a ceiling effect, it could be expected that the ratio between PPF under 5-HT and PPF under control conditions (PPF_5-HT/PPF_control) would increase monotonically with shorter interstimulus intervals. Investigating the mean PPF_5-HT/PPF_control across interstimulus intervals (Fig. 8G) revealed a sigmoidal relationship, which indicates that PPF is subject to saturation under both conditions. A second argument against a ceiling effect of PPF at the shortest interstimulus interval is provided by the observation in two cells that PPF under 5-HT is similar at different stimulus intensities (202 ± 13.6% vs. 201 ± 19.9% at 3 times higher stimulus intensity than that which evoked a similar PSP amplitude after a single pulse, observed for the lower intensity under control conditions; the interstimulus intervals of 10 ms was investigated). Having thus ruled out a postsynaptic saturation of PSP amplitudes, a possible presynaptic explanation is based on the known fact that the releasable transmitter pool is partly depleted at central synapses for a few 10s of milliseconds after transmitter release, i.e., the release sites are refractory for a short period in which the amplitude of a second PSP is negatively correlated to the amplitude of a preceding one (for references and discussion see Thomson 2000). Such a mechanism might convincingly explain two of the most salient results from our PPF experiments: 1) under control conditions the amount of PPF drops at very short interstimulus intervals possibly because the immediately releasable transmitter pool is still not fully recovered at the time the second pulse enters the terminals and 2) due to the inverse correlation of the amplitudes of consecutive PSPs during refractoriness, the 5-HT-induced reduction of the first PSP, reflecting a minor depletion of the releasable transmitter pool, may result in a stronger facilitation at very short interstimulus intervals as compared with control conditions. In summary, at short interstimulus intervals, facilitation is partly counterbalanced by a depletion-based depression. 5-HT might reduce depletion and therefore emphasize facilitation.

View larger version (29K): [in this window] [in a new window]   Fig. 8. Serotonergic modulation of paired-pulse facilitation (PPF). A: averaged postsynaptic response (n = 10) recorded in a PN neuron after paired stimulation within the cerebral peduncle (20 µA, 0.1-ms duration each, 20-ms interstimulus interval) under control conditions. The conditioning pulse strongly facilitated the response to the 2nd one. B: averaged (n = 10) control and isolated 2nd PSPs (top) and difference between control and isolated 2nd PSP (bottom) for all interstimulus intervals used. Recordings were from the same neuron in A. Isolated 2nd PSPs were obtained by subtracting the control PSP from the responses to paired stimuli. A substantial facilitation was observed for shorter interstimulus intervals. C: averaged postsynaptic response (n = 10) recorded during 5-HT application in the same neuron and using the same protocol as in A. The 5-HT-induced depolarization was compensated by current injection. As expected, the amplitude of the individual PSPs was reduced as compared with the control, but the pairing paradigm still elicited a strong facilitation of the second PSP. D: same neuron and conventions as in B during 5-HT application. Compared with the control, the facilitation of the second pulse was pronounced for short intervals. E and F: quantification of paired-pulse facilitation for the example shown in A-D (E) and for the entire sample (F). The amount of facilitation is expressed as percentage of the amplitude of the control PSP. As compared with control conditions, the maximum facilitation is increased and shifted to higher frequencies. The shift of facilitation to higher frequencies is reflected in the population data (F). The box-and-whisker plot shows medians by horizontal bars, 25-75% quartiles by boxes, and the range by vertical bars. Statistical analysis revealed no significant difference between both conditions for interstimulus intervals between 1,000 and 50 ms, but, as indicated by an asterisk, the amount of paired-pulse facilitation is significantly enhanced by 5-HT for interstimulus intervals of 20 and 10 ms. G: ratio between PPF during 5-HT application and during control conditions. Mean ratios ± SE are plotted vs. interstimulus intervals. The ratios are close to 1 for long interstimulus intervals (>100 ms) but increase for interstimulus intervals between 100-10 ms. Note however, that this increase is not monotonic. For those interstimulus intervals resulting in a significant increase in PPF during 5-HT application (20 and 10 ms; see G), the ratios are almost similar.

Pharmacology of 5-HT-induced modulation of PSPs

Modulation of PPF indicates a presynaptic 5-HT action within the PN. Presynaptic action of 5-HT has been shown to be mediated by 5-HT₁ receptors in other brain structures (Hwang and Dun 1999; Li and Bayliss 1998; Muramatsu et al. 1998; Schmitz et al. 1995a,b, 1998a,b; Wang et al. 1999). However, there is also evidence that 5-HT₂ receptors are located on nerve terminals (Jakab and Goldmann-Rakic 1998). To decide between these two possibilities, we coapplied 5-HT with cinanserin or cyanopindolol while electrically stimulating at sites within the cerebral peduncle. Representative examples of these experiments and their quantitative analysis are given in Fig. 6. As has been described before, 5-HT strongly but reversibly reduced the amplitude, maximal slope, and time integral in both cases. During blockade of 5-HT₁ receptors with 10 µM cyanopindolol, 5-HT did not exert similar effects on the PSP (Fig. 6, A and C). All of the PSP parameters, modulated by 5-HT application before, now paralleled the values determined from recordings after recovery from 5-HT action (Fig. 6C). As cyanopindolol is specific for 5-HT₁ receptors, the SMP depolarization, which is based on 5-HT₂ action, was present in these experiments and was compensated for by current injection into the soma. The successful blockade of 5-HT-induced decrement of PSPs under these conditions proves that this effect is due to genuine 5-HT action on synaptic transmission and cannot be explained by a reduced driving force due to lack of space-clamp control at the postsynaptic (dendritic) site. In contrast, blocking 5-HT₂ receptors with 20 µM cinanserin was ineffective in preventing serotonergic modulation of PSPs (Fig. 6, B and D). The PSPs were clearly reduced in magnitude compared with the control and recovery recordings, but similar to those during 5-HT application alone at any given stimulus current amplitude. Accordingly, the quantified values for amplitude, maximal slope, and time integral closely paralleled those obtained during 5-HT application (Fig. 6D). The observation of diminished PSPs in the presence of cinanserin provides further evidence against a role of depolarization in the 5-HT-induced reduction of PSPs. We conclude that 5-HT reduces synaptic transmission on PN neurons by acting on 5-HT₁ receptors. Population data are given in Table 2.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In this study, we demonstrate that information processing and information transfer in the PN is strongly modulated by 5-HT. First, activation of 5-HT₂ receptors increases the excitability of these neurons mainly based on depolarization of the somatic membrane potential, increases the apparent input resistance, increases inward rectification in depolarizing direction, and decreases the mAHP amplitude. Second, mediated by 5-HT₁ receptors, 5-HT decreases postsynaptic potentials in PN neurons. Third, the short-term facilitation of synaptic inputs on PN neurons is selectively enhanced for high-frequency inputs. In the following we will discuss the possible mechanisms of 5-HT action and argue that the PN may function as a high-pass filter gate during periods of serotonergic modulation.

Location and mechanisms of 5-HT action

5-HT has attracted a lot of interest since it has been assumed to play a crucial role in the regulation of behavioral states. Accordingly, a considerable amount of work has been done in many brain regions to unravel the molecular and ionic mechanisms underlying 5-HT action. The diversity of receptor subtypes, second messenger systems, and ionic conductances presently known to be involved is enormous, and yet, the net effects on individual neurons described so far may be assigned to only a few pharmacological categories. On the one hand, 5-HT either increases or decreases the excitability. On the other hand, it reduces or enhances synaptic transmission. Finally, it can change the firing pattern.

Increased excitability has been described in neocortical (Araneda and Andrade 1991; Davies et al. 1987; McCormick and Williamson 1989; Sheldon and Aghajanian 1990) and hippocampal (Andrade and Chaput 1991; Andrade and Nicoll 1987; Bijak and Misgeld 1997; Colino and Halliwell 1987; Gasparini and DiFrancesco 1999; Siarey et al. 1995) pyramidal and interneurons, thalamic neurons (McCormick and Pape 1990; McCormick and Wang 1991; Pape and McCormick 1989), inferior olive neurons (Placantonakis et al. 2000), and cranial neurons (Hsiao et al. 1997, 1998; Larkman et al. 1989), as well as spinal motoneurons (Berger and Takahashi 1990; Kjaerulff and Kiehn 2001). Commonly this increase is, as in our case, accompanied by depolarization. In the aforementioned studies, several mechanisms have been discussed to underlie depolarization: reduction of potassium conductances like the leak K⁺ conductance, inward rectifying K⁺ conductances, and I_M or enhancement of I_H and I_T. In PN neurons, there is no convincing evidence for the presence of I_M and I_T, and only a small portion of cells may possess I_H (Schwarz et al. 1997). Therefore these conductances most probably do not contribute substantially to the depolarization described here. A depolarization based on the reduction of K⁺ conductances would also be in accordance with the observed increase in R_in. Although we cannot rule out a modulation of I_Leak, the most likely candidates in PN neurons are the fast inwardly rectifying I_Kir and probably an unidentified outward rectifying K⁺ conductance. Both were previously described in PN neurons (Schwarz et al. 1997) and were shown in the present study to be affected by 5-HT. The latter one may also play an additional role in increasing excitability, since reduction of outward rectification in depolarizing direction could explain the decrease in rheobase observed even after correction for the increase in R_in. Finally, we have described a 5-HT-induced reduction in mAHP amplitude in PN neurons, which also adds to an enhanced excitability. Most studies on serotonergic modulation of AHPs report a reduction of the long-lasting sAHP due to a reduction of the Ca²⁺-dependent K⁺ current I_AHP (Andrade and Chaput 1991; Andrade and Nicoll 1987; Colino and Halliwell 1987; McCormick and Williamson 1989). However, Inoue and et al. (1999) showed that serotonergic action on a Ca²⁺-dependent K⁺ current could also reduce the amplitude of mAHPs. The mAHPs in PN neurons were shown to be composed of a Ca²⁺-dependent and a Ca²⁺-independent component (Schwarz et al. 1997). Therefore the mechanism described by Inoue et al. (1999) might account for the mAHP reduction in PN neurons.

The finding that cinanserin blocked all of the 5-HT effects on intrinsic membrane properties in PN neurons led us to assume that 5-HT₂ receptors mediate these effects. This conclusion is in line with many previous reports. In different cell types, activation of 5-HT₂ receptors was shown to be responsible for a reduction in K⁺ conductances, leading to increased R_in and depolarization and finally resulting in more excitable cells (Araneda and Andrade 1991; Davies et al. 1987; Hsiao et al. 1997; Newberry et al. 1999; Sheldon and Aghajanian 1990). In contrast, a decrease in excitability, caused by enhancement of conductance and accompanied by hyperpolarization and decrease in R_in, was often described to be linked to 5-HT₁ receptor activation (Andrade and Nicoll 1987; Araneda and Andrade 1991; Davies et al. 1987; Newberry et al. 1999; Okuhara and Beck 1994; Schmitz et al. 1995).

In addition to the serotonergic modulation of intrinsic properties, we observed a substantial reduction of PSPs in PN neurons. All of the PSPs investigated in this study were evoked by electrical stimulation within the cerebral peduncle and were of depolarizing nature. Although we have not routinely tried to block inhibitory PSPs (IPSPs) throughout our experiments, it is not likely that the PSP reduction in this study was generally due to enhancement of IPSPs contaminating excitatory PSPs (EPSPs) for the following reasons. First, evoking GABAergic IPSPs in PN neurons usually requires an extensive search for appropriate stimulation sites within the pontine tegmentum or the cerebral peduncle (Möck et al. 1997). Second, only in 4% of the experiments in the aforementioned study did a blockade of -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors unmask underlying IPSPs. We therefore assume that 5-HT suppresses EPSPs in PN neurons.

There are, in principle, two possible sites of action for 5-HT to reduce synaptic transmission: modulation of the presynaptic transmitter release or modulation of postsynaptic transmitter receptor. The latter case has been reported for spinal dorsal horn neurons (Lopez-Garcia 1998; Murase et al. 1990), but many other studies point to a presynaptic mechanism (Koyama et al. 1999; Li and Bayliss 1998; Schmitz et al. 1995a,b, 1998a, b; Zhou and Hablitz 1999). The latter studies provide strong support for a presynaptic mechanism by their finding that 5-HT did not affect postsynaptic currents evoked by exogenous glutamate application, reduce the frequency but not the amplitude of spontaneous miniature excitatory postsynaptic currents, or modulate PPF. The enhancement of PPF in our study, therefore, may be taken as indication of a presynaptic site of action. Further support for this notion is provided by the observation that the antagonist blocking 5-HT effects on synaptic transmission had no affect on the resting membrane properties of the postsynaptic neuron and vice versa. While cinanserin was potently blocking the 5-HT-induced increase in excitability, it was completely ineffective in preventing the reduction of PSPs. Therefore the presynaptic suppression of synaptic transmission in PN neurons is mediated by 5-HT receptors other than 5-HT₂. The evidence presented in this paper strongly favors the involvement of 5-HT₁ receptors, a finding that is in line with observations made in other brain regions. Reduction of excitatory synaptic transmission has been shown to be 5-HT₁ receptor-mediated in entorhinal cortex (Schmitz et al. 1998a,b), hippocampus (Schmitz et al. 1995a,b), amygdala (Wang et al. 1999), nucleus accumbens (Muramatsu et al. 1998), and brain stem (Hwang and Dun 1999; Li and Bayliss 1998).

Functional considerations

In this paper, we described opposing effects of 5-HT in the PN, namely an increase in excitability of the membrane and a reduction in excitatory synaptic transmission. As detailed in RESULTS, the amount of 5-HT-induced depolarization and single PSP amplitude reduction almost perfectly balance each other out at stimulus intensities high enough to drive the cell close to the firing threshold. On this basis it could be assumed that the net effect of serotonergic modulation in the PN is to increase the firing rate by reducing mAHPs and increasing R_in. An additional function of serotonergic modulation is, however, suggested by its effect on synaptic facilitation. Under control conditions in vitro, there is already a robust frequency-dependent facilitation for synaptic inputs arriving at intervals <200 ms. In this situation, the strongest facilitation was observed for intervals of 50 and 20 ms, and it dropped substantially for shorter ones (Möck et al. 1997). Thus synapses impinging on PN neurons possess a mechanism to gate inputs to these cells according to their temporal structure even under control conditions in vitro. Our finding of selective enhancement of synaptic facilitation for high-frequency input by 5-HT indicates a strengthening of the high-pass filter characteristics of the synaptic transmission onto PN neurons. Therefore during periods of 5-HT release, the effectiveness of high-frequency inputs to drive PN neurons may be increased compared with low-frequency inputs. Interestingly, a similar modulation of gating properties by acetylcholine and noradrenalin was recently described for corticothalamic synapses (Castro-Alamancos and Calcagnotto 2001).

To understand the functional significance of serotonergic modulation of PN neurons, one must consider the activity pattern within the sources of their serotonergic afferents. The PN receive serotonergic input from pontine as well as from medullary raphe nuclei (Mihailoff et al. 1989). The neurons in both groups are most active during periods of alertness and active behavior (Veasey et al. 1995, 1997). They differ, however, in their responses during active motor performance. Nearly all neurons located within the medullary raphe nuclei, which also provide input to the inferior olive (Bishop and Ho 1986; Compoint and Buisseret-Delmas 1988), display their maximal firing rates during motor activities (Veasey et al. 1995). In contrast, only about 25% of pontine raphe neurons increase their firing rates in the same situations, but most of them are activated by somatosensory stimuli to the face, head, and neck (Fornal et al. 1996; Veasey et al. 1997). Since many neocortical sensory and motor areas project to the PN (Legg et al. 1989; Mihailoff et al. 1989), it may be speculated that certain pontine neurons as well as the corresponding raphe neurons are activated in concert during specific sensory-motor integration tasks to facilitate the transmission of relevant information. In contrast, the serotonergic raphe neurons are virtually inactive during rapid eye movement (REM) sleep and slightly increase their firing rate during slow-wave sleep (Veasey et al. 1995, 1997). This low or even absent serotonergic activity may impair information transfer through the PN and thalamus (Pape and McCormick 1989) and therefore functionally uncouple the cerebrocerebellar loop and help to isolate the cerebral cortex during sleep.