Electrophysiological Properties of Rat Pontine Nuclei Neurons In Vitro. I. Membrane Potentials and Firing Patterns
Cornelius Schwarz,
Martin Möck, and
Peter Thier
Sektion für Visuelle Sensomotorik, Neurologische Universitätsklinik Tübingen, 72076 Tubingen, Germany
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ABSTRACT |
Schwarz, Cornelius, Martin Möck, and Peter Thier. Electrophysiological properties of rat pontine nuclei neurons in vitro. I. Membrane potentials and firing patterns. J. Neurophysiol. 78: 3323-3337, 1997. We used a new slice preparation of rat brain stem to establish the basic membrane properties of neurons in the pontine nuclei (PN). Using standard intracellular recordings, we found that pontine cells displayed a resting membrane potential of
63 ± 6 mV (mean ± SD), an input resistance of 53 ± 21 M
, a membrane time constant of 5.3 ± 2.4 ms and were not spontaneously active. The current-voltage relationship of most of the PN neurons showed the characteristics of inward rectification in both depolarizing and hyperpolarizing directions. A prominent feature of the firing of pontine neurons was a marked firing rate adaptation, which eventually caused the cells to cease firing. Several types of membrane conductances possibly contribute to this feature. For one, a medium and a slow type of afterhyperpolarization (AHP) control the pattern of firing. The medium AHP was partly susceptible to blockade of calcium influx, whereas it was abolished completely by blockade of potassium channels with tetraethylammonium, indicating that it is based on at least two conductances: a calcium-dependent and a calcium-independent one. The slow AHP was carried by potassium ions and could be blocked effectively by preventing calcium influx into the cell. It was present after single spikes but was strongest after a high-frequency spike train. Calcium entry into the cell was mediated by high-threshold calcium channels that were detected by the generation of calcium spikes under blockade of potassium channels. Furthermore, the early phase of the firing rate adaptation was shown to be related to the time course of a slow, tetrodotoxin (TTX)-sensitive, persistent sodium potential, which was activated already in the subthreshold range of membrane potentials. This potential was time dependent and imposed as a depolarizing "hump" with a maximum occurring in most cases between 50 and 100 ms after stimulus onset. In the suprathreshold range, it generated plateau potentials following fast spikes, if potassium channels were blocked. After the complete adaptation of the firing rate, PN neurons were observed to display irregular fluctuations of the membrane potential, which sometimes reached firing threshold thereby eliciting an irregular low-frequency spike train. As these fluctuations could be blocked with TTX, they probably are based on the persistent sodium currents. The opposing drive in hyperpolarizing direction may be provided by strong outward currents that generated a marked outward rectification in the current-voltage relationship under TTX. In conclusion, PN neurons show complex membrane properties that are reminiscent in many ways to cerebrocortical "regular firing" neurons.
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INTRODUCTION |
The projection from layer V of widespread regions of the cerebral cortex to the cerebellum via the pontine nuclei (PN) is one of the most massive within the brain, suggesting an important functional role. However, the functional properties of this projection system as a whole and in particular of the pontine relay are only poorly understood. In studies using intracellular and extracellular recordings from pontine neurons in vivo, a predominance of excitatory influences of cortical fibers on pontine neurons has been demonstrated (Allen et al. 1975
a,b 1977; Sasaki et al. 1970
; Tsukahara and Bando 1970
). In view of the massive number of cerebro-pontine fibers and its exclusive projection to the cerebellum, it is not surprising that the prevailing hypothesis of pontine information processing has considered the PN as a faithful relay of cerebral information to the cerebellum (see review in Allen and Tsukahara 1974
). However, more recent reports studying the pattern of connections of the cerebro-ponto-cerebellar pathway as well as other afferent systems of the PN have suggested that the role of the PN is much more integrative than hitherto has been thought. Morphological studies have revealed that both cerebro-pontine (Bjaalie 1989
; Brodal 1978
; Mihailoff et al. 1985
; Schwarz and Thier 1995
; Wiesendanger and Wiesendanger 1982
) and ponto-cerebellar fibers (Mihailoff 1983
; Mihailoff et al. 1981
; Nikundiwe et al. 1994
) show a compartmentalized, divergent pattern. Continuous cerebrocortical maps such as those found in primary sensory areas are not preserved within the PN. Rather, neighboring pontine compartments, as defined by cortical afferents, may receive information from distant sites of a single cerebrocortical area or even from regions of the cerebral cortex far apart. We previously have suggested that this reorganization of topological relationships may be the basis of the transformation of the continuous maps of the primary somatosensory cortex into the fractured somatotopy as present in the cerebellar granular layer (Schwarz and Thier 1995
). This representation is characterized by the fact that neighboring points on the map encode distant localizations in sensory space (Shambes et al. 1978a
,b
). Thus the compartmentalized organization of the PN might be used to allow for communication between different kinds of cortical information before it enters the cerebellum. Such communication could be mediated by the feedback projection from the cerebellar nuclei (Brodal et al. 1972
; Martin 1973
; Schwarz and Schmitz 1997
; Watt and Mihailoff 1983
), projections from subcortical sources, some of which contain inhibitory fibers (Border et al. 1986
; Mihailoff et al. 1989
), terminals from GABAergic local circuit neurons (Thier and Koehler 1987
), or axon collaterals of pontine projection neurons (Mihailoff 1978
; Möck et al. 1997
; Sasaki et al. 1970
). All these projections possibly influence several compartments. Furthermore, modulatory influences may be exerted by monoaminergic or acetylcholinergic fibers, which diffusely project to the PN (Aas et al. 1990
; Mihailoff et al. 1989
). In summary, the anatomic organization of the PN suggests that they serve as a computational device rather than a simple relay handing information over without significant processing.
To understand how pontine neurons process afferent information, their integrative properties, i.e., the synaptic mechanisms and membrane conductances involved, have to be known. Toward this goal we established a new slice preparation of rat brain stem containing the PN. This preparation allowed us to perform stable intracellular recordings for the study of the membrane properties of pontine neurons (this paper) and the characteristics of their postsynaptic responses (Möck et al. 1997
).
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METHODS |
Lister hooded rats (16-days to 5-wk old) were anesthetized deeply with ketamine and decapitated. During the dissection of the brain, care was taken not to impose mechanical stress onto the brain stem. In particular, the trigeminal nerves had to be cut before removing the brain from the scull. 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). The pia mater overlying the brain stem was removed gently using fine forceps. The block of tissue containing the PN was isolated by performing two frontal cuts: a rostral one at the level of the cerebral peduncles and another one just caudal to the pontine nuclei. The cerebellum was removed by a horizontal cut along the fourth ventricle. We prepared parasagittal slices by gluing the block of tissue with its lateral side down to an agar ramp. The agar ramp provided an angle of 45° with respect to a cubic block of agar, which was used to support the tissue from behind. Two agar blocks were placed on both sides of the tissue to reduce lateral vibrations during the slicing process. Using a vibrating microtome (Campden, London, UK), slices were cut to a thickness of 400 µm. They were stored in ACSF containing (in mM) 124 NaCl, 5 KCl, 1.2 KH2PO4, 1.3 MgSO4, 26 NaHCO3, 2.4 CaCl2, and 10 D-glucose, bubbled 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, Foster City, CA) in the bridge mode. The microelectrodes had a linear current-voltage relationship within
1.0-1.0 nA. Experimental drugs were added to the medium. In experiments using Co2+ or Mn2+, CaCl2 was substituted in equimolar concentration by the chloride salt of those ions while CdCl2 (100 µM) was added to the calcium containing ACSF. When using these divalent cations, chloride was substituted for phosphate to avoid precipitation. Tetrodotoxin (TTX) was used at a concentration of 1 µM and tetraethylammonium (TEA) at 15 mM in all cases. The voltage records were low-pass filtered (cutoff frequency 10 kHz) and digitized at a sample rate of 5 or 20 kHz using a PC with a 1401plus interface and Spike2 software (Cambridge Electronic Design, Cambridge, UK).
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RESULTS |
Pontine 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
(as computed from the membrane potential 190 ms after the onset of a current step of
0.1 nA amplitude). Furthermore, only neurons with spike amplitudes of >50 mV (as measured from the resting potential to the peak) and a spike width of maximally 1 ms (as measured at half-amplitude between resting potential and peak of the spike) were accepted. In a few experiments, we observed that PN neurons taken from animals younger than postnatal day 14 displayed action potentials and spiking patterns that differed from those of older animals. Spikes were broader and did not show the typical afterhyperpolarizing potentials (AHPs, see following text) as observed in older animals. Consequently, these cells were excluded from further analysis. In the range of age used for this study (16 days to 5 wk postnatally), the membrane properties were not dependent on the age of the animal. We therefore conclude that PN cells show adultlike membrane properties as early as 2 wk postnatally. Achieving stable intracellular recordings from pontine slices taken from rats older than 5 wk turned out to be hardly possible and was abandoned.
Applying the criteria given above, 179 pontine neurons were chosen for analysis. They displayed a resting potential of
63 ± 6 mV (mean ± SD), and none of them was spontaneously active. To avoid artifacts due to activation of voltage-dependent currents, the apparent membrane resistance and the membrane time constants were computed from the voltage response to the smallest current pulse in hyperpolarizing direction used (
0.1 nA). However, as most PN neurons showed a fast and sustained inward rectification in hyperpolarizing direction that was activated even by small currents (see further text and Fig. 1), these measurements are most probably underestimations of the passive membrane resistance and time constant at rest. With this regime, the PN neurons showed an apparent membrane resistance of53 ± 21 M
and a membrane time constant of 5.3 ±2.4 ms.

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| FIG. 1.
Subthreshold responses of 200-ms length current pulses in pontine neurons. A: typical current-voltage relationship of pontine nuclei (PN) neurons is characterized by inward rectification in hyperpolarizing and depolarizing direction. Different current-voltage relationships (right) in the depolarizing direction were gained with positive currents when measured at the peak of the hump (*) as compared with measurement 190 ms after stimulus onset ( ). In the current-voltage curve asterisks are not shown if identical to the measurements at 190 ms ( ). Addition of tetrodotoxin (TTX, 1 µM) to the medium abolished the depolarizing hump. Inward rectification in hyperpolarizing direction was left intact and an outward rectification in depolarizing direction was disclosed ( ). Resting potential: 73 mV. For both control and treatment with TTX a near threshold sweep (0.15 nA, uppermost of the voltage traces shown above) under control conditions and a comparable one reaching a similar potential under TTX (0.4 nA, not shown above) are shown enlarged. Depolarizing hump was almost totally blocked by TTX. Note, however, that in all cases a small time-dependent response remained (arrow, see also Fig. 8E). B: current-voltage relationship of 3 cells tested with a concentration of 5 mM ( ) and 0.5 mM ( ) extracellular potassium. Under 0.5 mM extracellular potassium the membrane potential prior to stimulation was adjusted to the resting membrane potential as observed with 5 mM potassium. The curve clearly deviate showing less inward rectification with lower potassium concentration. C: PN cell showing the slow but not the fast inward rectification. Current-voltage relationship was nearly linear with early measurement of the potential ( ) but showed an inwardly rectifying behavior if the potential was measured 190 ms after stimulus onset ( ). If present, the slow inward rectifying response was always accompanied by an overshoot of the membrane potential at stimulus offset ( ). Resting potential: 67 mV.
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Responses to hyperpolarizing stimuli
Typically, current-voltage relationships of PN neurons showed a marked inward rectification in hyperpolarizing direction when tested with current pulses of 200 ms length (Fig. 1A). This is shown by the significantly reduced apparent membrane resistance when using
1.0 nA pulses (40 ± 19 M
) for its measurement instead of
0.1 nA pulses (53 ± 21 M
; P < 0.000001, Student's t-test for dependent variables). On the basis of their time course, two different types of inward rectification, a slow and a fast one, could be discerned. The more common fast type of inward rectification already was activated when the potential reached the steady state after charging the membrane capacitance (Fig. 1A). Variation of the extracellular concentration of potassium changed the strength of the fast inward rectification. Lowering the extracellular potassium concentration from 5 to 0.5 mM (an action that moves the potassium equilibrium potential to more hyperpolarized potentials) resulted in an increased voltage response and thus a decreased strength of inward rectification (Fig. 1B). The slow type reached a steady state later than 100 ms after stimulus onset and always was characterized by a clear overshoot of the membrane potential after the stimulus offset (arrow in Fig. 1C). It therefore could be detected best if the potential was measured late in the sweep (circles) and was nearly absent early in the sweep (triangles). The inward rectification of most of the PN cells was of the fast type (75%, Fig. 1A), whereas only a minority of the cells were found to contain the slow one (9%, Fig. 1C). Both types of inward rectification could be found in 8% of the cells, whereas 67% showed exclusively the fast variant, 1% exclusively the slow variant, and 24% showed no inward rectification at all.
Subthreshold responses to depolarizing stimuli
Typically, PN neurons showed an inward rectification also in the depolarizing direction. This phenomenon was most obvious if the amplitude of the current step was close to rheobase (i.e., the minimal current amplitude needed to depolarize the membrane potential over the firing threshold). In these cases, a characteristic time-dependent depolarizing response ("depolarizing hump") was observed (Fig. 1A, enlarged voltage trace at bottom). It turned out that the current amplitude has to be adjusted to rheobase in small steps to see the hump consistently. Our routine paradigm to investigate the current-voltage relationship, which used current steps differing by 0.1 nA, detected the phenomenon in only 58% of the total sample. However, if the resolution of current was increased to 0.01 nA per step, a depolarizing hump was detected in all cells tested this way (n = 28). The maximum of the depolarizing hump occurred between 50 and 100 ms after stimulus onset in most cases. In some PN neurons, the hump reached its peak at latencies of <50 ms or >100 ms (Fig. 2A). The current-voltage relationship, therefore, depended critically on the time after stimulus onset at which the potential was measured. The point in time (190 ms after stimulus onset) chosen for the measurement of membrane potential in this study was after the decline of the depolarizing hump in all cells.

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| FIG. 2.
A: stimulation to threshold. The potential response of current pulses (length: 200 ms) which kept the membrane potential just subthreshold and the one elicited by an increased current (by 0.01 nA) to reach threshold. In all cases only 1 spike was elicited. The latency of the spike depended on the time course of the depolarizing hump. Cells with a short (left), medium (center), and long (right) latency of the peak potential of the depolarizing hump are shown. The earlier the peak of the hump, the earlier occurred the spike in the just suprathreshold stimulation. Note the membrane fluctuations that typically occurred in near threshold stimulations (arrows). B-D: slow spike and plateau potentials and their ionic basis. B and C: calcium-dependent action potentials. Addition of tetraethylammonium (TEA, 15 mM) to the medium revealed slow potentials following the fast sodium-dependent rise of the action potential. Time course of these potentials varied from cell to cell (compare cells in B and C). Blockade of sodium currents with TTX (1 µM) isolated a slow action potential with varying time course from cell to cell that could be blocked by subsequent blockade of calcium channels with Mn2+ (B). Note that the plateau potential shown in B bore typical spikelets ( ) under TEA that were blocked by TTX. Furthermore, TTX reduced the length of the slow potential. Resting potential of both cells: 60 mV. D: sodium-dependent plateau potentials. This cell showed a plateau potential following the fast sodium spike after blockade of calcium and potassium channels with Co2+ and TEA. Note the spikelet which is typically observed on top of plateau potentials under these conditions ( ). After addition of TTX these potentials were blocked in all cases (B). Resting potential: 62 mV.
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The ionic basis of the depolarizing response as revealed by pharmacological experiments is demonstrated in Fig. 1A. The PN neuron shown displayed the typical subthreshold responses as described before. As in all cases studied (n = 9), the depolarizing hump was reduced substantially by addition of TTX, indicating a persistent sodium conductance as its basis. In five of these cells, the hump was abolished by TTX after it persisted the treatment of divalent cations (not shown). Although the depolarizing hump was TTX sensitive in all cases, it must be pointed out that a slight time dependency in the voltage response typically persisted under TTX (Fig. 1A, arrow) as well as under additional blockade of voltage-gated calcium channels (Fig. 8E). It is difficult to explain this remaining time-dependent response by an incomplete blockade of the sodium conductance by TTX, as it always showed a different time course if compared with the sodium hump seen under control conditions. Therefore, instead of being caused by a remaining sodium current, it rather may reflect a sag generated by a slow outward current as previously shown for neostriatal cells (Nisenbaum and Wilson 1995
). In line with this notion, blockade of sodium channels always disclosed a strong outward rectification in depolarizing direction for higher stimulus amplitudes (Figs. 1A and 8E).

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| FIG. 8.
A: pontine neurons entered a characteristic steady-state firing after the initial spike train. The cell shown was depolarized by a 5-s pulse of 0.55 nA. Note the membrane fluctuations and spikelets during the pauses of firing (arrows). The membrane fluctuations irregularly reached the firing threshold and elicited a spike. B: same cell and stimulus protocol as in (A). Membrane potential taken from the end of the stimulus pulse is shown in higher resolution. Different current amplitudes were applied (0.2, 0.35, and 0.5 nA from left to right). Depolarization with 0.2 nA kept the membrane potential subthreshold and no membrane fluctuation was present. Higher currents led to long periods of membrane fluctuation without any spike (0.35 nA) and to irregular firing (0.5 nA). Note that the mean membrane potential (excluding spikes) showed a nonlinear increase with the current amplitude. Inset: autocorrelogram function of a period of subthreshold membrane fluctuations taken from the middle trace. There was a tendency of rhythmic occurrence, however the correlation coefficients of the side peaks were small (<0.15). C: addition of cobalt did not change the general pattern as seen under control conditions (B). Traces show depolarizations that match those in B (0.15, 0.25, 0.35 nA, from left to right). Fluctuations were observed to be somewhat broader than under control conditions. This tendency was reflected by the autocorrelogram although the coefficients were still small. D: addition of TTX abolished the spikes but also the subthreshold membrane fluctuations (current amplitude: 0.1, 0.3, and 0.5 nA from left to right). E: voltage responses of the same neuron to depolarizing currents in the presence of Co2+ and TTX to demonstrate the outward rectifying behavior of the membrane under these conditions (voltage measurement 190 ms after stimulus onset, circles, note the time-dependent deflection of the potential that remained after addition of TTX, see also Fig. 1A). The membrane potential could be depolarized by 21 mV using a stimulus amplitude of 1.0 nA. To compare this relationship to the mean potential between spikes under control conditions, the mean potential within long pauses of firing was assessed in the 2nd half of current pulses lasting 5 s excluding a period of 100 ms before and after spikes. The resulting current-voltage relationship showed an inward rectification in the subthreshold range of depolarization (rheobase 0.25 nA). Above threshold the curve turned to a marked outward rectifying behavior. The level of the membrane potential between spikes saturated at 0.4 nA to a level of 18 mV positive to rest. Resting membrane potential: 63 mV.
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Suprathreshold responses
Intracellular injection of positive currents, which drove the potential just above firing threshold (
42 ± 6 mV), elicited one single action potential in all PN neurons. The spikes had an amplitude of 71 ± 9 mV (defined by the difference between the resting membrane potential and the peak of spikes) and a width at half-amplitude of 0.76 ± 0.16 ms (as measured in those cells the responses of which were sampled at 20 kHz, n = 71) and were followed by a pronounced AHP. The time after stimulus onset at which the spike occurred depended on the temporal characteristics of the subthreshold depolarizing response. In Fig. 2A, three PN neurons are shown that displayed depolarizing responses of different time courses to near-threshold current pulses. The spike elicited with just suprathreshold stimulation occurred always at the peak of the underlying depolarizing hump.
Under control conditions, apart from the subthreshold depolarizing hump, PN cells did not show any obvious slow action
or plateau potentials. Pharmacological manipulation, however, revealed two additional suprathreshold responses (Fig. 2, B-D). Under blockade of repolarizing potassium channels with TEA, the fast rise of the action potential was followed by a more slowly decaying potential, which varied in its time course from cell to cell (range 20-450 ms, compare Fig. 2, B and C, n = 12). This slow component could be isolated by addition of the sodium channel blocker TTX (Fig. 2, B and C). It appeared in an all-or-none fashion and had a threshold of
23 ± 4 mV (n = 11). It was blocked by substitution of Ca2+ by Mn2+ or Co2+ in the medium (Fig. 2B, Mn2+: n = 6, Co2+: n = 1). These characteristics are consistent with a high-threshold calcium spike and were observed in all PN neurons tested. Under blockade of potassium conductances with TEA alone, the slow potential after the fast rise of the action potential was longer, more irregular, and, in many cases, bore small spikelets (arrows in Fig. 2C) when compared with the situation after addition of TTX (reduction in width between 14 and 70%). This fact suggested that sodium channels also may contribute to those slow potentials. To test this more rigorously, TEA was combined with the calcium channel blocker Co2+ in another set of experiments. Indeed, a high-amplitude, fast spike followed by an irregularly shaped plateau potential bearing spikelets was isolated (arrows in Fig. 2D, found in 6 of 7 PN neurons, Co2+: n = 4, Mn2+: n = 1, Cd2+: n = 1 of 2). These plateau potentials were blocked if TTX was present (Fig. 2B, n = 11), indicating that in addition to voltage-gated calcium channels, a persistent sodium current contributed to the long-lasting potential seen under TEA.
Medium AHP
Two types of AHPs with different time courses could be detected after a single spike or spike trains. We named these two parts of the AHP medium and slow, although the potential trace under control conditions did not allow to differentiate a fast AHP. This was done to ease comparison with other preparations like the cerebral cortex or hippocampus, where a similar set of conductances leads to AHPs that have been named correspondingly (Schwindt et al. 1988
; Storm 1987
) (see DISCUSSION). The medium AHP was observed in all PN neurons within our sample and lasted for ~50 ms. We quantified the amplitude and width of the medium AHP in all PN neurons within our sample using the first medium AHP elicited with the current pulse that was just sufficient to drive the membrane potential above firing threshold. Its amplitude (measured as the difference between firing threshold and the negative peak of the medium AHP) was 11 ± 3 mV. The negative peak was reached 6 ± 2 ms after the spike, and the total width from the spike to the point when the potential reached half the maximal amplitude again was 30 ± 17 ms. A typical example is shown in Fig. 3A (top left). To allow comparison of the AHP with the potential before the spike, we demonstrate a case where we used a short current pulse (3 ms) to elicit the spike. The arrow marks the transition to the slow AHP, which lasted for several hundreds of milliseconds in this case (see following text). After blockade of calcium channels with Co2+ (n = 6), the amplitude of the medium AHP was reduced substantially, suggesting a calcium-dependent outward current as its basis (Fig. 3A, bottom left). Cadmium produced inconsistent results. In three cases, Cd2+ blocked the medium AHP similar to Co2+, whereas it was unaffected in two other cases. Even in the cases where the medium AHP was blocked by divalent cations, spike repolarization remained intact and a small remnant of the medium AHP was visible. This was abolished only if the potassium channel blocker TEA was added to the medium (n = 3, see Fig. 3A, bottom right, for an example). In this case, the sodium-dependent plateau potential followed the spike. Figure 3A, inset, shows spikes of the same cell evoked by just threshold current pulses (length 200 ms, as in Fig. 2A) in higher temporal resolution. In these cases, the downstroke of the spike was not affected by addition of divalent cations (n = 13). Addition of TEA, however, increased the amplitude and width of the spike and gave rise to the sodium plateau potential (n = 4). These results indicate that the medium AHP was based on at least two membrane conductances: a faster calcium-independent one contributing to the spike repolarization (and thus to the width of the spike at half-peak) and a calcium-dependent one that controlled later parts of the medium AHP. The effects on the potential of these two conductances probably blend perfectly into each other and thus generate a smooth potential trace. Therefore, a hypothetical fast AHP reflecting the effect of the faster calcium-independent conductance was not discernible.

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| FIG. 3.
A: medium afterhyperpolarization (AHP) and spike repolarization. AHPs as observed after a single spike. Membrane potential was depolarized to 52 mV by continuous current injection (resting potential: 63 mV). A 3-ms current pulse was used to elicit a single spike. Under control conditions the fast repolarization of the action potential (···) was followed by the medium AHP lasting for ~50 ms and a slower one with low amplitude that lasted several hundreds of milliseconds (starting at the arrow). Both the medium and the slow AHPs were reduced substantially after blockade of calcium currents with Co2+. However, the spike repolarization was only abolished after addition of TEA (15 mM) revealing the long-lasting sodium plateau following the spike (a spikelet typically seen on top of these potentials is marked with an arrow, see Fig. 2D). Inset demonstrates the pharmacological effects on the spike repolarization on a higher timescale in the same cell. To avoid stimulus artifacts, single spikes elicited only by threshold stimulation using longer current pulses were used (200 ms, as in Fig. 2). Voltage trace under control conditions and under Co2+ deviated only during a later phase of the action potential. The spike width at half amplitude was unchanged. Addition of TEA increased the spike amplitude and substantially broadened the spike revealing the sodium plateau potential. B: medium AHP and membrane resistance. Current-voltage relationship of the negative peak potential during the interspike interval (ISI) using current ramps. The slope of a linear function fitted to the data points expressed this relationship in megohms as apparent membrane resistance. Under control conditions the negative peak potential was almost clamped to about 46 mV yielding an apparent membrane resistance of 1 M (3% of the apparent membrane resistance as measured just subthreshold). Blockade of calcium-dependent currents led to a more depolarized potential between spikes and to an increase of the apparent membrane resistance to 37 M (61% of the value just subthreshold). Additional blockade of voltage-activated potassium currents with TEA (15 mM) increased the value to 66 M (99% of the apparent membrane resistance just subthreshold). In each trial an offset was added to the triangular current of 1 nA amplitude in order to yield similar spike trains (Control: +0.3 nA; Co2+: 0; Co2+/TEA: 0.5 nA).
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The currents underlying the medium AHP were massive as its negative peak potential was not changed by different current amplitudes. This was best seen if the negative peak potential of the medium AHP was investigated using current ramps (Fig. 3B). The negative peak potential during the interspike intervals (ISIs) was almost clamped to a fixed potential despite the depolarizing current ramp. To demonstrate this more quantitatively, we assessed the current-voltage relationship of the negative peak potentials, which could be reasonably well fitted by a linear function (Fig. 3B, right). To allow a comparison to the apparent membrane resistance before reaching the firing threshold, another line (not shown) was fitted to 2,000 data points (corresponding to 0.14 nA on the x-axis) within the subthreshold current-voltage curve. To assure comparability with the measurements of the resistance during the medium AHPs, the window of membrane potentials from which these data points were taken was centered on the minimum membrane potential as seen during the first medium AHP. This window also was used to fit the line in later steps using pharmacological blockade of the medium AHPs. In the case shown, the apparent membrane resistance was 39 M
before the spike train, whereas the minimum potentials of the medium AHPs showed an apparent membrane potential of 1 M
(Fig. 3B). Thus the apparent membrane resistance during the medium AHP was only 3% of the one measured before the spike train. After blockade of the calcium-dependent component of the medium AHP with Co2+, the apparent membrane resistance at the negative peak potential increased with respect to the subthreshold value but was still only 61% of its value (subthreshold: 61 M
, minima of medium AHPs: 37 M
). This was expected from our finding that the medium AHP was made up by at least two outward conductances, one calcium dependent and the other not. Accordingly, it reached 99% of the value just subthreshold if potassium channels were blocked by TEA (subthreshold: 67 M
; minima of medium AHPs: 66 M
). In the population of cells analyzed that way, the apparent membrane resistance at the negative peak of the AHP was reduced to a value of 13 ± 9% (n = 9) as compared with the subthreshold value before the spike train. In the cells treated with a calcium channel blocker, the mean reduction was 47 ± 13% (n = 5), and in the cells in which calcium and potassium channels were blocked, it was 85 ± 14% (n = 3).
Slow AHP
As shown in Fig. 3A, a slow AHP could be observed frequently after a single action potential (n = 8 of 11). The slow AHPs were seen best with depolarized potentials and were barely visible at the resting potential. High-frequency spike trains more consistently revealed a slow AHP also at the resting potential (Fig. 4A), but its amplitude (as measured 100 ms after the spike train) was small (2.0 ± 0.9 mV, n = 10). This may be explained by the lack of driving force as the equilibrium potential of the slow AHP was close to the resting membrane potential. To study the effects of the slow AHP more quantitatively, we determined the minimal current needed to elicit a spike (referred to in short as minimal current in the following text) during the AHP as a measure of its strength (Fig. 4B). The use of a current as a measure had the advantage that shunting currents that occur near the equilibrium potential of the slow AHP (and thus not affecting the membrane potential) were assessed readily. To this end, conditioning current pulses of varying amplitudes (length 200 ms) were injected to elicit different numbers of spikes. The minimal current then was adjusted for a given amplitude of the conditioning pulse using a depolarizing test pulse of 10 ms length following 100 ms after the conditioning pulse. These experiments were performed using extracellular potassium concentrations of 5, 3, 1, and 0.5 mM. Both higher numbers of spikes as well as lower extracellular potassium concentrations clearly increased the minimal current during the slow AHP (Fig. 4B). Plotting the number of spikes (of all trials in all cells investigated) versus the respective minimal current during the slow AHP revealed that the relationship between the two parameters could be fitted significantly with a linear function (Fig. 4C). When we lowered the potassium equilibrium potential by using lower extracellular potassium concentrations (i.e., moved it away in hyperpolarizing direction from the resting membrane potential), the slope a of the fitted line (i.e., the amplitude of the minimal current adjusted with a certain number of spikes) increased (Fig. 4C, 0.5 mM: a = 2.98, r2 = 0.77, n = 2; 1 mM: a = 1.94, r2 = 0.76, n = 5; 3 mM: a = 1.44, r2 = 0.63, n = 8; and 5 mM: a = 0.96, r2 = 0.47, n = 9). We therefore conclude that the slow AHP is dependent on the number of preceding spikes and is carried at least partly by potassium ions.

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| FIG. 4.
A: slow AHP was observed after injection of a current pulse of 1 s length that evoked a high-frequency spike train. Voltage trace following the current pulse is enlarged to show the slow AHP more clearly. The example is the strongest found within our sample. B: minimal current needed to cross firing threshold during the slow AHP. Minimal current is shown (as adjusted with a 10 ms current pulse) 100 ms after a 200 ms depolarizing current pulse eliciting spike trains with different numbers of spikes. Following a spike train including many spikes, the minimal current is substantially higher than after a single spike. Furthermore, a concentration of 0.5 mM extracellular potassium increases the minimal current compared to 5 mM. Resting membrane potential 73 mV. C: relationship between the number of spikes during the long current pulse and the minimal current under 5 and 0.5 mM extracellular potassium. Data points could be fitted by a linear function (broken line, 0.5 mM: r2 = 0.77, n = 2; 5 mM: r2 = 0.47, n = 9). The amplitude of the slow AHP elicited by the same amount of spikes was clearly higher with the low extracellular potassium concentration.
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The effect of the slow AHP on the membrane potential was best shown by using depolarizing triangular current profiles that elevated the membrane potential above threshold (Fig. 5A). When plotting current versus potential as a Lissajou figure (Fig. 5B, left), a long-lasting (
2 s) hyperpolarization of the potential after the spike train (downward arrow) with respect to its value before the spike train (upward arrow) was evident. This difference could be observed only if spikes were elicited. It was close to zero if the membrane potential did not cross the firing threshold (Fig. 5B, middle, n = 12). Hence, it is likely to reflect exclusively the slow AHP rather than voltage-gated conductances. The slow AHP after spike trains clearly was prevented by blockade of calcium channels by Co2+ (Fig. 5B, right). Qualitatively the same results were obtained for nine PN neurons (Co2+: n = 6, Mn2+: n = 1, Cd2+: n = 2). These results indicate that the slow AHP in PN neurons is carried by a calcium-dependent potassium current.

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| FIG. 5.
Slow AHP. A: slow AHP was elicited by injection of suprathreshold triangular current ramps. PN neuron fired a high frequency spike train when reaching the firing threshold. B: plot of the current-voltage response as a Lissajou figure. Arrows indicate the upward and downward trace of the membrane potential during the triangular current injection. Suprathreshold and subthreshold stimulation under control conditions are shown. If a spike train is elicited (left) the membrane potential shows a slow AHP of >5 mV amplitude during the downward voltage trace. This is not observed if a negative offset is added to the stimulating current to keep the membrane potential subthreshold (middle). The slow AHP was blocked by Co2+ indicating an underlying calcium-dependent conductance (right). Resting potential: 72 mV.
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Firing rate adaptation
CONTRIBUTION OF INWARD CURRENTS.
Intracellular current pulses elevating the potential well above threshold elicited a spike train that showed a marked firing rate adaptation (Fig. 6). Thus the length of subsequent ISIs increased during the spike train. With just suprathreshold stimuli, the cells ceased to fire after the initial spike train, whereas stronger stimuli led to an irregular low-frequency firing pattern described in a following paragraph. This general feature was present in all PN neurons within our sample. More subtle differences of the firing rate adaptation found between different neurons depended on the time course of the subthreshold response. Figure 6 shows three PN neurons (of 22 studied selected according to the appearance of a prominent hump) with different latencies of the maximum of the depolarizing hump with respect to stimulus onset (tmax). In the PN neuron with a small tmax (Fig. 6A), the firing rate adaptation was most pronounced at the beginning of the sweep. This can be easily seen if the ISIs are plotted against their number of occurrence. The slope of the resulting curve was steepest during the first ISIs in the spike trains. Thereafter the curves tended to saturate. In the PN neuron with a medium tmax, the adaptation was slower and was distributed over more subsequent spikes (Fig. 6B). Thus the plot of ISIs against their number of occurrence had a positive slope also in later phases of the spike train. Finally, the cell with large tmax showed a comparatively small but fairly regular increase of ISIs over long sequences of spikes (Fig. 6C). To quantify this effect, we compared the cells with early depolarizing hump (tmax < 50 ms, n = 8) to those with late ones (tmax > 100 ms, n = 5; Fig. 6D). To match the cells according to firing frequency, we considered only those current pulses (duration 200 ms) that elicited four to seven spikes corresponding to an average firing rate of 20-35 Hz. The instantaneous strength of adaptation was expressed as the quotient of a pair of subsequent ISIs (the later divided by the earlier, In+1/In). These quotients were >1 if the spike trains showed firing rate adaptation. The first two quotients in the cells with early maxima indicated a significantly stronger firing rate adaptation. The quotient made up by the fourth and third ISI was no longer significantly different (Fig. 6D; I2/I1: median early hump 1.64, late hump 1.11, P = 0.003; I3/I2: median early hump 1.32, late hump 1.07, P = 0.003; I4/I3: median early hump 1.18. late hump 1,05, P = 0.46, Mann-Whitney U test). In contrast to cells with an early maximum of the depolarizing hump, those with a late maximum showed only a small variance in the instantaneous strength of adaptation, indicating that the firing rate adaptation in the latter cases was more regular.

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| FIG. 6.
Firing rate adaptation and depolarizing hump. A-C: voltage response of current pulses (length: 200 ms) that kept the membrane potential just subthreshold and the one elicited by a current eliciting 4-7 spikes. In all cases a marked firing rate adaptation was present. The adaptation was related to the time course of the subthreshold depolarizing hump. Cells with a short (A), medium (B), and long (C) latency of the peak potential of the depolarizing hump are shown. The earlier the peak of the depolarizing hump the stronger was the firing rate adaptation in the early phase of the spike train. Membrane fluctuations near the firing threshold are marked with arrows. The adaptation seen with different current amplitudes (0.1 nA steps) is demonstrated by plotting the ISI with respect to the number of its occurence (right). The sweeps marked with a star are those shown on the left. Resting potential: 63 mV (A), 60 mV (B), 57 mV (C). D: comparison of the strength of adaptation in the cases with a latency of the peak potential of the depolarizing hump (tmax) earlier than 50 ms to those with tmax >100 ms. Quotients of subsequent intervals (In+1/In) up to the 4th interval are shown as a box-and-whisker plot. Medians are given by horizontal bars, 25-75% quartiles by boxes, and the range by vertical bars. Values higher than 1 indicate firing rate adaptation. Cells with an early hump (tmax < 50 ms) showed significant higher quotients but also more variance as compared to the cells with tmax > 100 ms in the 1st 2 quotients (P < 0.003 in both cases, Mann-Whitney U test). The 2 populations no longer differed significantly in the 3rd quotient(P > 0.46).
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CONTRIBUTION OF CALCIUM-DEPENDENT OUTWARD CURRENTS.
Conductances underlying afterhyperpolarizing potentials have been reported to contribute to firing rate adaptation in other preparations (Schwindt et al. 1988
; Storm 1987
). Because part of the conductances underlying the AHPs in PN neurons were calcium dependent, we tested which influence the blockade of calcium channels had on the firing rate adaptation. In Fig. 7A, spike trains of a representative PN neuron are shown. The firing frequency increased with higher stimulus currents, but in all cases, subsequent ISIs became increasingly longer during the spike train thus leading to a marked firing rate adaptation. Blockade of calcium channels with Cd2+ increased the firing frequency elicited with a given current amplitude. With most stimulus amplitudes, spike trains reached a constant steady state frequency early during the stimulus; this was not the case under control conditions. It must be pointed out, however, that although the firing rate adaptation thus was reduced substantially, it was still present in the early phases of the spike train and in the traces with low stimulus currents (see the top traces under Cd2+, current amplitude 0.5 and 0.6 nA). Figure 7B shows the quantification of these effects. The firing rate adaptation is demonstrated by plotting the length of the ISIs against the number of their occurrence. The resulting curves all show a positive slope under control conditions, whereas those observed under Cd2+ reached a constant level early during the stimulus (with the exception of the trace elicited by 0.6 nA). The apparent membrane resistance under Cd2+ was somewhat higher than under control conditions as seen with all divalent cations (Fig. 7C). Therefore, it may be argued that the effect of divalent cations on the firing rate adaptation was due to an unspecific increment of the membrane resistance, causing the firing frequency to saturate with the current amplitudes used. To exclude this possibility we plotted the current-frequency relationship (as computed from the inverse of the 1st 5 ISIs). Under control conditions, it showed a fairly linear relationship for currents
1 nA (Fig. 7B). Blockade of calcium influx increased the firing frequency of about five times, but the current-frequency relationship showed only a modest degree of saturation even with the highest stimulus amplitude. We therefore conclude that the absence of firing rate adaptation in the late phase of the spike train was due to the blockade of the calcium-dependent slow AHP rather than reflecting saturation of firing frequency. Qualitatively, the same results were obtained for 28 PN neurons (Co2+: n = 16, Mn2+: n = 5, Cd2+: n = 7). In two of seven cases, Cd2+ did not block the calcium-dependent medium AHP as mentioned above. The effects on the firing rate adaptation, however, were readily visible.

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| FIG. 7.
Effect of blockade of calcium channels on the firing rate adaptation. A: suprathreshold voltage traces of a PN neuron in response to current pulses of different amplitudes under control conditions and after blockade of calcium channels with Cd2+. The neuron displayed a marked firing rate adaptation over the total length of the stimulation. Cd2+ increased the firing rate and reduced the adaptation in a later phase of the stimulation, although with lower stimulation, the adaptation was still observed. Resting potential: 71 mV. B: plots demonstrating the effects as seen in A. Under control conditions the positive slope observed when plotting the ISIs against their number of occurrence indicate firing rate adaptation up to the highest current amplitude used. Addition of Cd2+ reduced the positive slope of this function in later phases of the spike trains that were stimulated with higher stimulus amplitudes. Note, however, that the firing rate adaptation remained in earlier phases of the spike train and with just suprathreshold stimulation (0.6 nA). Plotting the instantaneous frequency assessed by the inverse of the ISIs demonstrated that the current-frequency relationship was linear for the 1st to 5th ISI. Treatment with Cd2+ increased the firing frequency about 5-fold and the current-frequency curves (most visible in the one computed from the 1st ISI) showed a slight deflection towards lower frequencies with high stimulus currents but did not saturate within the range of current amplitudes used. C: current-voltage relationship in depolarizing direction of the PN neuron shown in A and B. The apparent membrane resistance is somewhat increased under Cd2+. D: effect of the medium AHP of another PN cell during the ISIs of spike trains elicited with a current pulse of 0.7 nA amplitude and 200 ms length. Under control conditions the negative peak potential reached 55 mV independent of the current amplitude. The voltage trace was somewhat more depolarized during the 1st ISI, a phenomenon seen in many pontine neurons. During the subsequent ISIs the potential trace immediately following the action potential was very similar. The increment of the ISIs was generated in a later phase after the potential displayed a typical bend (arrows). The later phase of the ISIs showed a more hyperpolarized voltage trace thus reaching the firing threshold later. After blockade of the medium and slow AHPs with Co2+ the potential was more depolarized during the ISIs causing a higher firing frequency. The potential was more susceptible to the current amplitude: the negative peak potential became more positive with higher current amplitudes. Furthermore, the characteristic bend dividing the ISI in 2 parts under control conditions was missing. Resting potential: 62 mV.
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The increment of firing frequency observed under blockade of calcium channels partly may be due to the blockade of the calcium-dependent conductance underlying the medium AHP. To illustrate this in more detail, the course of the membrane potential between subsequent spikes under different pharmacological conditions in another cell is demonstrated in Fig. 7D. The potential trace of the ISIs elicited with 0.7 nA was superimposed using the preceding spike as a starting point. ISIs recorded under control conditions and under Co2+ with the same stimulus current amplitude are plotted using the same axes to allow direct comparison. Under control conditions, the negative peak potential during the ISIs reached about
55 mV. Typically the voltage traces displayed a bend (arrow) that led to a smaller slope after ~20 ms after the spike. The later the interval occurred during the spike train, the smaller was the slope, thus leading to longer ISIs. Adding Co2+ to the medium led to a more depolarized potential during the ISI (negative peak potential
36 mV). Furthermore, the ISIs were shorter and did not show the characteristic bend as seen in control medium. Similar qualitative effects were observed in all 28 cells in which spike trains were elicited under treatment with divalent cations.
Steady state firing pattern
Long current pulses (duration 5 s) that depolarized the PN neurons well above threshold elicited a spike train that showed a strong firing rate adaptation as described above. A typical example is shown in Fig. 8A. After the spike train, a level of membrane potential was reached that was characterized by spontaneous fluctuations. Spikelets of
5 mV amplitude were observed (arrows in Fig. 8A). Eventually, one of these fluctuations reached the firing threshold again and elicited another action potential, thus generating an irregular low frequency spike train. Some PN cells actually paused for seconds after the initial high-frequency spike train before entering the steady state firing (not shown).
In Fig. 8B, the cell was subjected to long current pulses (5 s) of different amplitudes. Small depolarizations, which elevated the membrane potential to a level between the resting potential and ~10 mV negative to the firing threshold, did not elicit obvious membrane fluctuations (Fig. 8B, left). Levels of membrane potential in the range of 10 mV below the firing threshold clearly elicited spikelets that eventually reached the firing threshold (Fig. 8B, middle). To address the question of whether these fluctuations are regular (i.e., oscillatory), we computed standard autocorrelograms (AC; MATLAB software package, The MathWorks) from the subthreshold membrane potential. The periods chosen for this purpose were 1.3-2 s long and excluded 100 ms before and after a spike to prevent contamination of the ACs by the waveform of spikes or the medium AHPs. The AC showed only a low degree of oscillatory patterns as indicated by the fact that the correlation coefficients outside the central peak were all <0.15. This was true for all cases tested (n = 9). With higher current amplitudes, the membrane potential fluctuated around the firing threshold thus eliciting an irregular spike train (Fig. 8B, right).
To gain insight into the ionic basis of the steady state membrane fluctuations giving rise to this characteristic firing pattern, we blocked the voltage-dependent calcium currents with Co2+. As expected from the results reported before, blockade of calcium channels blocked the medium AHP, increased the firing rate, and substantially suppressed the slower component of the firing rate adaptation. However, the general pattern of membrane fluctuations resisted this treatment (Fig. 8C, n = 4). Compared with the control, the fluctuations were observed to be somewhat slower as shown by the AC. But still the coefficients of the side peaks were low, indicating a substantial irregularity of the pattern. As shown in Fig. 5, calcium-gated potassium currents are blocked under these conditions. Therefore, they seem not to be necessary for the generation of membrane fluctuations and the irregular pattern of steady state firing. Finally, the membrane fluctuations were blocked effectively by additional blockade of sodium channels with TTX when tested with depolarizations comparable with those elicited under control conditions. (Fig. 8D, n = 3). Accordingly, the AC was essentially flat under these conditions. The same effect was observed in two more cells that were treated with TTX alone.
The state of membrane potential giving rise to the irregular firing pattern was reached with a wide range of suprathreshold current amplitudes, suggesting an activation of strong outward currents that opposed the depolarization made up by the electrode current and voltage-activated inward currents. The mean potential between spikes under control conditions saturated at a current amplitude of 0.45 nA to a value of 18 mV above rest and was not further elevated by higher current amplitudes (stars in Fig. 8E, right, a period of 100 ms after a spike was excluded from the computation of the mean potential to exclude effects of the medium AHP). The conventional current-voltage relationship as assessed under Co2+ and TTX (Fig. 8E, left) is plotted in the same graph (circles). It showed a marked outward rectification under Co2+ and TTX, indicating voltage-activated outward currents. Nevertheless, the potential could be driven to more depolarized levels (21 mV above rest at 1.0 nA) as compared with that reached by the potential between spikes under steady state firing. Therefore, another outward current, probably the calcium-dependent current underlying the slow AHP, seemed to contribute to the level of membrane potential, which is adjusted between spikes during steady state firing.
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DISCUSSION |
The membrane potentials and their pharmacological analysis shown in this study indicate that cells in the PN possess a rich equipment of membrane conductances. In addition to the sodium and potassium conductances necessary for the generation of fast spikes, these neurons displayed slow potentials based on persistent sodium and calcium channels. However, these slow action and plateau potentials were observed only if potassium channels were blocked. Furthermore, PN neurons are endowed with a complex set of AHPs after single spikes or spike trains and strong inward and outward rectifying responses. In the following discussion, we first will give a detailed discussion of the single potentials found and the conductances possibly underlying them and compare them with other structures in the CNS. We then will discuss the possible interplay of these conductances in PN neurons and speculate on their functional roles.
Inwardly rectifying responses
The majority of the PN neurons showed a strong inwardly rectifying current-voltage relationship in hyperpolarized direction. The fast activation, the lack of a rebound excitation, and its dependency to extracellular concentration of potassium suggest that it is generated by an anomalous potassium current similar to that known as IKir (Kandel and Tauc 1966
). In 9% of the PN neurons, a slower inward rectifier was apparent. In contrast to the first one, the potential in these neurons reached a steady state only after ~100 ms and led to a rebound depolarization after the offset of the hyperpolarizing current pulse. These characteristics resemble those described for a mixed cation current activated by hyperpolarization named IH (for review, see Pape 1996
). However, more detailed pharmacological experiments are needed for the secure classification of these conductances. The two variants of inward rectification behavior do not allow to put the PN neurons into groups. In some neurons that showed the slow inward rectifying conductance, the fast variant also was present, although there was the tendency that its action was weak in these neurons. Furthermore, the fact that PN neurons that showed only one of the two inwardly rectifying responses did not differ in any other membrane property argues against a classification based on these conductances.
Slow potentials based on sodium conductances
Pontine neurons possessed slow depolarizing plateau potentials, which persisted the blockade of calcium channels with divalent cations but were blocked by TTX. Potentials with similar temporal and pharmacological properties have been described in several brain regions, including cerebral cortex, hippocampus, and cerebellum. They have been reported to be based on persistent sodium currents (INaP) (for review, see Crill 1996
). The effect of TTX-sensitive sodium currents on the membrane potential varies consistently among different neuronal cell types depending on their interaction with other conductances. In Purkinje cells and in the cells of the deep cerebellar nuclei, the potentials based on a TTX-sensitive INaP were shown to generate high-amplitude plateau potentials carrying fast spikes under control conditions (Jahnsen 1986a
,b
; Llinás and Mühlethaler 1988
; Llinás and Sugimori 1980
). This is in marked contrast to the effects of persistent sodium channels of cerebrocortical Betz pyramidal cells (Stafstrom et al. 1982
, 1984
, 1985
) and hippocampal cells (French et al. 1990
). In these cerebral cells, the effects of INaP are similar to those found by us in the PN. INaP is clearly active in the subthreshold range of potentials but does not lead to suprathreshold plateau potentials under control conditions. Suprathreshold sodium-dependent plateau potentials are only visible after pharmacological blockade of potassium channels, suggesting that these potentials are masked by strong outward potassium currents under physiological conditions (Stafstrom et al. 1985
). Our observation of strong outward rectification under blockade of sodium channels with TTX in PN cells is in accord with this hypothesis.
Potentials based on calcium conductances
Our results indicate that PN neurons possess calcium conductances being active in the suprathreshold range of membrane potentials. Similar to the sodium plateau, the high-threshold calcium spike was not visible under control conditions. As suggested by its unmasking by TEA and already discussed with respect to the sodium potentials, this is most probably due to strong outward currents based on potassium. Again these properties closely match those found in cerebrocortical cells (Connors et al. 1982
; Stafstrom et al. 1984
, 1985
) and are distinct from those of Purkinje cells and cells in the cerebellar nuclei, which show calcium plateaus under control conditions (Jahnsen 1986a
,b
; Llinás and Mühlethaler 1988
; Llinás and Sugimori 1980
).
AHPs
The temporal and pharmacological characteristics of AHPs in PN neurons indicate that they are based on at least three different currents. The slow component was seen most prominently after spike trains. Its increase in strength with the number of spikes preceding it most probably reflects the accumulation of intracellular calcium due to calcium influx during the action potential. Such a mechanism is well established for slow AHPs in other regions of the brain (Schwindt et al. 1988
; Storm 1987
; for review, see Sah 1996
). Furthermore, an AHP of medium length after each spike was shown to be calcium dependent, whereas a fast component contributing to spike repolarization persisted the blockade of calcium channels and was blocked only by TEA. AHPs have been found in a variety of neuronal cell types in different species. The composition of the underlying currents, however, has been reported to differ between different cell types (see for review Blatz and Magleby 1987
; Sah 1996
). The time course and the pharmacological characteristics of AHPs in PN cells may be comparable with those found in cerebrocortical Betz pyramidal cells (Schwindt et al. 1988
, 1992
). As in the PN, three conductances have been reported to contribute to the AHPs in these cells of which the slow and medium component were calcium dependent. The fast calcium-independent component of cerebrocortical AHPs was reported to be based on voltage-sensitive potassium currents (Schwindt et al. 1988
). The characteristics of outward rectification in the PN (Fig. 1) suggests that fast voltage-activated potassium currents exist also in PN neurons and thus may well contribute to the AHP.
Possible interaction of conductances and repetitive firing
We suggest that the firing rate adaptation observed in PN neurons depolarized by current steps is based on a complex interplay of different conductances rather than being determined by a single one. First, we showed that the early phase of the firing rate adaptation is related to the time course of the depolarizing hump (as seen in the subthreshold range of potentials; Fig. 6) and was not abolished by calcium channel blockers (Fig. 7). In line with this observation, INaP in the cerebral cortex has been reported to contribute to the potential between spikes thus modulating the firing pattern (Stafstrom et al. 1982
). Second, later parts of the firing rate adaptation could be reduced by blocking calcium influx into the cells (Fig. 7). This most probably is due to the blockade of the slow AHP as it is activated cumulatively by the number of spikes (Fig. 4) and is calcium dependent. Because the apparent membrane resistance is very low immediately after the spike due to the effect of the medium AHP (Fig. 3B), the action of the slow AHP is expected to be restricted to a later phase of the ISI. Indeed, the potential during this late period was less depolarized in successive ISIs, leading to a delay in the arrival at the firing threshold, a feature that was blocked altogether by divalent cations (Fig. 7D). Similarly, slow AHPs in the cerebral cortex and the hippocampus have been suggested to contribute to firing rate adaptation (Hotson et al. 1979
; Lancaster and Adams 1986
; Madison and Nicoll 1984
; Schwindt et al. 1988
). Furthermore, others than the aforementioned components may contribute to the firing rate adaptation as well. Particularly, firing rate adaptation was seen under divalent cations during longer periods if the cells were stimulated with current amplitudes driving the membrane potential just over the threshold (Fig. 7, A and B, stimulation with 0.5-0.6 nA) This fact at present is unresolved by our analysis. A possible candidate contributing to the remaining adaptation may be the slow component of the outward rectification detected under blockade of sodium currents (Figs. 1B and 8E).
The initial high-frequency spike train was followed by irregular sodium-dependent membrane fluctuations counteracted by voltage-dependent outward currents (Fig. 8). A similar interplay of inward sodium currents and outward currents has been reported in guinea pig entorhinal cortex layer 2 (Alonso and Llinás 1989
), rat sensorimotor cortex layer 5 (Silva et al. 1991
), and guinea pig frontal cortex layer 4 (Llinás et al. 1991
). In contrast to our finding in the PN, the membrane fluctuations in the cerebrocortical cells were highly rhythmic with constant current injections. Although there was a slight tendency of oscillatory activity in our PN neurons, the coefficients within the ACs were never as high as those observed in the frontal cortex (Llinás et al. 1991
). It must be kept in mind, however, that the temporal pattern of subthreshold membrane potential and that of the firing may be highly artificial when rectangular current pulses are used for stimulation. Dynamic somatic currents generated by synaptic inputs in vivo may generate quite different output patterns as has been shown for cerebrocortical cells (Mainen and Sejnowski 1995
).
The dynamic state of the membrane in the near threshold range of potentials is likely to have important consequences for the integration of signals performed by PN neurons in vivo. Studies using extracellular recording from pontine neurons in anesthetized and awake animals reported spontaneous activity in the PN (Kosinski et al. 1988
; P. W. Dicke and P. Thier, unpublished observations). As we found no spontaneous activity in pontine neurons in the slice, resting activity in vivo must be generated by tonic excitatory inputs. Thus relevant signals from the cerebral cortex in vivo are likely to arrive at pontine neurons, which are in a similar state as the ones injected by us with depolarizing current in vitro (Fig. 8). In this situation, activation and inactivation of INaP and its interplay with the voltage-dependent outward currents will very much determine how the synaptic inputs influence the output of the pontine neuron. In addition, action potentials themselves will set the time structure of the firing by means of their AHPs.
In summary, the PN neurons show a set of membrane properties that resemble the one characterizing regular firing neurons, a subtype of cerebrocortical neurons consisting of pyramidal and spiny stellate cells (Connors and Gutnick 1990
; Connors et al. 1982
; McCormick et al. 1985
). As discussed in detail above, they share the lack of spontaneous activity, complex AHPs, slow sodium and calcium potentials after blockade of outward currents, strong rectifying responses, firing rate adaptation, and dynamic behavior near firing threshold. On the other hand, they distinctly differ from cells in the cerebellum. Purkinje cells and cells in the deep cerebellar nuclei are spontaneously active in vitro, do not show a slow afterhyperpolarization and firing rate adaptation, and show plateau potentials based on calcium and sodium channels already under control conditions. Nevertheless, the PN cannot be interpreted as a "seventh layer of the cerebral cortex" specialized for output to the cerebellum as the mapping of information has been shown to be entirely different from that in the cerebral cortex. The PN are organized in compartments, thus possibly generating the fractured somatotopy found in the granular layer of the cerebellar cortex (Bower et al. 1981
; Schwarz and Thier 1995
). Therefore, we may offer the prospect that the PN integrate information that is mapped for the purposes of the cerebellum using membrane properties typical for the cerebral cortex.
 |
ACKNOWLEDGEMENTS |
We thank U. Grosshennig for excellent technical assistance. D. Placantonakis is thanked for improving our English.
This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 307-A1. M. Möck was supported partly by fortüne (Medical School, University of Tubingen, Germany).
 |
FOOTNOTES |
Address for reprint requests: C. Schwarz, Sektion für visuelle Sensomotorik, Neurologische Universitätsklinik Tübingen, Hoppe-Seyler Strasse 3, 72076 Tubingen, Germany.
Received 9 October 1996; accepted in final form 29 July 1997.
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REFERENCES |
-
AAS, J.-E.,
BRODAL, P.,
BAUGHMAN, R. W.,
STORM-MATHISEN, J.
Projections to the pontine nuclei from choline acetyltransferase-like immunoreactive neurons in the brainstem of the cat.
J. Comp. Neurol.
300: 183-195, 1990.[Medline]
-
ALLEN, G. I.,
KORN, H.,
OSHIMA, T.
The mode of synaptic linkage in the cerebro-ponto-cerebellar pathway of the cat. I. Responses in the brachium pontis.
Exp. Brain Res.
24: 1-14, 1975.[Medline]
-
ALLEN, G. I.,
KORN, H.,
OSHIMA, T.,
TOYAMA, K.
The mode of synaptic linkage in the cerbro-ponto-cerebellar pathway of the cat. II. Responses of single cells in the pontine nuclei.
Exp. Brain Res.
24: 15-36, 1975.[Medline]
-
ALLEN, G. I.,
OSHIMA, T.,
TOYAMA, K.
The mode of synaptic linkage in the cerebro-ponto-cerebellar pathway investigated with intracellular recording from pontine nuclei cells of the cat.
Exp. Brain Res.
29: 123-136, 1977.[Medline]
-
ALLEN, G. I.,
TSUKAHARA, N.
Cerebrocerebellar communication systems.
Physiol. Rev.
54: 957-1006, 1974.[Free Full Text]
-
ALONSO, A.,
LLINÁS, R. R.
Subthreshold Na+ theta-like rhythmicity in stellate cells of entorhinal cortex layer II.
Nature
342: 175-177, 1989.[Medline]
-
BJAALIE, J. G.
The corticopontine projection from area 20 and surrounding areas in the cat: terminal fields and distribution of cells of origin as compared to other visual cortical areas.
Neuroscience
29: 81-93, 1989.[Medline]
-
BLATZ, A. L.,
MAGLEBY, K. L.
Calcium-activated potassium channels.
Trends Neurosci.
10: 463-467, 1987.
-
BORDER, B. G.,
KOSINSKI, R. J.,
AZIZI, S. A.,
MIHAILOFF, G. A.
Certain basilar pontine afferent systems are GABA-ergic: combined HRP and immunocytochemical studies in the rat.
Brain Res. Bull.
17: 169-179, 1986.[Medline]
-
BOWER, J. M.,
BEERMANN, D. H.,
GIBSON, J. M.,
SHAMBES, G. M.,
WELKER, W.
Principles of organization of a cerebro-cerebellar circuit.
Brain Behav. Evol.
18: 1-18, 1981.[Medline]
-
BRODAL, A.,
DESTOMBES, J.,
LACERDA, A. M.,
ANGAUT, P. A
cerebellar projection onto the pontine nuclei. An experimental anatomical study in the cat.
Exp. Brain Res.
16: 115-139, 1972.[Medline]
-
BRODAL, P.
The corticopontine projection in the rhesus monkey. Origin and principles of organization.
Brain
101: 251-283, 1978.[Medline]
-
CONNORS, B. W.,
GUTNICK, M. J.
Intrinsic firing patterns of diverse neocortical neurons.
Trends Neurosci.
13: 99-104, 1990.[Medline]
-
CONNORS, B. W.,
GUTNICK, M. J.,
PRINCE, D. A.
Electrophysiological properties of neocortical neurons in vitro.
J. Neurophysiol.
48: 1302-1320, 1982.[Abstract/Free Full Text]
-
CRILL, W. E.
Persistent sodium current in mammalian central neurons.
Annu. Rev. Physiol.
58: 349-362, 1996.[Medline]
-
FRENCH, C. R.,
SAH, P.,
BUCKETT, K. J.,
GAGE, P. W. A
voltage-dependent persistent sodium current in mammalian hippocampal neurons.
J. Gen. Physiol.
95: 1139-1157, 1990.[Abstract]
-
HOTSON, J. R.,
PRINCE, D. A.,
SCHWARTZKROIN, P. A.
Anomalous inward rectification in hippocampal neurons.
J. Neurophysiol.
42: 889-895, 1979.[Abstract/Free Full Text]
-
JAHNSEN, H.
Extracellular activation and membrane conductances of neurones in the guinea-pig deep cerebellar nuclei in vitro.
J. Physiol. (Lond.)
372: 149-168, 1986a.[Abstract]
-
JAHNSEN, H.
Electrophysiological characteristics of neurones in the guinea-pig deep cerebellar nuclei in vitro.
J. Physiol. (Lond.)
372: 129-147, 1986b.[Abstract]
-
KANDEL, E. R.,
TAUC, L.
Anomalous rectification in the metacerebral giant cells and its consequences for synaptic transmission.
J. Physiol. (Lond.)
183: 287-304, 1966.[Medline]
-
KOSINSKI, R. J.,
AZIZI, S. A.,
MIHAILOFF, G. A.
Convergence of cortico-and cuneopontine projections onto components of the pontocerebellar system in the rat: an anatomical and electrophysiological study.
Exp. Brain Res.
71: 541-556, 1988.[Medline]
-
LANCASTER, B.,
ADAMS, P. R.
Calcium-dependent current generating the afterhyperpolarization of hippocampal neurons.
J. Neurophysiol.
55: 1268-1282, 1986.[Abstract/Free Full Text]
-
LLINÁS, R.,
MÜHLETHALER, M.
Electrophysiology of guinea-pig cerebellar nuclear cells in the in vitro brain stem-cerebellar preparation.
J. Physiol. (Lond.)
404: 241-258, 1988.[Abstract]
-
LLINÁS, R.,
SUGIMORI, M.
Electrophysiological properties of in vitro Purkinje cell somata in mammalian cerebellar slices.
J. Physiol. (Lond.)
305: 171-195, 1980.[Abstract]
-
LLINÁS, R. R.,
GRACE, A. A.,
YAROM, Y.
In vitro neurons in mammalian cortical layer 4 exhibit intrinsic oscillatory activity in the 10- to 50-Hz frequency range.
Proc. Natl. Acad. Sci. USA
88: 897-901, 1991.[Abstract]
-
MADISON, D. V.,
NICOLL, R. A.
Control of the repetitive discharge of rat CA1 pyramidal neurones in vitro.
J. Neurophysiol.
354: 319-331, 1984.
-
MAINEN, Z. F.,
SEJNOWSKI, T. J.
Reliability of spike timing in neocortical neurons.
Science
268: 1503-1506, 1995.[Medline]
-
MARTIN, G. F.
Projections of the cerebellum to the pre-cerebellar relay nuclei in the opossum.
Anat. Rec.
175: 384, 1973.
-
MCCORMICK, D. A.,
CONNORS, B. W.,
LIGHTHALL, J. W.,
PRINCE, D. A.
Comparative electrophysiology of pyramidal and sparsely spiny stellate neurons of the neocortex.
J. Neurophysiol.
54: 782-806, 1985.[Abstract/Free Full Text]
-
MIHAILOFF, G. A.
Principal neurons of the basilar pons as the source of a recurrent collateral system.
Brain Res. Bull.
3: 319-332, 1978.[Medline]
-
MIHAILOFF, G. A.
Intra- and interhemispheric collateral branching in the rat pontocerebellar system, a flourescence double-label study.
Neuroscience
10: 141-160, 1983.[Medline]
-
MIHAILOFF, G. A.,
BURNE, R. A.,
AZIZI, S. A.,
NORELL, G.,
WOODWARD, D. J.
The pontocerebellar system in the rat: an HRP study. II. Hemispheral components.
J. Comp. Neurol.
197: 559-577, 1981.[Medline]
-
MIHAILOFF, G. A.,
KOSINSKI, R. J.,
AZIZI, S. A.,
BORDER, B. G.
Survey of noncortical afferent projections to the basilar pontine nuclei: a retrograde tracing study in the rat.
J. Comp. Neurol.
282: 617-643, 1989.[Medline]
-
MIHAILOFF, G. A.,
LEE, H.,
WATT, C. B.,
YATES, R.
Projections to the basilar pontine nuclei from face sensory and motor regions of the cerebral cortex in the rat.
J. Comp. Neurol.
237: 251-263, 1985.[Medline]
-
MÖCK, M.,
SCHWARZ, C.,
THIER, P.
Electrophysiological properties of rat pontine nuclei neurons in vitro. II. Postsynaptic potentials.
J. Neurophysiol.
78: 3338-3350, 1997.[Abstract/Free Full Text]
-
NIKUNDIWE, A. M.,
BJAALIE, J. G.,
BRODAL, P.
Lamellar organization of pontocerebellar neuronal populations. A multi-tracer and 3-D computer reconstruction study in the cat.
Eur. J. Neurosci.
6: 173-186, 1994.[Medline]
-
NISENBAUM, E. S.,
WILSON, C. J.
Potassium currents responsible for inward and outward rectification in rat neostriatal spiny projection neurons.
J. Neurosci.
15: 4449-4463, 1995.[Abstract]
-
PAPE, H.-C.
Queer current and pacemaker: the hyperpolarization-activated cation current in neurons.
Annu. Rev. Physiol.
58: 299-327, 1996.[Medline]
-
SAH, P.
Ca2+-activated K+ currents in neurones: types, physiological roles and modulation.
Trends Neurosci.
19: 150-154, 1996.[Medline]
-
SASAKI, K.,
KAWAGUCHI, S.,
SHIMONO, T.,
PRELEVIC, S.
Electrophysiological studies of the pontine nuclei.
Brain Res.
20: 425-438, 1970.[Medline]
-
SCHWARZ, C.,
SCHMITZ, Y.
The projection from the cerebellar lateral nucleus to precerebellar nuclei in the mossy fiber pathway is glutamatergic. A study combining anterograde tracing with immunogold labeling in the rat.
J. Comp. Neurol.
381: 320-334, 1997.[Medline]
-
SCHWARZ, C.,
THIER, P.
Modular organization of the pontine nuclei: dendritic fields of identified pontine projection neurons in the rat respect the borders of cortical afferent fields.
J. Neurosci.
15: 3475-3489, 1995.[Abstract]
-
SCHWINDT, P. C.,
SPAIN, W. J.,
CRILL, W. E.
Calcium-dependent potassium currents in neurons from cat sensorimotor cortex.
J. Neurophysiol.
67: 216-226, 1992.[Abstract/Free Full Text]
-
SCHWINDT, P. C.,
SPAIN, W. J.,
FOEHRING, R. C.,
STAFSTROM, C. E.,
CHUBB, M. C.,
CRILL, W. E.
Multiple potassium conductances and their functions in neurons from cat sensorimotor cortex in vitro.
J. Neurophysiol.
59: 424-449, 1988.[Abstract/Free Full Text]
-
SHAMBES, G. M.,
BEERMANN, D. H.,
WELKER, W. I.
Multiple tactile areas in cerebellar cortex: another patchy cutaneous projection to granule cell columns in the rat.
Brain Res.
156: 123-128, 1978a.
-
SHAMBES, G. M.,
GIBSON, J. M.,
WELKER, W. I.
Fractured somatotopy in granule cell tactile areas of rat cerebellar hemispheres revealed by micromapping.
Brain Behav. Evol.
15: 94-140, 1978b.[Medline]
-
SILVA, L. R.,
AMITAI, Y.,
CONNORS, B. W.
Intrinsic oscillations of neocortex generated by layer 5 pyramidal neurons.
Science
251: 432-435, 1991.[Medline]
-
STAFSTROM, C. E.,
SCHWINDT, P. C.,
CHUBB, M. C.,
CRILL, W. E.
Properties of persistent sodium conductance and calcium conductance of layer V neurons from cat sensorimotor cortex in vitro.
J. Neurophysiol.
53: 153-170, 1985.[Abstract/Free Full Text]
-
STAFSTROM, C. E.,
SCHWINDT, P. C.,
CRILL, W. E.
Negative slope conductance due to a persistent subthreshold sodium current in cat neocortical neurons in vitro.
Brain Res.
236: 221-226, 1982.[Medline]
-
STAFSTROM, C. E.,
SCHWINDT, P. C.,
FLATMAN, J. A.,
CRILL, W. E.
Properties of subthreshold response and action potential recorded in layer V neurons from cat sensorimotor cortex in vitro.
J. Neurophysiol.
52: 244-263, 1984.[Abstract/Free Full Text]
-
STORM, J. F.
Action potential repolarization and a fast afterhyperpolarization in rat hippocampal pyramidal cells.
J. Physiol. (Lond.)
385: 733-759, 1987.[Abstract]
-
THIER, P.,
KOEHLER, W.
Morphology, number and distribution of putative GABA-ergic neurons in the basilar pontine gray of the monkey.
J. Comp. Neurol.
265: 311-322, 1987.[Medline]
-
TSUKAHARA, N.,
BANDO, T.
Red nuclear and interposate nucle ar excitation of pontine nuclear cells.
Brain Res.
19: 295-298, 1970.[Medline]
-
WATT, C. B.,
MIHAILOFF, G. A.
The cerebellopontine system in the rat. I. Autoradiographic studies.
J. Comp. Neurol.
215: 312-330, 1983.[Medline]
-
WIESENDANGER, R.,
WIESENDANGER, M.
The corticopontine system in the rat. II. The projection pattern.
J. Comp. Neurol.
208: 227-238, 1982.[Medline]