Activity-Dependent Depression of Excitability and Calcium Transients in the Neurohypophysis Suggests a Model of “Stuttering Conduction”

Optical recordings of voltage changes from the neurohypophysis

Fast potentiometric dyes provide optical signals that are linearly proportional to changes in transmembrane voltage (Cohen and Salzberg, 1978). Figure 1A shows a representative trace of the fluorescence change recorded from the intact mouse neurohypophysis in response to a single stimulus using the fluorescent voltage-sensitive dye JPW 1114 (Antic et al., 1992; Antic and Zecevic, 1995). This optical record represents the voltage response of the axonal membrane (axon segments, neurosecretory swellings, and terminals) together with any other cell structures that become depolarized. After the action potential, a prominent afterdepolarization develops, which peaks ∼50 msec after the action potential. A comparable afterdepolarization in optical recordings of the membrane potential from the molecular layer of the skate cerebellum has been identified as the glial cell response to local potassium accumulation (Konnerth et al., 1987). Two lines of evidence support the same interpretation here. First, similar to the observations in skate (Konnerth et al., 1987), potentiometric dyes with different-length alkyl side chains tend to yield different relative amplitudes for the afterdepolarization and the action potential (data not shown). This suggests that the optical signal arises from two distinct types of membranes having differential affinities for different potentiometric dyes. Independently, we were able to stain the axonal membrane (axon segments, varicosities, and nerve terminals) selectively by means of anterograde loading of the dye through the cut axons of the infundibular stalk (Fig. 1B). Internal loading reverses the orientation of this membrane-impermeant dye molecule in the cell membrane, thereby changing the sign of the fast fluorescence signal (Davila et al., 1974). Superimposing the external and internal signals (Fig. 1C), it is readily apparent that selective staining of the axonal membrane replaces the afterdepolarization with an afterhyperpolarization. Because pituicytes are the only other cell type contributing a significant fraction of the membrane area in the neurohypophysis (Nordmann, 1977), we interpret the afterdepolarization as the voltage response of this glial cell type to extracellular potassium accumulation.

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Figure 1.

Optical recording of membrane potential in the neurohypophysis: external versus internal dye staining. A, Individual population action potential from the intact mouse neurohypophysis, recorded optically with a large-area photodiode after external staining with the fast potentiometric dye JPW 1114. The prominent afterdepolarization (ADP) peaks 50 msec after the action potential transient and decays within 1 sec. B, Same signal as in A after selective staining of the nerve terminals and axons by anterograde loading of JPW 1114 through the cut axons of the infundibular stalk. Because JPW 1114 is membrane-impermeant, internal loading versus external staining lead to opposite orientations of the dye molecule in the cell membrane, thereby reversing the signal polarity. C, Superposition of A and B. After internal staining, the prominent afterdepolarization of the optical signal is replaced by an afterhyperpolarization (AHP), demonstrating that the afterdepolarization arises from the voltage response of nonaxonal membranes, most likely pituicytes (see Results). For details of data acquisition and processing, see Materials and Methods.

Although eliminating the contaminating glial signal represented by the afterdepolarization, anterograde staining substantially increased phototoxicity and typically reduced signal-to-noise ratios. To avoid these problems we used external dye staining for most subsequent experiments. Fortunately, both the slow rise time (dye-independent) and the small amplitude (dye-dependent) of the pituicyte signal relative to the action potential limited the distortion of the action potential waveform to the late phase (>10 msec) of repolarization (Fig. 1C). During trains of stimuli, saturation of the glial response (Konnerth et al., 1987) further decreases the contribution of the afterdepolarization. Thus, to quantify overall excitability changes, we have used the amplitude of the extrinsic transient optical changes, ΔV_tr, as measured from the onset of the voltage signal to the peak of the action potential. This approach to the separation of the glial from the neuronal signal is further validated by the observation that dye loss during optical measurement of membrane potential was selective, reducing the glial afterdepolarization much more rapidly than the fast optical signal from the neuronal membrane. This loss of glial signal, however, had no effect on the changes in ΔV_tr peak amplitudes detailed below.

Voltage versus calcium transients during repeated stimulation

One objective was to determine whether and how transmembrane voltage responses change during physiologically relevant episodes of repetitive firing. Also, if there are changes in the action potential, we wanted to explore how these changes might contribute to the observed frequency-dependent modulation of the associated calcium transients (Muschol and Salzberg, 2000). To this end, we measured and compared the paired pulse ratios for the tissue-averaged ΔV and Δ[Ca²⁺] transients. We also compared the amplitude modulation of each of these signals as a function of stimulus frequency during prolonged trains of stimuli.

Paired pulse modulation of ΔV and Δ[Ca²⁺] transients

Figure 2 displays representative optical recordings of membrane potential (Fig. 2A) and calcium transients (Fig. 2B) after pairs of stimuli at 4 of the 15 different pulse separations (T_sep) used for these measurements. The amplitudes of the fast transients of each optical trace were determined from the difference between the signal level immediately preceding the stimulus and its subsequent peak value. This procedure effectively eliminates the contribution from the slow component present in each of the optical signals (pituicyte afterdepolarization for ΔV_tr and residual calcium rise and decay for ΔCa_tr) to the stimulus-induced amplitude changes.

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Figure 2.

Paired pulse recordings of transmembrane voltage and calcium changes in the intact neurohypophysis. A, Spatially integrated voltage response of the neurohypophysis to a pair of stimuli at various pulse separations (T_sep = 50, 100, 300 msec and 1 sec). The peaks of the first transients, ΔV_tr, in each pair are matched to one another and superimposed. Except for the T = 1 sec trace, all records were truncated shortly after the second peak. The optical voltage signals have been inverted so that depolarization is shown as an upward deflection. Dye: JPW 1114, external staining. B, Calcium transients, Δ[Ca²⁺]_tr, in response to a pair of stimuli with the same pulse separations as in A. The fast-rising calcium transient, Δ[Ca²⁺]_tr, is followed by a bimodal decay phase. The elevated baseline in the calcium signal originates from a very slow decay component not well resolved on this time scale (for details, see Muschol and Salzberg, 2000). All traces are peak-matched, superimposed, and truncated as in A. Dye: magfluo-4 AM. All records are low-pass filtered at 500 Hz and digitized at 1 kHz. C, Facilitation and depression of the paired pulse ratios of the ΔV (filled squares) and Δ[Ca²⁺] (open circles) transients as a function of the pulse separation, T_sep. The displayed ratios represent averages from several measurements (ΔV, n = 6; ΔCa, n = 3). Error bars indicate 1 SD from the mean. For details of data processing, see Materials and Methods. D, Same data as in C on a logarithmic time scale. ΔV_tr and Δ[Ca²⁺]_tr display different correlations in each of the three indicated time windows, I-III (see Results).

Figure 2, C and D, shows the paired pulse ratios of ΔV_tr and Δ[Ca²⁺]_tr versus pulse separation, T_sep, on linear and logarithmic time scales. For clarity, we used a logarithmic time axis in Figure 2D and divided it into three separate time domains. ΔV_tr paired pulse ratios facilitate only slightly (<3%) in region III (200 msec ≤ T_sep ≤ 1 sec) but depress noticeably in regions II (60 msec ≤ T_sep ≤ 200 msec) and I (T_sep ≤ 60 msec). In contrast, ratios of Δ[Ca²⁺] transients show prominent and persistent paired pulse facilitation in region III, peaking at ∼20%. In region II, ΔCa_tr and ΔV_tr ratios deteriorate at equal rates with decreasing pulse separation. For T_sep in region I, the rate of ΔCa_tr deterioration accelerates significantly as pulse separation decreases, eventually exceeding ΔV_tr depression for T_sep of <30 msec.

The separation of time scales for the onset of ΔCa_tr facilitation versus depression implies that these two processes are activated by separate mechanisms. This conclusion is consistent with the observation that the recovery rates for ΔCa_tr facilitation and depression during burst stimulation are equally distinct (Muschol and Salzberg, 2000). The absence of ΔV_tr facilitation in region III indicates that ΔCa_tr facilitation is not directly related to changes in the population spike amplitude. The tight coupling of the onset and the rate of deterioration for ΔCa_tr and ΔV_tr ratios in region II (60 msec ≤ T_sep ≤ 200 msec), in turn, suggest that ΔV_tr changes are responsible for the decline in ΔCa_tr facilitation and the eventual onset of ΔCa_tr depression in region I.

The decline in the population spike amplitude with decreasing pulse separation, although surprisingly large, could indicate either a reduction in the peak amplitude of the action potential (partial refractoriness) or a loss of excitable membrane area resulting from changes in tissue excitability. Similarly, the correlation between ΔV_tr depression and the decline of ΔCa_tr is suggestive but might not reflect a causal connection. We have shown previously (Muschol and Salzberg, 2000), however, that ΔCa_tr amplitudes deteriorate by as much as 80% during trains of stimuli at 40 Hz. A comparable decline in ΔV_tr amplitudes during repeated stimulation could no longer be reconciled with a decline in the amplitude of individual action potentials. Instead, it would strongly support the idea that the loss of excitable membrane area attributable to excitability and invasion changes dominates ΔV_tr (population) amplitude depression, and this decline contributes significantly to the depression of tissue-averaged calcium transients.

ΔV_tr and ΔCa_tr modulation during trains of action potentials

A characteristic voltage signal from the neurohypophysis in response to a train of 40 stimuli at 15 Hz is shown in Figure 3A. Each stimulus evokes a population action potential superimposed on a slowly increasing fluorescence background. The transients related to the action potential, ΔV_tr, rise rapidly from this background and can be readily separated from it (Fig. 3A). The background increase consists of at least two components: the glial afterdepolarization, dominant at low frequencies, and activity-dependent dye internalization (Rohr and Salzberg, 1994), prominent at higher frequencies (also see Materials and Methods). Measurements of calcium transients, ΔCa_tr, corresponding to the same stimulation conditions as in Figure 3A are displayed in Figure 3B. Again, the rapid rising phase of ΔCa_tr is well separated from the slow increase in residual calcium.

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Figure 3.

Frequency-dependent modulation of voltage and calcium transients during trains of action potentials. A, Representative trace of the voltage signal from the neurohypophysis in response to a train of 40 stimuli delivered at 15 Hz. The rapid voltage transients rise from a slowly modulated increase in background fluorescence. The background increase consists of the glial afterdepolarization and contributions from activity-dependent dye internalization (e.g., membrane reuptake). B, Calcium changes recorded with the calcium indicator dye mag-fluo-4 AM under the same stimulation conditions as in A. C, Amplitude modulation of transmembrane voltage transients, relative to the first transient in a given train, at five different frequencies (n = 6). D, Modulation of the amplitude ratios of successive calcium transients, relative to the first transient in a given train (data from Muschol and Salzberg, 2000; error bars below symbol size). For details of measurements and data processing, see Materials and Methods.

The modulation of ΔV_tr and ΔCa_tr amplitudes with ordinality, and at various stimulation frequencies, is summarized in Figure 3, C and D, respectively. At the lowest stimulation frequencies (≤5 Hz), there is a modest facilitation of ΔV_tr during the first 20 stimuli. Beginning at 15 Hz, the dominant effect is the pronounced depression of the population spike. The depression deepens throughout the stimulus train and reaches values as high as 75% after 40 stimuli at 40 Hz. The dramatic decline in the population spike amplitude implies that a loss of excitable membrane area, and not a reduction in the peak amplitude of individual action potentials, dominates the observed ΔV_tr modulation. The modulation of ΔCa_tr is identical to that reported previously (Muschol and Salzberg, 2000). Comparing the variation of the two signals, several features are evident. The facilitation of ΔV_tr at low frequencies is likely to contribute to but cannot fully explain the magnitude and persistence of the pronounced facilitation of ΔCa_tr (past n_stim = 20) in that frequency range. In contrast, the prominent depression of ΔV_tr correlates very well with the corresponding depression of ΔCa_tr at frequencies of >10 Hz (at n_stim ≥ 20). Similar to the paired pulse data, the rate of depression with ordinality of ΔCa_tr at high frequencies exceeds the depression of ΔV_tr, particularly early in the train. Nevertheless, the loss of excitability at moderate to high frequencies and during prolonged stimulus trains can readily account for a significant fraction of the depression of calcium transients.

Spatial properties of excitability changes

Although the single-photodiode recordings establish the importance of ΔV_tr modulation, these data do not ascribe responsibility to any specific mechanism. Indeed, a variety of distinct mechanisms could account for the observed changes in the population action potential. On the basis of the actual axonal morphology (Bourque, 1990, his Fig. 10; Jackson, 1993, his Fig. 4), we have considered four different models, represented schematically in Figure 4. Axons (including their varicosities and terminals) might become partially refractory during repetitive stimulation, leading to a uniform decrease in action potential amplitude (Fig. 4, model A). Alternatively, the action potential may invade the whole axonal arbor (including varicosities) but may fail to evoke active responses locally in nerve terminals (Fig. 4, model B). An overall increase in the action potential threshold, with whole axons becoming refractory to spike initiation at the site of stimulation, provides yet another possible explanation (Fig. 4, model C). Finally, action potential propagation might be blocked in distal parts of the ramified axonal arbor (Obaid and Salzberg, 1996), with the rate of propagation failures depending on stimulation frequency (Fig. 4, model D).

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Figure 4.

Models for the amplitude modulation of the population action potential: diagram representing the essential morphological features of the neurohypophysial axonal arbor (for actual axonal morphology displays, see Nordmann, 1977; Bourque, 1990; Jackson, 1993). Straight segments, Axon; V, in-line secretory varicosities; Bp, axonal branch points; T, nerve terminals. The waveforms above the axon indicate the local response (spike, active propagation; exponential or line, passive spread and decay) to a stimulus at the left margin. Shading indicates the spatial spread of inactivation: white, fully active; black, completely passive. A, Increased refractoriness. The sodium channels in the axons do not completely recover in between stimuli, thereby reducing the amplitude of the action potential. B, Local loss of excitability. Only nerve terminals become inexcitable (e.g., through the activation of a localized Ca-dependent K-conductance; see Results). The action potential invades the nerve terminals passively. C, Partial failure of spike initiation. A fraction of the axonal branches becomes completely refractory to spike initiation at the site of stimulation, whereas others remain active (indicated by a split between black, white). D, Propagation failure and block. The action potential is blocked from invading the distal parts of the axon, by failing either at passivated varicosities or at branch points. Note that the axonal segments, nerve terminals, and varicosities are distributed approximately uniformly throughout the intact neurohypophysis. Therefore, models A-C predict a uniform decline of locally averaged recordings of the transmembrane voltage, whereas model D predicts increasing failures with distance from the stimulation site. E, Model of stuttering conduction (see Discussion). Varicosities and nerve terminals lose their excitability after repeated stimulation. The action potential invades these inactive structures passively but carries enough current to elicit an active response downstream. Each time the action potential passes through a passive varicosity, it picks up a small but finite delay; i.e., it stutters. Stuttering conduction readily accounts for both the spatially uniform decline in the amplitude of the locally averaged population action potential and the increase in temporal dispersion and latency with location and stimulus ordinality.

Ideally, to distinguish between these different models, one should image action potential propagation in individual axonal branches. Because fast potentiometric dye signals are small in amplitude, and only dye molecules associated with the cell surface contribute to the signal, multiple-site optical recording of transmembrane voltage, in an intact tissue, with subcellular resolution (Rohr and Salzberg, 1994) is technically feasible only under special circumstances [e.g., intracellular injection of single somata (Antic et al., 1999) or extracellular staining of two-dimensional networks (Obaid et al., 1999)]. These models do, however, predict distinct spatial patterns of action potential modulation that can be tested on a macroscopic scale. Because varicosities and terminals are distributed approximately uniformly along the axonal arbors in the neurohypophysis, and because even individual camera pixels record the average response from many of these structures, increased refractoriness (Fig. 4, model A), failures of spike initiation (Fig. 4, model C), or local loss of excitability at axonal terminals (Fig. 4, model B), should all result in a spatially uniformly reduction of the population spike amplitude. In Figure 4, model D, the number of in-line varicosities and branch points encountered by the action potential increases along the axon. Thus, failures will accumulate, causing ΔV_tr depression to intensify with distance from the stimulation electrode.

Using a high-speed CMOS camera developed recently by one of us (M.I.), with 100 × 100 pixels and frame rates of up to 10 kHz, we set out to determine the spatial pattern of ΔV_tr modulation to distinguish between these different mechanisms. Although morphological variations in pituitary size and shape preclude the averaging of results from multiple preparations, the image data discussed below represent the typical behavior seen with five different preparations. Figure 5A, panel 1, shows the resting fluorescence of the preparation stained by bath application of JPW 1114. The stimulation electrode is located slightly outside the recording area, as indicated by the bar. Figure 5A, panels 1-11, are the false-color images of the change in resting fluorescence (ΔF) as the first population action potential sweeps from the top to the bottom across the preparation. As is evident from the sequence in Figure 5A, the first action potential spreads over the entire tissue.

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Figure 5.

Optical recordings of the spatial pattern of action potential invasion. A, Panel 1, Resting fluorescence of the neurohypophysis after staining with JPW 1114. The location of the stimulation electrode is indicated. Panels 1-11, Consecutive frames (1 msec spacing) of the change from resting fluorescence, representing the propagation of the population action potential induced by a single stimulus. The fluorescence background (panel 1) has been subtracted from panels 1-11. The maximal fluorescence change shown represents 2.5% of the resting fluorescence of the dye. B, Fluorescence change at the peak of the population action potential after the 2nd, 5th, 10th, 20th, 30th, and 40th stimulus at three different frequencies: 10, 25, and 40 Hz. With persistent stimulation, the amplitudes of the local fluorescence changes decline uniformly throughout the preparation. This spatially uniform decline in spike amplitude increases with both the ordinality (left to right) and frequency (top to bottom) of stimulation. All differential false-color images have been corrected for several slow components of fluorescence changes not related to the population action potential (see Materials and Methods). Also, see supplemental material at http://www.med.upenn.edu/nscience/neuro_muschol.

The data permit us to obtain an approximate estimate for the propagation speed of the action potential under our recording conditions. The peak of the action potential traverses the ∼800 μm distance from the top to the bottom of the preparation in 5 msec, corresponding to a conduction velocity of 160 mm/sec at room temperature. This is approximately half the value determined from extracellular recordings of the compound action potential at 36°C (Nordmann and Stuenkel, 1986) and compares very well with the value of 190 mm/sec obtained from numerical simulations of action potential propagation in this preparation (Jackson and Zhang, 1995).

The fluorescence difference images provide an optical record of the spatiotemporal pattern of action potential invasion. To discern changes in the spatial invasion of the action potential during repeated stimulation, we compared the frames representing the peak intensity of the spatially averaged action potential after the 2nd, 5th, 10th, 20th, 30th, and 40th stimuli. Figure 5B shows this sequence of frames for 10, 25, and 40 Hz stimulus trains. All three sequences illustrate the overall decline in depolarization with ordinality (repeated stimulation), with the rate of decline increasing with stimulus frequency. This result confirms the behavior observed in the single photodiode recordings shown in Figure 3, A and C. More importantly, within the limits of spatial resolution for these recordings (8 μm²/pixel), the reduction in action potential amplitude appears spatially uniform. There is, however, some residual spatial heterogeneity in the optical records, with apparent hot spots lateral to the midline on both sides of the preparation (e.g., Fig. 5B, frame 1). These spatial variations in the ΔF records of the first stimulus are not readily interpretable because they can arise from a combination of nonuniform dye staining and variations in the local density of excitable membrane area (axons and nerve terminals).

To confirm or disprove the visual impression of spatially uniform amplitude modulation, we analyzed quantitatively traces of local ΔV_tr changes. As indicated in Figure 6A, we extracted fluorescence versus time traces at three different locations on the preparation: close to the stimulation electrode (top), near the center of the preparation (center), and at the end farthest from the stimulation electrode (bottom). To improve the signal-to-noise ratio, the image data were locally averaged over an area of 10 × 10 pixels. As with the single photodiode data, we determined the ΔV_tr amplitude ratios of the nth to the first stimulus, ΔV_tr,n/ΔV_tr,1, for all three stimulation frequencies but now at each of the three locations independently (Fig. 6B). This analysis intrinsically accounts for variations in the local density of excitable membrane. Within the noise, the amplitude modulation of ΔV_tr transients is completely independent of location. This remains true for all three frequencies investigated here. A comparable analysis of local ΔV_tr modulation at the two apparent hot spots in Figure 5B (left, right) yielded identical results (Fig. 6B). Therefore, the weak residual heterogeneity of the optical response seen in Figure 5, A and B, arises from density variations of stained membrane, excitable membrane, or both.

Changes in waveform of the population spike

Besides the amplitude modulation within a train, the waveform of the population action potential also changes significantly with stimulus frequency, ordinality, and recording location. These changes fall into two distinct categories: altered temporal dispersion, which modifies the summation of individual action potentials into the population spike, and changes in the waveform of the underlying action potentials themselves.

For any given ordinality, the width of the ΔV_tr spike is much narrower proximal to the stimulation electrode (Fig. 7A, top) than at the two distal locations (Fig. 7A, center, bottom). Broadening at the distal locations is approximately symmetric around the peak of the population spike, increases further with stimulus ordinality, and is accompanied by a temporal shift (increased latency) of the peak position. These properties suggest that distal broadening originates from the temporal dispersion of the propagating excitation wave, traveling via different axonal pathways to the recording location. The increase in temporal dispersion of the locally recorded population spike with distance is already evident in the first response to a stimulus train (Fig. 7A, compare first spike, top to bottom). The morphological substrate for this initial rise in temporal dispersion is undoubtedly the intrinsic axonal branching pattern in the neurohypophysis.

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Figure 7.

Changes in action potential waveform: action potential broadening versus temporal dispersion of the population action potential. A, Superposition of the waveform of the 1st (solid line), 10th (dotted line), 20th (solid line), 30th (dashed, dotted line), and 40th (solid line) transients of locally extracted traces from a 25 Hz stimulus train. The amplitudes of the transients have been matched to one another. The asymmetric broadening of the population action potential at the top is indicative of a small broadening of the individual action potentials, which develops gradually with ordinality. In contrast, the increase in peak latency and symmetric broadening of the peak at the more distal locations is indicative of temporal dispersion of the transients. With increasing distance from the stimulation electrode, temporal dispersion rapidly comes to dominate the changes in the population spike waveform. B, Temporal dispersion versus location. Changes in the rise time (τ_rise) of the local ΔV_tr transients in A at a location near (open symbols) and far (filled symbols) from the recording electrode are shown. The difference in rise time for n_stim = 1 indicates the intrinsic difference in temporal dispersion attributable to the axonal branching morphology (see Results). The dynamic increase of rise times with n_Stim is approximately constant at the proximal location (overlapping open traces) but accelerates noticeably with frequency at the more distant location. Rise times were obtained from Gaussian fits to the rising phase and peak region of individual optical transients. Straight lines represent least-squares fits through the rise time data.

The temporal dispersion of the population action potential is not constant in time, either; the latency and the width of the ΔV_tr peaks increase throughout any given stimulus train (Fig. 7A,B). The fixed axonal branching morphology cannot account for this dynamic component of temporal dispersion. Furthermore, the dynamic broadening at more distal locations is augmented by raising stimulation frequency (Fig. 7B). As a consequence of the frequency and ordinality dependence, the time to peak of the population spike can easily increase by several milliseconds at the more distal locations, reducing the speed of action potential propagation appreciably. The spatial variation in temporal dispersion of the population action potential is in stark contrast to its spatially homogeneous amplitude modulation.

The optical traces also suggest that there are changes in the waveform of individual action potentials. The optical transients, ΔV_tr, at the most proximal location (Fig. 7B, top) have quite similar rise times but broaden noticeably at the falling flank of the action potential (Fig. 7A, top). As previously noted (Gainer et al., 1986), this behavior suggests broadening of the individual action potential waveform and is equivalent to the spike broadening observed in electrophysiological recordings from the neurohypophysis (Bourque, 1990; Jackson et al., 1991). Broadening of the individual action potential is masked at the more distal locations by the rapidly increasing temporal dispersion of the population spike.

Calcium dependence of ΔV_tr amplitude modulation

The spatially homogeneous modulation of ΔV_tr amplitudes is clearly inconsistent with failures of action potential propagation (Fig. 4D) but is compatible with several of the alternative models. One intriguing mechanism is the reported failure of nerve terminals and in-line varicosities to support active responses after repeated stimulation (Bourque, 1990; Bielefeldt and Jackson, 1993). This local loss of excitability is apparently related to a slowly activating, Ca²⁺-dependent K⁺ conductance (Bielefeldt and Jackson, 1993). No selective toxin for this K⁺ channel has been identified. However, interference with Ca²⁺ influx strongly affects the rate of these local excitability failures (Bourque, 1990; Bielefeldt and Jackson, 1993). Figure 8 quantitates the effects of the Ca²⁺ blocker CdCl₂ and the L-type Ca²⁺ channel agonist (S-)Bay-K 8644 on the macroscopically observed modulation of ΔV_tr amplitudes. Strikingly, 100 μm CdCl₂ nearly completely abolishes both the facilitation and depression of ΔV_tr amplitudes. Only during 40 Hz stimulation does a residual component of depression persist (Fig. 8C). The CdCl₂ result implies that Ca²⁺ influx is required for both the facilitation and depression of ΔV_tr. Augmenting Ca²⁺ entry with Bay-K 8644 does not alter facilitation but, as expected (Bielefeldt and Jackson, 1993), enhances depression of ΔV_tr amplitudes, particularly at intermediate frequencies. The depicted sensitivity of ΔV_tr amplitudes to both CdCl₂ and Bay-K 8644 supports the idea that the local loss of active responses in nerve terminals and varicosities, mediated by a Ca-activated K conductance, generates the macroscopic changes in the population action potential seen in our optical data.

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Figure 8.

Calcium dependence of ΔV_tr modulation. Modulation of ΔV_tr amplitude ratios, ΔV_tr,n/ΔV_tr,1, recorded from a single preparation before (open circles) and after (filled squares) application of CdCl₂ (100 μm; A) or the L-type Ca²⁺ agonist (S-)Bay-K8644 (10 μm; B) is shown. The amplitudes of the individual transients used to generate these data were extracted from optical recordings of voltage changes as shown in Figure 3A. To quantify the effect of the drugs, we compared their impact on the maximum of the facilitation and the depression of ΔV_tr amplitudes during a continuous train of 40 stimuli at three different frequencies. Each pair of bars in the charts below summarizes the average values (n = 4; error bar indicates 1 SD) of maximal facilitation (upward, open bars) and depression (downward, filled bars) in control or after exposure to either CdCl₂ (100 μm; C) or (S-)Bay-K 8644 (10 μm; D). The effects of the drugs are shown separately for each of the three indicated frequencies.

ΔV_tr and Δ[Ca²⁺]_tr amplitude modulation and the role of action potential broadening

The results of our optical experiments indicate that both the amplitude and the waveform of the population action potential are noticeably altered during repetitive stimulation. Particularly surprising is the strong amplitude modulation. During repeated stimulation at low frequencies, some facilitation emerges. The dominant effect, however, is amplitude depression, which develops after persistent stimulation even at low frequencies and accelerates dramatically with increasing stimulation frequency (see Fig. 3C). The combined paired pulse recordings of ΔV and ΔCa²⁺ transients reveal that ΔV_tr depression is bound to contribute to ΔCa_tr depression. At the same time, the lack of ΔV_tr facilitation and onset of ΔV_tr depression during ΔCa_tr facilitation (see Fig. 2D, regions III, II, respectively) indicate that ΔV_tr modulation cannot account for all aspects of ΔCa_tr variation. Other mechanisms involved in Δ[Ca²⁺]_tr depression might include calcium channel inactivation (Branchaw et al., 1997) or buildup of extracellular potassium (Leng and Shibuki, 1987).

In addition to the amplitude modulation, the optical data reveal significant changes in the waveform of the population action potential. The activity- and site-dependent rise in temporal dispersion dominates these waveform changes. Dispersion and increasing latency are robust features of action potential invasion in the neurohypophysis, observed consistently in our optical experiments, during electrophysiological recordings of the compound action potential (Nordmann and Stuenkel, 1986), and in action potentials recorded from individual nerve terminals (Bourque, 1990). Any model of the amplitude modulation of the population action potential also has to account for this dynamic increase in latency.

Could action-potential broadening give rise to the large facilitation of ΔCa²⁺ transients? Increased calcium influx caused by action potential broadening was one of the earliest mechanisms invoked to explain the frequency dependence of hormone release (Gainer et al., 1986; Bourque, 1990; Jackson et al., 1991). It has recently received renewed attention at hippocampal synapses (Geiger and Jonas, 2000). We have previously argued that the rapid onset of ΔCa²⁺ facilitation with ordinality does not agree well with the gradual development of action potential broadening in the neurohypophysis seen in optical and electrophysiological recordings (Gainer et al., 1986; Bourque, 1990; Jackson et al., 1991). Consistent with our reservations, broadening of the optically recorded ΔV_tr waveform is most noticeable toward the end of stimulation trains and at high frequencies (Fig. 7A, top), similar to the results from electrophysiological measurements of broadening (Bourque, 1990, his Fig. 6). High stimulation frequencies and stimulus ordinality, however, are both conditions associated with large depression, not facilitation, of ΔCa²⁺ transients (Muschol and Salzberg, 2000, their Fig. 3).

Mechanisms of ΔV_tr modulation

It was our a priori expectation that the dramatic decline in ΔV_tr amplitudes (up to 75%; see Fig. 3C) reflected failures and block of action potential propagation at axonal varicosities or branch points (Fig. 4, model D). This expectation was based on three previous observations. First, invasion can be altered significantly in the amphibian neurohypophysis (Obaid and Salzberg, 1996). Second, during action potential propagation in the rat neurohypophysis, sharp electrode recordings from nerve terminals revealed a sudden transition from active responses to catastrophic failure (Bourque, 1990, his Fig. 9). Third, local current injection into nerve terminals frequently failed to evoke active responses during repeated stimulation (Bielefeldt and Jackson, 1993). Block of propagation, however, should preferentially decrease the amplitude of the population action potential at locations distal to the stimulation electrode (Fig. 4, model D). Clearly, this does not occur; instead, the amplitude of the population action potential decreased uniformly throughout the neurohypophysis (see Figs. 5, 6).

In principle, failures of action potential initiation at the site of simulation (Fig. 4, model C), combined with enhanced refractoriness to account for the increase in latency (Fig. 4, model A), could produce the pronounced amplitude depression of the population action potential. Two observations argue against this interpretation in the neurohypophysis. Most significantly, Bourque (1990) noted that, although local loss of excitability was readily observed in nerve terminals and varicosities, action potentials recorded from the axons of the infundibular stalk showed no signs of refractoriness or propagation failure. In addition, the increased latency with stimulus ordinality, far from the stimulation electrode (Fig. 7A,B), seems to imply that distal locations would be more prone to remain below the threshold for action potential generation. Therefore, failures should arise there preferentially and not at the stimulation site. This is at variance with the spatially uniform decline in the population action potential seen in our optical data (Fig. 6B).

The effects of CdCl₂ and Bay-K 8644 on ΔV_tr modulation (Fig. 8), conversely, argue for a significant role for local excitability failures in the macroscopically observed modulation of ΔV_tr amplitudes. Given the limited resolution of our optical recordings (8 μm²) and the uniform distribution of axons, nerve terminals, and varicosities within the neurohypophysis, local loss of excitability is consistent with the spatially homogeneous change in the amplitude of the population action potential. The hypothesis of selective excitability loss in terminals and varicosities is further buttressed by evidence that neurohypophysial nerve terminals have populations of K⁺ and Ca²⁺ channels that are electrophysiologically and pharmacologically different from those in their magnocellular somata (Fisher and Bourque, 1995; Dopico et al., 1999). The distinctive populations of Ca²⁺ and K⁺ channels in terminals and varicosities would then provide the macromolecular machinery underlying local loss of excitability. The restriction of local excitability failures to axonal end points (Fig. 4, model B), however, is inconsistent with morphological studies that indicate that a substantial fraction of the secretory sites are in-line varicosities (Jackson, 1993), which do inactivate (Bourque, 1990; Bielefeldt and Jackson, 1993). Therefore, the action potential has to propagate through passive varicosities without failing.

These considerations suggest to us the modified version of “local loss of excitability” shown schematically in Figure 4E. Consistent with existing electrophysiological evidence (Bourque, 1990; Bielefeldt and Jackson, 1993), we assume that repeated stimulation eventually fails to evoke active responses in some in-line varicosities and nerve terminals. The combined cable properties of axonal segments and varicosities, however, permit the action potential to spread passively through varicosities and, yet, to evoke an actively propagated response after emerging distal to the swellings. The latter conjecture is supported by numerical simulations by Jackson and Zhang (1995), which indicate that action potentials can propagate through in-line varicosities in the neurohypophysis, even when the active response in the varicosity itself is suppressed by inhibitory input. We have termed this model “stuttering conduction” because it also provides a straightforward explanation for the dynamic retardation of the action potentials. The active propagation of the action potential is progressively retarded by the increasing number of passive structures that the voltage signal has to negotiate. This activity-dependent rise in local latency should increase dispersion with distance from the stimulating electrode, consistent with our optical data (Fig. 7A). Latency will also tend to increase during sustained stimulation, as more and more secretory varicosities become inactivated. (Nordmann and Stuenkel, 1986; Bourque, 1990; this study, Fig. 7B). In short, stuttering conduction provides a compelling, self-consistent explanation for all the experimental observations on action potential propagation and local loss of excitability in the neurohypophysis.

Conclusions

Sustained stimulation of the mammalian neurohypophysis induces a frequency-dependent modulation of the population action potential. The magnitude of ΔV_tr depression, in particular, establishes the loss of excitable membrane area as the dominant contribution to the modulation. The onset and magnitude of the depression in excitability is closely related to concurrent changes in intraterminal calcium transients. Excitability changes, therefore, play a significant role in the short-term depression of peptide release from the mammalian neurohypophysis during physiologically relevant episodes of stimulation. The spatial pattern of excitability depression reveals that the amplitude of the population action potential decays uniformly everywhere, whereas temporal dispersion increases with both the distance from the stimulation site and the stimulus ordinality. To account for these two specific and robust features of the optical data and the existing electrophysiological evidence we have proposed a model of stuttering conduction.

It remains to be seen whether local excitability changes as a mechanism of short-term plasticity are limited to the neurohypophysis or to peptide release. For example, the observed retardation of the action potential could be considered a peculiarity of the specialized morphology of secretory varicosities arrayed linearly along the axons. This morphology is, however, strongly reminiscent of the en passant synapses between parallel fibers and Purkinje neurons in the cerebellum (Xu-Friedman et al., 2001) and could alter the spatial and temporal summation of synaptic inputs from cerebellar granule cells. Finally, the calcium-dependent regulation of the local excitability of nerve terminals and secretory swellings suggests yet another role for calcium in neuropeptide release. The combination of Ca²⁺ channels and Ca²⁺-dependent K⁺ channels implicated in these local excitability changes (Bourque, 1990; Bielefeldt and Jackson, 1993), however, is commonly found in cells with Ca²⁺-triggered exocytosis (Jackson, 1995). Local regulation of presynaptic excitability, thus, could emerge as a natural and possibly widespread mechanism for evoking short-term plasticity in other areas of the CNS.