Exp Brain Res (1993) 97:325-333

Experimental BrainResearch 9 Springer-Verlag 1993

Membrane potential oscillations in CA1 hippocampal pyramidal neurons in vitro: intrinsic rhythms and fluctuations entrained by sinusoidal injected current Antonia Garcia-Mufioz, Luis C. Barrio*, Washington Bufio Instituto Cajal, CSIC, Av. Doctor Arce 37, E-28002 Madrid, Spain Received: 17 December 1992 / Accepted: 15 July 1993

Abstract. The mechanisms mediating intrinsic and en-

Key words: CA1 pyramidal neurons - Na + plateau po-

trained CA1 pyramidal neuron rhythmic membrane potential oscillations were investigated in rat hippocampal slices. Intrinsic oscillations (6-14Hz, <10mV) were evoked by long duration (2 s), depolarizing current pulses in 42% of the cells. Oscillations were also evoked by imposing sinusoidal transmembrane currents at 2, 7, and 14 Hz, adjusted at 7 Hz to imitate the synaptically mediated in vivo "intracellular theta". Slow all-or-none events (40 mV, 55 ms) - reminiscent of the rhythmic, high threshold slow spikes observed in vivo - were evoked and entrained by the sine wave current cycles with large, imposed depolarization in 35% of the cells. Intrinsic oscillations were insensitive to Ca2+-free, Co 2+ (2 mM) and Mn 2+ (2 raM) solutions, but were blocked by tetrodotoxin (TTX; 5 gM), illustrating that they were Na+-mediat ed. Tetraethylammonium (TEA; 15 mM) unmasked slow all-or-none events (40-50 mV, 20-55 ms) and plateau potentials (40-60 mV, 100-700 ms). Plateaus were Co 2+ and Mn 2+ resistant and were abolished by TTX, hence suggesting that the underlying persistent conductance was Na+-mediated. Plateaus were entrained one-to-one at all sinusoidal current frequencies in Ca 2+-free, TEA + Co 2+, or TEA + Mn 2+ solutions. However, the high threshold Ca 2+ spikes uncovered in TEA + TTX could only follow sinusoidal currents of less than 7 Hz. In conclusion, the high threshold Ca 2+ and persistent Na + conductances coexist in CA1 pyramidal cells. The persistent Na + conductance mediated the intrinsic oscillations, and fluctuated at all the sine wave current frequencies used. The more sluggish high-threshold Ca 2+ conductance exclusively oscillated at frequencies of less than 7 Hz and did not support the intrinsic rhythm. Therefore, the findings suggest that the Na+-mediated oscillations may contribute to the high-frequency, type I, hippocampal theta rhythm present in vivo, whereas the high threshold Ca 2+ conductance may take part in the low-frequency, type II rhythm.

tentials High threshold Ca 2+ spikes - Persistent Na + conductance - Theta rhythm - Rat

*Present address: Depto. de Investigacidn, Hospital "Ram6n y CajaI', Ctra. de Colmenar Km 9, E-28034 Madrid, Spain Correspondence to: W. Bufio

Introduction In specified behavioral states the hippocampus displays a characteristic, sinusoidal-like, rhythmic slow activity at 4-12 Hz, called theta (0) rhythm (e.g., Bufio et al. 1978; Bufio and Velluti 1977; Garcia-Sfinchez et al. 1978; Green and Arduini 1954; Macadar et al. 1970; Steriade et al. 1990). Although 0 rhythm is believed to occur as a result of the activity of the septohippocampal cholinergic pathway (Alonso et al. 1987; Apostol and Creutzfeldt 1974; Gaztelu and Bufio 1982; Lewis and Schute 1967; Macadar et al. 1970; Pestche et al. 1962), the underlying cellular mechanisms remain controversial. It has been suggested that the dominant mechanism for the generation of 0 rhythm is the synchronous driving of hippocampal pyramidal neurons by rhythmic excitatory postsynaptic potentials (EPSPs) arising from medial septal neurons and neurons of the diagonal band of Broca (Fujita and Sato 1964; Nufiez et al. 1987, 1990a). However, inhibitory postsynaptic potentials (IPSPs) may also participate (Andersen and Eccles 1962; Artemenko 1973; Fox 1989; Leung and Yim 1986). Subsequently, it has been determined that rhythmicity resembling 0 rhythm could be induced intrinsically in the hippocampus in vitro following carbachol superfusion (Konopacki et al. 1987). This rhythmicity has been interpreted as resulting from synaptic circuitry intrinsic to the hippocampus (MacVicar and Tse 1989). Furthermore, conclusive evidence has been provided that endogenously generated rhythmic slow spikes in identified CA1-CA3 pyramidal neurons participate in "intracellular 0 " genesis in vivo (Nufiez et al. 1987). The rhythmic, highthreshold, slow all-or-none events were interpreted as high threshold Ca 2+ spikes (e.g., Benardo et al. 1982; Schwartzkroin and Slawsky 1977; Wong and Prince

326 1978). Support in favor of their Ca 2+ nature was later provided by Nufiez and Bufio (1992) in QX-314 loaded pyramidal cells in vivo. Another voltage-gated current which m a y take part in pyramidal neuron 0 rhythm genesis is the slowly inactivating or persistent N a + conductance (e.g., French et al. 1990; Garcia-Mufioz et al. 1991). There is evidence that this conductance accounts for the slow prepotentials (MacVicar 1985) and the low-amplitude ( < 6 mV), rhythmic 2- to 10-Hz oscillations observed in vitro in depolarized pyramidal neurons (Leung and Yim 1991) and in entorhinal cells (Alonso and LlinAs 1989). Consequently, 0 rhythm does not exclusively originate from network properties but is the outcome of interactions between septal and hippocampal oscillators. A general principle governing the interactions between oscillators is that one oscillator can be driven by another (e.g., Barrio and Bufio 1990; G u e v a r a et al. 1988). Interestingly, this behavior, called "entrainment" or "phase-locking" occurs in CA1 hippocampal pyramidal neurons in vivo where high-threshold Ca 2+ spikes are entrained by septal EPSPs (Nufiez and Bufio 1992; Nufiez et al. 1987, 1990a,b). In the present study entrainment was examined under forced driving with sinusoidal currents at 0 rhythm and other frequencies - imitating the rhythmic septal synaptic drive - summed with long-duration depolarizing current pulses, a situation which closely imitates the in vivo state, where sustained depolarization and synaptic oscillations m a y be present during 0 rhythm (e.g., Fujita and Sato 1964; Nufiez et al. 1987). Therefore, this stimulation provides a procedure to evaluate the participation of m e m brane mechanisms mediating the innate rhythmic potential oscillations of CA1 pyramidal neurons. These results have already been presented in abstract form (GarciaMufioz et al. 1991).

long duration pulses, except Figs. 1, 5, and insets in Fig. 8. Data were plotted with a digital oscilloscope or a desk-top PC/AT computer with a Tecmar analog-digital/digital-analog board and plotter systems. When drug application modified the membrane potential, a constant current was applied to drive it to its original value (see Figs. 1-8). Intracellular injections with biocytin-filled (5% in a 0.05 mM TRIS and 0.5 M KC1 solution) pipettes (80-150 M~ ) and subsequent histological manipulations were according to the technique described by Horikaba and Armstrong (1988), except that sections made from the original 350-gm slice were 100 pm thick. Data are expressed as means _+SD.

Results

Control characteristics The 71 CA1 pyramidal neurons analyzed in resting conditions and without constant current injection had m e m brane potentials and input resistances of - 67 • 8.0 mV (range - 5 5 to - 8 0 m V ) and 49+_18 Mf~ (range 20 to 80 M~), respectively. Fast spike amplitude and duration were 84_+ 12 mV (55-100 mV) and 1.4+0.5 ms (range 0.71-1.76 ms), respectively. The current threshold, estimated with 10 ms pulses, was 0.16 nA • 0.10; occasionally it was as low as 0.02 hA. The 16 biocytin-injected cells showed the characteristic apical and basilar dendritic arborization profiles (Fig. IA) of C A I pyramidal neurons (Ram6n y Cajal 1893), and were electrophysiologically identical to all the other neurons, which were also considered pyramidal cells. All cells fired an initial fast Na § spike burst lasting 300-600 ms, triggered by a slow depolarization (e.g., Figs. 1B-C, 2B). However, different cells varied with regard to

B

A

Materials and methods Wistar rats (20-30 days old) were decapitated and the brains were rapidly removed. Transverse 350-1am thick slices of the hippocampus were cut with a vibratome and preincubated in control solution for about 1 h at room temperature. Slices were then transferred to the recording chamber and superfused continuously at 5 ml/min and 34-37 ~ The control solution contained NaC1 124 mM, KC1 3 raM, KH2PO4 1.25 raM, MgSO 4 2 mM NaHCO 3 26 mM, CaC12 2 raM, and glucose 10 raM. The pH was adjusted to 7.4 by continuous bubbling with 95% 02 and 5% CO2. Tetraethylammonium chloride (TEA), 15 raM, and CoC12, 2 raM, solutions were made by equimolarly substituting with NaC1 and CaC12, respectively in the control solution. In 2 mM MnC12 solutions, CaC12 was omitted, KH2PO4, and MgSO 4 were excluded to prevent precipitation, and divalent cations were maintained by adjusting the Mg2+ concentration. Tetrodotoxin (TTX) was added to the other solutions at 5 pM. All chemicals were purchased from Sigma. Glass pipettes filled with 3 M KC1 (60-90 M~ ) and the single electrode current-clamp technique, with an Axoclamp-2A amplifier in the bridge mode, were used. Impalements were made under direct visualization of the CA1 pyramidal soma layer with a dissecting microscope. Neurons were subject to de-hyperpolarizing 200-ms pulses, brief 10 ms depolarizing pulses, and long duration 2-s depolarizing pulses (0.01-2.0 nA) presented in isolation or summed with sinusoidal currents at 2, 7, and 14 Hz (0.1-1.5 nA). Figures usually show the first portion of

C

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Fig. l. Histologically identified CA1 neuron and long-duration, pulse-evoked responses in control solution. A Biocytin-injected neuron; B responses of same cell. Cells fired regularly at low rate after an initial burst (B) or were silent for a prolonged interval after the burst in response to long-duration pulse stimuli (C). Note the subthreshold oscillations (asterisk in C). Membrane potentials and input resistances were - 8 0 m V and -75 mV, and 44M~ and 80 MD for B and C, respectively. Some spikes were truncated by the relatively low sampling rate

327

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Fig. 2. Control responses evoked by long-duration pulses and superimposed sinusoidal currents. A Low-intensity, long-duration, pulse-evoked responses without (top) and with (bottom three traces) added sinusoidal currents. B Same cell as in A and identical sinusold, but higher intensity pulse. Note slow all-or-none events at 2 and 7 Hz (asterisks) and lower amplitude, slow depolarization at 2 Hz (arrow). Membrane potential and input resistance -55 mV and 65 Mr2, respectively. Truncated spikes the subsequent response when stimulated with long-duration 2-s pulses. Most cells (n--41 or 58%) showed regular firing with a gradual rate reduction (e.g., Fig. 1B). In the rest of the neurons (n = 30 or 42%) a prolonged interval lasting 200 ms-l.5 s interrupted the firing after the initial burst (asterisk, Fig. 1C; Fig. 5, top left; Lanthorn et al. 1984). These cells usually resumed firing at low rates after the silence, and showed rhythmic subthreshold potential oscillations (6-14 Hz, < 10 mV; Leung and Yim 1991). No other electrophysiological differences were found between the two cell types (see also below). There is evidence indicating that the high-threshold Ca 2+ spikes recorded in hippocampal pyramidal neurons in vivo are entrained by rhythmic EPSPs (Nufiez et al. 1987, 1990a) of septal origin during 0 rhythm. Therefore, we examined entrainment evoked by long-duration pulses and added sinusoidal currents. Intrinsic slow all-or-none events entrained by sinusoidal currents

Typical responses evoked by low- and high-intensity depolarizing long-duration pulses are illustrated in Fig. 2A and B, respectively (top). Periodic de-hyperpolarization and fast Na + spikes were evoked by the low-intensity,

~120mY I1nA

200ms Fig. 3. Effects of Ca2+-free, tetraethylammonium + Co 2+ (TEA + Co) solution on responses elicited by imposed oscillations. Fast Na § spikes in control solution and plateau potentials in TEA + Co solution, respectively (same cell). Fast spikes and slow all-or-none events (asterisk) were elicited by each sinusoidal cycle at 2 and 7 Hz, and occasionally at 14 Hz in TEA + Co solution. Membrane potential and input resistance - 8 0 mV and 33 M~2, respectively. The cell was hyperpolarized by - 0 . 1 5 nA constant current injection in TEA + Co solution

long-duration pulses and the added sine wave currents (Fig. 2A). Sinusoidal currents were adjusted, at membrane potential levels and at 7 Hz, to evoke potential oscillations between 5 and 20 mV, imitating the intracellular 0 (e.g., Artemenko 1973; Fox 1989; Fujita and Sato 1964; Leung and Yim 1986; Nufiez et al. 1987). A larger depolarization and fast Na § spikes followed by a slow all-or-none event (Fig. 2B, asterisk; about 40 mV amplitude, 55 ms duration) were elicited by each sine wave current cycle at 2 and 7 Hz when the higher intensity, long-duration pulse was added (Fig. 2B). Slow all-ornone events were greatly reduced in amplitude at 14 Hz (Fig. 2B). A second lower amplitude depolarization was also elicited by the 2-Hz stimulus (Fig. 2B, arrow). It is noteworthy that with smaller imposed oscillation amplitudes (e.g., 5 mV) the same behavior was observed but higher intensity pulses were needed (data not shown). Twenty five out of the 71 (or 35%) neurons showed fast Na + spikes and slow all-or-none events, 9 cells (or 36 %) were of the regularly firing type, and 16 (or 64%) were of the type that showed a silence with subthreshold oscil-

328 CONTROL

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lations after the initial burst. The rest of the cells (n = 46 or 65%) displayed fast spike inactivation and pronounced rectification and no slow all-or-none events (data not shown). The above findings indicate that at least three different response types were evoked, depending on imposed depolarization. To establish the ionic nature of the oscillations, the same stimulation was presented during block of Na-- or Ca 2§ conductances with T T X or with Ca2§ Co 2+, or Mn 2+ solutions, respectively. TEA was used to unmask the responses evoked by inward ionic currents.

Na + plateau potentials entrained by sinusoidal currents

Figure 3 shows responses evoked by the long-duration pulse and added sine wave currents in control and Ca 2§ free, T E A + C o 2+ solutions. A fast spike followed by a plateau was elicited by the long-duration pulse in TEA + Co 2§ solution (upper trace, Fig. 3). An initial fast spike or a doublet followed by a single, slow, depolarizing event (Fig. 3, asterisks) were triggered by each sine wave cycle at 2 and 7 Hz in TEA + Co 2+ solution. Smaller depolarizing events were evoked at 14 Hz, except at cycle 3 after the long-duration pulse, which evoked a response comparable with those elicited at lower frequencies (Fig. 3, asterisks). The duration of the slow events decreased as a function of sinusoidal current frequency. The results suggest that a plateau potential was evoked and subsequently aborted by each de-hyperpolarizing swing at every sine wave current cycle during the long-duration pulse. A similar behavior was observed in 27 of the 34 (or 80%) pyramidal neurons studied in these conditions, and no significant differences were found between regularly

Fig. 4. Na+-mediated plateau potentials and intrinsic oscillations. Responses evoked by 200- (0.4, 0.6, and 0.8 nA) and 10-ms pulses in control solution. The same stimulation triggered widened, initial fast Na § spikes, followed by plateau potentials with superimposed oscillations in Ca2+-free, tetraethylammonium + Co2+ (TEA+ Co) solution. Plateaus and fast spikes were suppressed in Ca2+-free, TEA + tetrodotoxin + Co (TEA + 77X + Co) solution. All records from the same cell. Membrane potential and input resistance - 70 mV and 50 M~, respectively. The cell was hyperpolarized by a -0.2-nA constant current injection in TEA + Co. Control responses elicited by a 200-ms pulse, and spikes and plateaus with superimposed oscillations evoked by 200- and 10-ms pulses in Caa+-free, TEA + Mn2+ solution (TEA + Mn) in another cell; membrane potential and input resistance -65 mV and 47 Ms respectively; a constant current of -0.05 nA was injected in TEA + Mn

firing cells, subthreshold oscillating cells, and cells which displayed pronounced inactivation and rectification in control solution. To further test the above suggestions, the responses evoked by 200- (at different intensities) and 10-ms pulses in different solutions were investigated in the absence of sinusoidal currents. Figure 4 shows typical responses in control solution. Initial fast Na + spikes followed by plateau potentials were elicited in Ca2+-free, T E A + C o 2+ solution (Garcia-Mufioz et al. 1991). Plateaus lasted about 100-700 ms (even in response to the brief 10-ms pulse), initially overshot or were close to the zero potential value, and decayed gradually to amplitudes of about 50 mV from the firing threshold (see also Fig. 3). Plateaus could be aborted by brief hyperpolarizing pulses (data not shown) and were evoked at all the sine wave current frequencies in all cells tested. Interestingly, plateaus showed superimposed oscillations at about 6-14 Hz and 10-20 mV in all cells studied. They were reminiscent of those displayed in control solution in 42% of the cells (cf. Fig 1C). The delay to the plateau onset and the amplitude of the superimposed rhythmic oscillations decreased with increasing pulse intensities. Plateaus and fast spikes were blocked and a large depolarization was evoked when T T X was added to the T E A + C o 2+ solution (Fig. 4). Identical plateaus were evoked in Ca2+-free, T E A + Mn 2+ solutions (e.g., Fig. 4) in five cells tested; they were also entrained at all sinusoidal current frequencies (data not shown). A neuron of the type that fired an initial fast spike burst followed by a silence with low-amplitude potential oscillations in response to long-duration pulses is illustrated in Fig. 5, top left. Oscillation amplitude increased gradually with time after the initial burst and eventually

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Fig. 5. Responses evoked by long-duration pulses at different intensities in control, TEA + Co, and TEA + TTX + Co solutions. The initial fast spike burst was followed by oscillations (top/eft: insets on right, taken between arrows) that increased in frequency with current pulse intensity (0.5, 0.75 and 1 nA) in control solution. Plateau potentials with superimposed oscillations (top right: insets on right, taken between arrows) were evoked in Ca2+-free, TEA+Co solution. Oscillations increased in frequency and decreased in amplitude with pulse intensity in control and TEA + Co solutions. Spikes, plateaus, and oscillations were blocked in TEA + TTX + Co solution. Membrane potential and input resistance -68 mV and 50 M ~ , respectively; a current of -0.15 nA was injected in TEA + TTX + Co solution. Some spikes were truncated by the low sampling rate

reached a maximum at about 7 mV (Fig. 5, top left, right). Fluctuation frequency increased with pulse intensity from about 6-12 Hz (Fig. 5, top left, lower trace and upper trace, respectively) and decreased in amplitude with even higher pulse intensities (data not shown). Oscillations were usually absent in the regularly firing neuron type (e.g., Fig. 1B), but the slow prepotentials preceding spikes (Lanthorn et al. 1984) may represent the oscillations in this cell type. A fast spike followed by a plateau with superimposed oscillations at about 14 Hz was evoked by the low-intensity, long-duration pulse in the same cell in Ca2+-free, T E A + Co 2+ solution (Fig. 5, top right, lower trace). The superimposed fluctuations decayed and then increased in amplitude during the plateau (see inset, Fig. 5). The plateau ended before the cessation of the pulse. Oscillations decreased in amplitude and increased somewhat in frequency with increasing pulse intensity (Fig. 5, top right, middle and upper traces). Similar responses to those in Figs. 4 and 5 were elicited in 27 out of the 34 (or 80%) neurons studied in these conditions. Interestingly, the three cell types, i.e., those which fired regularly (n = 10), the ones that showed a prolonged silence with oscillations (n = 12), and those which displayed inactivation with pronounced rectification (n = 5), could show oscillations in T E A + Co 2+ solution. Fast spikes, plateaus, and oscillations were abolished in all cells tested when T T X was added (Fig. 5, bottom). However, Hoehen et al. (1993) have provided conclusive evidence indicating that a high threshold TTX-resistant, slow activating-deactivating, Na +-K +-mediated current participates in plateau potential genesis in hippocampal pyramidal cells. The current needed to evoke the TTX-insensitive plateaus

was in the order of 3-4 nA (Hoehen et al. 1993), intensities which we could not deliver successfully with our high impedance electrodes (see Materials and methods). The above findings illustrate that a persistent Na + conductance mediated both plateau potentials and intrinsic oscillations and could be driven at all the imposed sinusoidal current frequencies.

Sinusoidal current driving of high threshold Ca + spikes

Figure 6 shows long-duration pulse- and sine wave current-evoked responses in control and in TEA + T T X solutions at two pulse intensities. The high threshold Ca z+ spikes evoked in T E A + T T X could only follow sinusoidal current frequencies of less than 7 Hz, even at the higher pulse intensity. Plateaus and intrinsic oscillations were absent in TEA + T T X solution. Similar effects were observed in all 20 cells studied in those conditions. The dynamic oscillating behavior may be affected by the block of most of the opposing K + conductances both by T E A and by Co 2+ or Mn 2+. To test T E A effects, the entrained behavior was compared in T T X solution - in the few cases (4 out of 26 cells) when high-threshold Ca 2+ spikes were evoked in those conditions - and in T T X + T E A solutions. Although high threshold Ca 2+ spikes increased in amplitude and changed in shape when T E A was added, the entrained behavior did not change much (data not shown). Indeed, high-threshold Ca 2+ spikes could only follow frequencies of less than 7 Hz in both solutions, indicating that the block of most of the K + conductances by T E A did not significantly modify the oscillating behavior in our experimental conditions.

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Furthermore, two independent lines of evidence imply that the blocking agents used did not introduce important changes in the oscillating behavior of the Na § ated responses. First, responses in T E A and T E A + TTX solutions were compared in the same cell. Second, the responses in control and Caa+-free, Co 2+, or Mn 2+ solutions in the absence of T E A were compared in the same neuron. As illustrated in Figs. 7 and 8, any difference in behavior in the first condition must be due to the lack of Na+-mediated responses, while any difference in the second condition must be due to the block of both the Ca 2+ current (Ica) and the Ca2+-dependent K + current (Ilqca/) (Lancaster and Adams 1986; see below).

TEA

Fig. 6. Effects of TEA + TTX on responses evoked by imposed sinusoidal currents. Left, responses in control solution. Right, highthreshold Ca2+ spikes without a clear relationship with sinusoidal cycles were evoked in TEA + TTX solution. Ca2+ spikes failed to follow the 7- and 14-Hz sinusoid even at low (right, left traces) and high (right, right traces) pulse intensity; same cell. Membrane potential and input resistance - 72 mV and 42 M~, respectively; a current of -0.1 nA was injected in TEA+TTX. solution

TEA§

Imposed oscillations in TEA and TEA + 7 T X solutions Figure 7 illustrates responses evoked by long-duration pulses and added sinusoidal currents in T E A and T E A + TTX solutions. Fast N a § spikes and slow all-ornone events followed all sine wave current frequencies in TEA, but the high threshold Ca 2§ spikes in T E A + TTX solutions could not in the 20 cells tested. Therefore, the lively Na + and the sluggish Ica conductances account for the dissimilarities. The larger responses in T E A + TTX were probably due to the increased membrane resistance owing to the N a § conductance block by TTX.

200ms Fig. 7. Responses in TEA and TEA+TTX solutions. Sinusoidal current-evoked responses in TEA solution show slow all-or-none depolarization following all sinusoidal frequencies. Note also the lower amplitude depolarization at 2 Hz (as in Fig. 2B). Highthreshold Ca2+ spikes in TEA+TTX solutions could not follow the sinusoids. All records, same cell. Membrane potential and input resistance - 7 0 mV and 39 M~, respectively; currents of -0.15 nA were injected in TEA and TEA + TTX solutions.

Entrainment in Ca2+-free, Co 2+ and M n 2+ solutions Figure 8 shows long-duration pulse- and sinusoidal current -evoked responses in control and Ca2+-free, Co ~+ solutions. Whereas the control showed the characteristic

response with a brief initial burst followed by regular firing at a lower rate (Fig. 8, left), the initial portion of the pulse response displayed a slightly higher discharge rate and repeated, fast spike doublets triggered on slow depo-

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Discussion

Our central objective was to analyze in vitro the membrane potential oscillations present in CA1 pyramidal neurons in an attempt to determine the voltage-gated conductances participating in vivo during 0 rhythm. Theta rhythm was absent in the de-afferented slice prepaA ration, thus, the in vitro analysis was performed by imitating the rhythmic synaptic drive with injected sinusoidal currents. 2Hz The control sine wave current-evoked responses were either low amplitude oscillations at resting potential levels or rhythmic, slow all-or-none events when cells were depolarized. Therefore, important initial evidence was 60ms that the in vitro entrained and the in vivo 0 rhythm behaviors were similar, since during natural 0 rhythm pyramidal neurons either showed: (a) small oscillations, representing the septal synaptic drive, at resting levels (e.g., 7Hz Fujita and Sato 1964; Leung and Yim 1986; Nufiez et al. 1987, 1990a,b); or (b) rhythmic, high-threshold Ca 2+ spikes when depolarized during 0 rhythm (e.g., Nufiez et al. 1987; Nufiez and Bufio 1992). Other major findings were that: (a) TEA unmasked slow all-or-none events and plateau potentials; (b) plateaus were Co 2§ and Mn2+-resistant and were abolished by TTX, hence suggesting that the underlying persistent conductance was 14Hz Na+-mediated; (c) whereas plateaus were entrained oneto-one at all the sinusoidal current frequencies used in Ca2+-free T E A + C o 2+ solutions, the high-threshold Ca 2+ spikes uncovered in TEA + TTX solution could only follow sinusoidal currents under 7 Hz; (d) intrinsic osI 40rnV 5nA cillations were insensitive to Co 2§ and Mn 2+, but were blocked by TTX, evidencing that they were Na+-mediat 300ms ed (Barrio et al. 1991; Garcia-Mufioz et al. 1991; MacViFig. 8. Effects of Ca 2+-free, C o 2 + (Co) solution. Responses in control car 1985). and in Co solutions. Note slow plateau-like depolarizations (arrowHowever, Hoehen et al. (1993) have recently provided heads; see inset). All records same neuron. Membrane potential and input resistance - 8 0 mV and 60 Mf~, respectively; no current inconclusive evidence that a high-threshold, slowly activatjection. Some spikes were truncated by the low sampling rate ing-deactivating Na +- and K+-mediated, TTX-resistant current participates in plateau genesis. Whereas the Na +mediated plateau potentials uncovered in T E A + Co 2+ or TEA + Mn 2+ could follow sinusoidal current frequenlarizing events (Fig. 8, arrowheads; see below) in Ca 2§ cies of at least 14 Hz in the present experiments (e.g., Fig. free, Co 2§ solution (Fig. 8, right). Larger periodic re3), the slow, on-off kinetics of the TTX-resistant composponses with one or two slow plateau-like superimposed nent, with time constants larger than 100 ms, would limit depolarizations (Fig. 8, arrowheads) were evoked at all the sine wave entrained oscillations to frequencies below frequencies by the added sinusoidal currents in Co 2+ so5 Hz. Consequently, TTX either slows down the kinetics lution (Fig. 8, right, inset at 2 Hz) in the 12 cells tested. of the persistent Na + current or more than two persistent Similar effects were observed in Ca2+-free, Mn 2§ soluNa + conductances participate in plateau genesis. Indeed, tion in the five ceils tested (data not shown). Although of French et al. (1990) have shown a persistent, low smaller amplitude, the plateau-like depolarizations were threshold, TTX-sensitive Na + current with fast on-off reminiscent of the Na +-mediated responses in kinetics in CA1 pyramidal cells, suggesting that there is T E A + C o 2§ solution (e.g., Fig. 3, right). Therefore, they probably more than one persistent Na + component conwere probably due to the persistent Na + conductance tributing to plateaus. In the slowly adapting stretch rewhich was unmasked by the block of the opposing IK(Ca) ceptor of crayfish there is a higher TTX-sensitivity of fast by the added Co 2§ or Mn 2+. Consequently, the persisNa + spikes than of Na+-mediated plateaus, again sugtent Na + conductance mediating both the sustained regesting two different conductances with different TTX sponses and the intrinsic oscillations was able to follow sensitivities (Barrio et al. 1991). at all frequencies used even in the absence of TEA. Whereas three different neuron types could be distinguished according to the responses evoked by long-duration pulses and by superimposed sinusoidal currents in control solution, their behavior was essentially identical

332 when Ca 2t or Na t conductances were blocked under TEA. Although the mechanisms underlying the differences in control solution are difficult to pinpoint, the behavioral similarity in conditions of block suggest that all cells posses similar persistent Na n and Ca 2§ conductances. The present results indicate that while the persistent Na t conductance had the appropriate on-off kinetics (e.g., Alonso and Llinfis 1989; French et al. 1990; Schwindt and Crill 1980) to oscillate throughout the 0 rhythm frequency range, the high threshold Ica could only fluctuate at less than 7 Hz in our in vitro conditions. This may be also true in vivo where high-threshold Ca 2§ spikes were synaptically entrained during 0 rhythm at frequencies of less than 7 Hz, but could not follow higher frequencies even when driven by imposed transmembrahe sinusoidal currents (Nufiez et al. 1990a; Nufiez and Bufio 1992). 0 rhythm has been classified into type I, with frequencies between 6-12 Hz, related to movement and blocked by barbiturate anesthetics, and type II, which oscillates at 4-7 Hz, related to alert immobility and blocked by atropine, and interpreted as cholinergic and muscarinic (Vanderwolf 1969). Therefore, the in vitro-in vivo similarities suggest strongly that whereas the persistent Na + conductance may be influential at the higher type I 0 rhythm frequencies, the more sluggish high threshold Ca 2t conductance (e.g., Brown and Griffith 1983; Halliwell 1983) may participate in the genesis of the slower cholinergic type II rhythm. It is generally accepted that intrinsic oscillations are produced by a combination of both inward and outward voltage-gated currents, and that reducing K § conductances may alter the cell's oscillating ability. Nevertheless, several lines of evidence from our results suggest that the outward currents blocked by TEA and indirectly by Co 2+ or Mn 2+ were not important in controlling the oscillations. It may be argued, however, that the sinusoidal current-evoked potential fluctuations were essential for eliciting the rhythmic slow all-or-none events since imposed depolarization would activate inward currents while hyperpolarization would deactivate those currents, thus at least partly duplicating the K +-mediated hyperpolarization. The sustained depolarization that combines with the rhythmic septal synaptic drive in vivo (Nufiez et al. 1987) - imitated in the present experiments by long duration depolarizing pulses - may be due to the activation of cholinergic synapses that block several opposing outward K § conductances (e.g. Cole and Nicoll 1983, 1984; Halliwell and Adams 1982). The in vivo high-amplitude oscillations and rhythmic, high-threshold Ca 2t spikes were triggered during sustained depolarization, but not at resting potentials (Nufiez et al. 1987). Therefore, they were probably due to the unmasking of the high threshold Ca 2+ spikes by the muscarinic block of the opposing K + conductance during 0 rhythm. However, acetylcholine (Ach) is apparently not spontaneously active in vitro because it is hydrolyzed by acetylcholinesterase (Cole and Nicoll 1984). Therefore, it is not surprising that whereas slow all-or-none depolarizing events were evoked in only 35% of the cells in control

solution, they were unmasked in most cells when some of the opposing K t were blocked by TEA in vitro (Fig. 7), a situation which probably resembled the in vivo condition when Ach was synaptically released. It may be argued that in vitro superfusion with carbachol is more physiological than the sinusoidal current method, since one type of natural 0 rhythm is generated by muscarinic mechanisms (Konopacki et al. 1987). However, our aim was to analyze the oscillations mediated by voltage-gated currents, and the carbachoMnduced rhythm occurs with conductance and potential oscillations resulting from local network activity (MacVicar and Tse 1989), thus making the pharmacological isolation of the different ionic conductances more difficult. Important also are the possible functional implications of slow depolarizing events. The high-threshold Ica and also probably the persistent Na + conductance are concentrated at the soma and dendrites (e.g., Benardo et al. 1982; Westenbroek et al. 1990) and are supposedly absent in the axon, as occurs in other systems (e.g., Barrio et al. 1991). Furthermore, while excitatory synapses are mainly dendritic, inhibitory ones are primarily somatic (e.g., Blackstad and Kjaerheim 1961). Therefore, when cholinergic inputs are active during 0 rhythm, the resulting somatic depolarization, increased membrane resistance and space constant would assist the conduction of the rhythmic, dendritic, slow all-or-none events to the axonic trigger, whereas when tonic inhibition is operative, dendrites may be functionally disconnected from the axonic trigger by the resulting somatic hyperpolarization, current shunt and decreased space constant. Therefore, tonic cholinergic excitation summed with rhythmic septal EPSPs would generate slow spikes and bursts, while inhibition would shunt the dendritic currents, thus preventing bursts. However, it is conceivable that if, as suggested by others, synaptic inhibition participates in 0 rhythm genesis (Artemenko 1973; Fox 1989; Leung and Yim 1986). IPSPs could also entrain slow dendritic events as occurs with pacemaker activity in other systems (e.g., Barrio and Bufio 1990; Perkel et al. 1964). Furthermore, the periodic de-hyperpolarizations due to EPSP-IPSP interactions may participate concurrently by activating and deactivating, respectively, the persistent Na + conductance in the natural 0 rhythm situation, as was mimicked by the imposed sinusoidal currents in our in vitro conditions. Finally, further support is provided for the growing body of evidence indicating that 0 rhythm is not exclusively generated by network attributes but that it partly depends on the intrinsic regenerative properties of CA1 pyramidal neurons.

Acknowledgements. This work was supported by DGICYT (Spain) and CEC DGXII/PVD (Europe) grants to W.B.; A.G.-M. and L.C.B were Areces Foundation doctoral and CSIC postdoctoral fellows, respectively.Many thanks are due to AlfonsoAraque for his valuable suggestions in the correction of this manuscript. References

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