176
Brain Research, 533 (1990) 176-179
Elsevier BRES 24383
Sy
ic contribu
to 0 rhythm in rat CA1-CA3 hippocampal pyra ,neurons in vivo
A n g e l N t i f i e z 1'*,
Elio Garcia-Austt 1'** and Washington B u f i o 2
l Departamento de lnvestigaci6n, Hospital Ramdn y Cajal, and 2Neurofisiologia, lnstituto Cajal, CS1C, Madrid (Spain)
(Accepted 7 August 1990) Key words: 0 Rhythm; Hippocampus; Pyramidal neuron; Rhythmic excitatory postsynaptic potential; In vivo; Membrane conductance
The effects of intracellular CI- diffusion and hyperpolarizing current pulses on inhibitory postsynaptic potentials (IPSPs) and the transmembrane theta rhythm of CA1-CA3 pyramidal neurons were tested in urethanized and curarized rats. CI- diffusion and hyperpolarizing currents decreased the amplitude of IPSPs evoked by fornix stimulation without modifying the 0 rhythm amplitude and phase. The membrane conductance was typically 22-46% higher at the positive than negative intracellular 0 peaks. Results indicate that in curarized rats excitatory postsynaptic potentials were the main components of intraceUular 0 without an important participation of IPSPs in 0 rhythm genesis. Evidence obtained with in vivo intracellular recordings in hippocampal pyramidal neurons during theta rhythm (0) has suggested two different mechanisms underlying the rhythm's generation, either rhythmic inhibitory postsynaptic potentials (IPSPs) I"3"7'8'13or rhythmic excitatory postsynaptic potentials (EPSPs) 1°'15"16. The participation of IPSPs has been deduced by: (1) the reduced intracellular 0 amplitude and the decreased peak amplitude of synaptically evoked IPSPs at more negative membrane potentialsS'13; (2) the IPSP amplitude reduction and eventual reversal during intracellular CI- diffusion8"13; (3) the synchronism of negative extracellular with positive intracellular 0 rhythm waves 3 and (4) the magnitude and localization of the conductance change 7. On the other hand, Fujita and Sato 1°, Mufioz et al. 15 and Nufiez et a1.16 indicated that rhythmic EPSPs play a prominent role in 0 genesis. The evidence consisted in increases and decreases of intracellular 0 amplitudes with hyperpolarizing and depolarizing currents, respectively, without changes of the intracellular 0 phase; i.e. the behavior expected of EPSPs. The participation of rhythmic slow spikes, probably high threshold Ca 2÷ spikes, in 0 genesis was also suggested 4'14"16. In the present report we show in identified (by antidromic fornix driving) CA1 and CA3 pyramidal neurons the effects of hyperpolarizing current injections and CI- diffusion, Both conditions decreased the IPSP amplitude and eventually reversed the initial C1--dependent IPSP component without modifying the intracellular
0 amplitude nor its phase relationship with the extracellular E E G rhythm. As previously described in detail 16, threphine holes were drilled in the skull at preselected stereotaxic coordinates 11 in 22 urethane-anesthetized (1.5 g/kg i.p.) and curarized (1-2 mg/kg i.p.) Sprague-Dawley rats. Doses of anesthetic and curare were given as necessary during the experiment while maintaining an optimal 0 rhythm in the hippocampal field recording. The dorsal hippocampal E E G was recorded through a macroelectrode in the CA1 layer (A 3.5, L 3.0, H 2.5) and an indifferent electrode in the frontal lobe. The dorsal fornix was stimulated through bipolar electrodes (A 5.7, L 1.5, H 3.0). Micropipettes were filled with 3 M potassium acetate or 2 M KCI (40-90 MJ2), and intracellular recordings were obtained through a bridge amplifier. Conductance changes were measured with brief hyperpolarizing current pulses. Data were stored on FM tape for later analysis. Statistical analysis were performed 'off-line' in a P D P / l l computer with at least 3 rain of continuously stored data (2 KHz sampling rate). Calculations included averages of the hippocampal E E G and of the transmembrane potentials using fornix stimuli or the E E G zero crossings as reference events. Intracellular recordings from 37 cells, identified as pyramids by antidromic driving through fornix stimulation, and held between 10 and 30 min, were considered. All neurons displayed a stable membrane potential < - 5 0
* Present address: Depto. de Morfologia, Facultad de Medicina, UAM, c/Arzobispo Morcillo s/n, E-28029, Madrid, Spain. ** Present address: Neurofisiologia, Instituto "C. Estable", Av. Italia 3318, Montevideo, Uruguay. Correspondence: W. Bufio, Neurofisiologfa, Instituto Cajal CSIC, Av. Doctor Arce 37, E-28002 Madrid, Spain. 0006-8993/90/$03.50 ~ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
177
!
2
lI015 ~0mV nA
t
l
B~
200 ms
Fig. I. Hyperpolarizing current pulse effects during reset of 0 rhythm by fornix stimulation. A: fornix stimulation (arrows), 1, averages (n = 8) of extracellular and 2, intracellular records. B: hyperpolarizing pulses synchronized with fornix stimulation, 1 and 2 as in A. Lower traces, transmembrane current. CA3 pyramidal neuron,
mV (mean -61.3 mV, S.D. 4.3) overshooting spikes (mean 65.7 mV, S.D. 9.2) and input resistances >20 MD (mean 28 MD, S.D. 7.1). We found no electrophysiologicai difference between the 11 CA1 and the 26 CA3 pyramidal cells considered. Bufio et al. 5 described that the E E G 0 rhythm and rhythmic hippocampal unit discharges tended to occur at a fixed phase following electrical stimulation of hippocampal afferents; an effect termed 'reset' of the 0 rhythm. Reset was exploited here to simultaneously analyze, with averaging procedures, the phase relationship between intra- and extracellular 0 and the IPSP and intracellular 0 amplitude modifications induced by long duration inward current pulses. Fig. 1 shows the effects of fornix stimulation on the CA1 E E G and the membrane potential of a CA3 pyramidal cell. Antidromic spikes were evoked synchronized with brief negative deflections in the hippocampal E E G average corresponding to the 'population spike '2. The intracellular spike was somewhat widened by the averaging procedure. In the control A2, a long-lasting IPSP followed the antidromic spike. The periodic poststimulus waves which followed the IPSP in both averages indicate that afferent stimulation reset the extra- and intracellular 0 rhythms (A1, A2, respectively). Both intra- and extracellular 0 rhythms were close to 180° out of phase, as in all other cases with +30 ° fluctuations 7' 8,10.13,16 There were no pre-stimulus 0 waves in the averages, although they were present in the not shown raw records. This occurred because stimuli were delivered with identical probability at all 0 phases and therefore, 0 waves cancelled out in the prestimulus portion of the averages. When hyperpolarizing current pulses (800 ms, 0.4 nA)
A ' ~ I ~ - - l[,~r0V
[20mv I 1
"tSms
200ms
Fig. 2. Intracellular C1- diffusion during 0 rhythm, and effects of fornix stimulation. A: hippocampal EEG (upper) and intracellular record (lower) 30 s after impalement with a KCI filled pipette, B: same as A, but 10 rain after impalement. C: expanded initial portion of superimposed intracelluar traces A and B. CA3 cell.
were injected synchronized with fornix stimulation in B1 and B2, although more negative potential values were reached due to the imposed current, the amplitude and slope of the brief, initial, IPSP component were greatly reduced (B2). The late IPSP component was not affected by hyperpolarization, suggesting a remote origin for that component 9. The intracellular 0 amplitude increased during hyperpolarizing current pulses and the phase remained unchanged (B1,B2); i.e. the behavior expected of EPSPs. The mean peak-to-peak intracellular 0 amplitude measured in 16 neurons changed from 7.1 (S.D. 1.0) to 11.0 mV (S.D. 1.2) during hyperpolarizing pulses of 0.5 nA without significant phase changes. Similar results were obtained with inward current pulses of up to 1.0 nA. The larger peak at the fornix stimulus on in B2, was due to the combination of the reversed IPSP plus an artifactual peak at the hyperpolarizing pulse on. Only the artifact was present at pulse off, and could be erased. The effect of intracellular CI- diffusion were tested in 6 CA1 and 7 CA3 pyramidal neurons. Fig. 2 illustrates a representative example of the hippocampal E E G (upper) and the intracellular records (lower) obtained 30 s (A) and 10 min (B) after impalement of a CA3 cell with a KCI electrode. The intracellular CI- diffusion reduced the initial IPSP hyperpolarization without changing the intracellular 0 rhythm's amplitude nor phase (A,B). The mean peak-to-peak amplitude of the intracellular 0 measured over 20 successive cycles in the 13 tested neurons was 8.2 (S.D. 1.6) and 7.8 mV (S.D. 1.3) immediately after impalement and at least 10 min later, respectively. The late K+-mediated IPSP component 17 was not affected. The effects of CI- diffusion are clearer in the enlarged superimposed traces in Fig. 2C. Phase
178
~ ~ v " ' ~ .
Io.5~v
A
12o.,v ~.
u
u
[1
nA
250ms
B
C
D
I00 ms Fig. 3. Membrane conductance modifications at the negative and positive peaks of the intracellular 0 wave. A: hippocampal EEG (upper) and intracellular (middle) records during injection of brief hyperpolarizing current pulses (lower; 0.3 nA, 40 ms). Asterisks indicate larger pulse responses at the negative intracellular 0 waves peaks. B and C: hippocampal EEG (upper) and membrane potential (lower) averages (n = 14) with EEG zero crossing as references (horizontal lines). Hyperpolarizing pulses were delivered at a fixed delay from crossings at the positive or negative peaks of extracellular 0 (B and C, respectively). Note averaged spike at pulse off in B. D: expanded superimposed version of pulse responses B and C. CA1 pyramidal neuron.
relationship variations were as those in control conditions both in Cl--loaded neurons and in cells hyperpolarized by current pulses. Membrane conductance changes were measured in 3 CA1 and 5 CA3 cells at different phases of the intracellular 0 waves by injection of brief hyperpolarizing pulses (0.3 nA, 40 ms; Fig. 3A) delivered at a fixed delay from the positive or negative E E G 0 zero crossings. Averages of extra- and intracellular 0 waves using the E E G zero crossing as reference events revealed that the conductance was larger (38%) at the more depolarized intracellular 0 peaks (Fig. 3B,C). The pulse response difference is clearer in the enlarged superimposed traces in Fig. 3D. Moreover, the conductance was similar at the negative peak than during periods without 0 (data not shown). The mean conductance difference, measured in the 8 cells considered, ranged between 22 and 46%. These results provide strong evidence in favor of depolarizing 0 peaks, and contradict an important participation of synaptic inhibition during negative peaks. The above findings indicate that manipulations which modify the amplitude of the initial C1- IPSP component 1 Andersen, P. and Eccles, J.C., Inhibitory phasing of neuronal discharges, Nature (Lond.), 196 (1962) 645-647. 2 Andersen, P., Bliss, T.V.P. and Skrede, K.K., Unitary analysis
do not affect the intracellular 0. The present results agree with those obtained in the dentate gyrus 15, but are in contradiction with the previously reported 0 phase shift during CI- diffusion 13 or during hyperpolarizing current injection 7. Although it has been proposed that rhythmic depolarizing IPSPs were a component of the intracellular 0 rhythm 7, fornix evoked IPSPs were always hyperpoiarizing in our conditions. Therefore, the present findings provide no conclusive evidence indicating that IPSP (either hyper- or depolarizing) participate in intracellular 0 genesis. It has been previously suggested that a possible cause for the above discrepancies was the use of curare 7. This suggestion requires that curare is able to cross (or bypass) the blood-brain barrier TM. The intracellular 0 amplitude, the input resistances, the membrane potential, and the firing rate were typically higher in our sample than those found in urethanized, non-curarized rats. Although these differences may be attributed to the drug, there are other possible explanations. It has been shown that curare reduced the amplitude of GABA-mediated responses in vitro 12, and that intracerebral administration of D-tubocurarine increased the hippocampal E E G 0 amplitude in vivo 6. The above findings indicate that 0 rhythm amplitude increased when IPSPs were presumably reduced by curare. Therefore, although the effects of curare are difficult to pinpoint, the findings strongly suggest that IPSPs are not the main component of the rhythm. The 0 amplitude differences in both conditions may be due to higher input resistances in our sample which would increase the depolarization generated by EPSP currents. The more negative membrane potential would also increase EPSP amplitude by increasing the driving force for the ionic species involved. The larger EPSP depolarizations would in turn evoke a higher firing rate. Our experiments strongly suggest that the initial Cl--dependent IPSPs are not an important component of the intracellular 0 rhythm. The findings also indicate that in our experimental conditions EPSPs were the main synaptic events in the pyramidal neuron's intracellular 0 rhythm, and therefore, represent the decisive factor in the genesis of the hippocampal E E G rhythm 1°J6. The participation of the late IPSP component in 0 genesis, which was not modified by our manipulations, is unlikely since in the dentate gyrus when it is blocked by intracellular Cs ÷ diffusion no 0 modifications were observed 15. Work supported by DGICYT grant to W.B. of hippocampal population spikes, Exp. Brain Res., 13 (1971) 208-221. 3 Artemenko, D.P., Participation of hippocampal neurons in
179 theta-wave generation, Neurophysiology, 4 (1973) 409-415. 4 Bland, B.H., Colom, L.V., Konopacki, J. and Roth, S., Intracellular records of carbachol-induced theta rhythm in hippocampal slices, Brain Research, 447 (1988) 364-368. 5 Bufio, W., Garcia-S~inchez,J.L. and Garcia-Austt, E., Reset of hippocampal rhythmical activities by afferent stimulation, Brain Res. Bull., 3 (1978) 21-28. 6 Dajas, E, Gaztelu, J.M., Rodriguez Zavalla, C., Macadar, O. and Garcia-Austt, E., Effects of intraventricular curarimimetics on hippocampal electrical activity, Exp, Neurol., 79 (1983) 160-167. 7 Fox, S.E., Membrane potential and impedance changes in hippocampal pyramidal ceils during theta rhythm, Exp. Brain Res., 77 (1989) 283-294. 8 Fox, S.E., Wolfson, S. and Ranck, J.B., Investigating the mechanism of hippocampal theta rhythms: approaches and progress. In W. Siefert (Ed.), Neurobiology of the Hippocampus, Academic Press, New York, 1983, pp. 303-319. 9 Fujita, Y., Evidence for the existence of inhibitory postsynaptic potentials in dendrites and their functional significance in hippocampal pyramidal cells of adult rabbits, Brain Research, 175 (1979) 59-69. 0 Fujita, Y. and Sato, T., Intracellular records from hippocampal pyramidal cells in rabbit during theta rhythm activity, J. Neurophysiol., 27 (1964) 1011-1026.
11 Konig, J.ER. and Klippel, R.A., The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Williams and Wilkins, Baltimore, MD, 1963. 12 Lebeda, L.S., Hablitz, J.J. and Johnston, D., Antagonism of GABA-mediated responses by D-tubocurarine in hippocampal neurons, J. Neurophysiol., 48 (1982) 622-632. 13 Leung, L.W.S. and Yim, Ch.Y., Intracellular records of theta rhythm in hippocampal CA1 cells of the rat, Brain Research, 367 (1986) 323-327. 14 MacVicar, B.A. and Tse, EW.Y., Local neuronal circuitry underlying cholinergic rhythmical slow activity in CA3 area of rat hippocampal slices, J. Physiol. (Lond.), 417 (1989)197-212. 15 Mufioz, M.D., Nfifiez, A. and Garcia-Austt, E., In vivo intracellular analysis of rat dentate granule cells, Brain Research, 509 (1990) 91-98. 16 Ntifiez, A., Garcia-Austt, E. and Bufio, W., Intracellular theta rhythm generation in identified hippocampal pyramids, Brain Research, 416 (1987) 289-300. 17 Proctor, W.R., Mynlieff, M. and Dunwiddie, W., Facilitatory action of etomidate and pentobarbital on recurrent inhibition in rat hippocampal pyramidal neurons, J. Neurosci., 6 (1986) 3161-3168. 18 Winson, J., Hippocampal theta rhythm. I. Depth profiles in the curarized rat, Brain Research, 103 (1976) 57-70.
Brain Research, 533 (1990) 176-179
Elsevier BRES 24383
Sy
ic contribu
to 0 rhythm in rat CA1-CA3 hippocampal pyra ,neurons in vivo
A n g e l N t i f i e z 1'*,
Elio Garcia-Austt 1'** and Washington B u f i o 2
l Departamento de lnvestigaci6n, Hospital Ramdn y Cajal, and 2Neurofisiologia, lnstituto Cajal, CS1C, Madrid (Spain)
(Accepted 7 August 1990) Key words: 0 Rhythm; Hippocampus; Pyramidal neuron; Rhythmic excitatory postsynaptic potential; In vivo; Membrane conductance
The effects of intracellular CI- diffusion and hyperpolarizing current pulses on inhibitory postsynaptic potentials (IPSPs) and the transmembrane theta rhythm of CA1-CA3 pyramidal neurons were tested in urethanized and curarized rats. CI- diffusion and hyperpolarizing currents decreased the amplitude of IPSPs evoked by fornix stimulation without modifying the 0 rhythm amplitude and phase. The membrane conductance was typically 22-46% higher at the positive than negative intracellular 0 peaks. Results indicate that in curarized rats excitatory postsynaptic potentials were the main components of intraceUular 0 without an important participation of IPSPs in 0 rhythm genesis. Evidence obtained with in vivo intracellular recordings in hippocampal pyramidal neurons during theta rhythm (0) has suggested two different mechanisms underlying the rhythm's generation, either rhythmic inhibitory postsynaptic potentials (IPSPs) I"3"7'8'13or rhythmic excitatory postsynaptic potentials (EPSPs) 1°'15"16. The participation of IPSPs has been deduced by: (1) the reduced intracellular 0 amplitude and the decreased peak amplitude of synaptically evoked IPSPs at more negative membrane potentialsS'13; (2) the IPSP amplitude reduction and eventual reversal during intracellular CI- diffusion8"13; (3) the synchronism of negative extracellular with positive intracellular 0 rhythm waves 3 and (4) the magnitude and localization of the conductance change 7. On the other hand, Fujita and Sato 1°, Mufioz et al. 15 and Nufiez et a1.16 indicated that rhythmic EPSPs play a prominent role in 0 genesis. The evidence consisted in increases and decreases of intracellular 0 amplitudes with hyperpolarizing and depolarizing currents, respectively, without changes of the intracellular 0 phase; i.e. the behavior expected of EPSPs. The participation of rhythmic slow spikes, probably high threshold Ca 2÷ spikes, in 0 genesis was also suggested 4'14"16. In the present report we show in identified (by antidromic fornix driving) CA1 and CA3 pyramidal neurons the effects of hyperpolarizing current injections and CI- diffusion, Both conditions decreased the IPSP amplitude and eventually reversed the initial C1--dependent IPSP component without modifying the intracellular
0 amplitude nor its phase relationship with the extracellular E E G rhythm. As previously described in detail 16, threphine holes were drilled in the skull at preselected stereotaxic coordinates 11 in 22 urethane-anesthetized (1.5 g/kg i.p.) and curarized (1-2 mg/kg i.p.) Sprague-Dawley rats. Doses of anesthetic and curare were given as necessary during the experiment while maintaining an optimal 0 rhythm in the hippocampal field recording. The dorsal hippocampal E E G was recorded through a macroelectrode in the CA1 layer (A 3.5, L 3.0, H 2.5) and an indifferent electrode in the frontal lobe. The dorsal fornix was stimulated through bipolar electrodes (A 5.7, L 1.5, H 3.0). Micropipettes were filled with 3 M potassium acetate or 2 M KCI (40-90 MJ2), and intracellular recordings were obtained through a bridge amplifier. Conductance changes were measured with brief hyperpolarizing current pulses. Data were stored on FM tape for later analysis. Statistical analysis were performed 'off-line' in a P D P / l l computer with at least 3 rain of continuously stored data (2 KHz sampling rate). Calculations included averages of the hippocampal E E G and of the transmembrane potentials using fornix stimuli or the E E G zero crossings as reference events. Intracellular recordings from 37 cells, identified as pyramids by antidromic driving through fornix stimulation, and held between 10 and 30 min, were considered. All neurons displayed a stable membrane potential < - 5 0
* Present address: Depto. de Morfologia, Facultad de Medicina, UAM, c/Arzobispo Morcillo s/n, E-28029, Madrid, Spain. ** Present address: Neurofisiologia, Instituto "C. Estable", Av. Italia 3318, Montevideo, Uruguay. Correspondence: W. Bufio, Neurofisiologfa, Instituto Cajal CSIC, Av. Doctor Arce 37, E-28002 Madrid, Spain. 0006-8993/90/$03.50 ~ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
177
!
2
lI015 ~0mV nA
t
l
B~
200 ms
Fig. I. Hyperpolarizing current pulse effects during reset of 0 rhythm by fornix stimulation. A: fornix stimulation (arrows), 1, averages (n = 8) of extracellular and 2, intracellular records. B: hyperpolarizing pulses synchronized with fornix stimulation, 1 and 2 as in A. Lower traces, transmembrane current. CA3 pyramidal neuron,
mV (mean -61.3 mV, S.D. 4.3) overshooting spikes (mean 65.7 mV, S.D. 9.2) and input resistances >20 MD (mean 28 MD, S.D. 7.1). We found no electrophysiologicai difference between the 11 CA1 and the 26 CA3 pyramidal cells considered. Bufio et al. 5 described that the E E G 0 rhythm and rhythmic hippocampal unit discharges tended to occur at a fixed phase following electrical stimulation of hippocampal afferents; an effect termed 'reset' of the 0 rhythm. Reset was exploited here to simultaneously analyze, with averaging procedures, the phase relationship between intra- and extracellular 0 and the IPSP and intracellular 0 amplitude modifications induced by long duration inward current pulses. Fig. 1 shows the effects of fornix stimulation on the CA1 E E G and the membrane potential of a CA3 pyramidal cell. Antidromic spikes were evoked synchronized with brief negative deflections in the hippocampal E E G average corresponding to the 'population spike '2. The intracellular spike was somewhat widened by the averaging procedure. In the control A2, a long-lasting IPSP followed the antidromic spike. The periodic poststimulus waves which followed the IPSP in both averages indicate that afferent stimulation reset the extra- and intracellular 0 rhythms (A1, A2, respectively). Both intra- and extracellular 0 rhythms were close to 180° out of phase, as in all other cases with +30 ° fluctuations 7' 8,10.13,16 There were no pre-stimulus 0 waves in the averages, although they were present in the not shown raw records. This occurred because stimuli were delivered with identical probability at all 0 phases and therefore, 0 waves cancelled out in the prestimulus portion of the averages. When hyperpolarizing current pulses (800 ms, 0.4 nA)
A ' ~ I ~ - - l[,~r0V
[20mv I 1
"tSms
200ms
Fig. 2. Intracellular C1- diffusion during 0 rhythm, and effects of fornix stimulation. A: hippocampal EEG (upper) and intracellular record (lower) 30 s after impalement with a KCI filled pipette, B: same as A, but 10 rain after impalement. C: expanded initial portion of superimposed intracelluar traces A and B. CA3 cell.
were injected synchronized with fornix stimulation in B1 and B2, although more negative potential values were reached due to the imposed current, the amplitude and slope of the brief, initial, IPSP component were greatly reduced (B2). The late IPSP component was not affected by hyperpolarization, suggesting a remote origin for that component 9. The intracellular 0 amplitude increased during hyperpolarizing current pulses and the phase remained unchanged (B1,B2); i.e. the behavior expected of EPSPs. The mean peak-to-peak intracellular 0 amplitude measured in 16 neurons changed from 7.1 (S.D. 1.0) to 11.0 mV (S.D. 1.2) during hyperpolarizing pulses of 0.5 nA without significant phase changes. Similar results were obtained with inward current pulses of up to 1.0 nA. The larger peak at the fornix stimulus on in B2, was due to the combination of the reversed IPSP plus an artifactual peak at the hyperpolarizing pulse on. Only the artifact was present at pulse off, and could be erased. The effect of intracellular CI- diffusion were tested in 6 CA1 and 7 CA3 pyramidal neurons. Fig. 2 illustrates a representative example of the hippocampal E E G (upper) and the intracellular records (lower) obtained 30 s (A) and 10 min (B) after impalement of a CA3 cell with a KCI electrode. The intracellular CI- diffusion reduced the initial IPSP hyperpolarization without changing the intracellular 0 rhythm's amplitude nor phase (A,B). The mean peak-to-peak amplitude of the intracellular 0 measured over 20 successive cycles in the 13 tested neurons was 8.2 (S.D. 1.6) and 7.8 mV (S.D. 1.3) immediately after impalement and at least 10 min later, respectively. The late K+-mediated IPSP component 17 was not affected. The effects of CI- diffusion are clearer in the enlarged superimposed traces in Fig. 2C. Phase
178
~ ~ v " ' ~ .
Io.5~v
A
12o.,v ~.
u
u
[1
nA
250ms
B
C
D
I00 ms Fig. 3. Membrane conductance modifications at the negative and positive peaks of the intracellular 0 wave. A: hippocampal EEG (upper) and intracellular (middle) records during injection of brief hyperpolarizing current pulses (lower; 0.3 nA, 40 ms). Asterisks indicate larger pulse responses at the negative intracellular 0 waves peaks. B and C: hippocampal EEG (upper) and membrane potential (lower) averages (n = 14) with EEG zero crossing as references (horizontal lines). Hyperpolarizing pulses were delivered at a fixed delay from crossings at the positive or negative peaks of extracellular 0 (B and C, respectively). Note averaged spike at pulse off in B. D: expanded superimposed version of pulse responses B and C. CA1 pyramidal neuron.
relationship variations were as those in control conditions both in Cl--loaded neurons and in cells hyperpolarized by current pulses. Membrane conductance changes were measured in 3 CA1 and 5 CA3 cells at different phases of the intracellular 0 waves by injection of brief hyperpolarizing pulses (0.3 nA, 40 ms; Fig. 3A) delivered at a fixed delay from the positive or negative E E G 0 zero crossings. Averages of extra- and intracellular 0 waves using the E E G zero crossing as reference events revealed that the conductance was larger (38%) at the more depolarized intracellular 0 peaks (Fig. 3B,C). The pulse response difference is clearer in the enlarged superimposed traces in Fig. 3D. Moreover, the conductance was similar at the negative peak than during periods without 0 (data not shown). The mean conductance difference, measured in the 8 cells considered, ranged between 22 and 46%. These results provide strong evidence in favor of depolarizing 0 peaks, and contradict an important participation of synaptic inhibition during negative peaks. The above findings indicate that manipulations which modify the amplitude of the initial C1- IPSP component 1 Andersen, P. and Eccles, J.C., Inhibitory phasing of neuronal discharges, Nature (Lond.), 196 (1962) 645-647. 2 Andersen, P., Bliss, T.V.P. and Skrede, K.K., Unitary analysis
do not affect the intracellular 0. The present results agree with those obtained in the dentate gyrus 15, but are in contradiction with the previously reported 0 phase shift during CI- diffusion 13 or during hyperpolarizing current injection 7. Although it has been proposed that rhythmic depolarizing IPSPs were a component of the intracellular 0 rhythm 7, fornix evoked IPSPs were always hyperpoiarizing in our conditions. Therefore, the present findings provide no conclusive evidence indicating that IPSP (either hyper- or depolarizing) participate in intracellular 0 genesis. It has been previously suggested that a possible cause for the above discrepancies was the use of curare 7. This suggestion requires that curare is able to cross (or bypass) the blood-brain barrier TM. The intracellular 0 amplitude, the input resistances, the membrane potential, and the firing rate were typically higher in our sample than those found in urethanized, non-curarized rats. Although these differences may be attributed to the drug, there are other possible explanations. It has been shown that curare reduced the amplitude of GABA-mediated responses in vitro 12, and that intracerebral administration of D-tubocurarine increased the hippocampal E E G 0 amplitude in vivo 6. The above findings indicate that 0 rhythm amplitude increased when IPSPs were presumably reduced by curare. Therefore, although the effects of curare are difficult to pinpoint, the findings strongly suggest that IPSPs are not the main component of the rhythm. The 0 amplitude differences in both conditions may be due to higher input resistances in our sample which would increase the depolarization generated by EPSP currents. The more negative membrane potential would also increase EPSP amplitude by increasing the driving force for the ionic species involved. The larger EPSP depolarizations would in turn evoke a higher firing rate. Our experiments strongly suggest that the initial Cl--dependent IPSPs are not an important component of the intracellular 0 rhythm. The findings also indicate that in our experimental conditions EPSPs were the main synaptic events in the pyramidal neuron's intracellular 0 rhythm, and therefore, represent the decisive factor in the genesis of the hippocampal E E G rhythm 1°J6. The participation of the late IPSP component in 0 genesis, which was not modified by our manipulations, is unlikely since in the dentate gyrus when it is blocked by intracellular Cs ÷ diffusion no 0 modifications were observed 15. Work supported by DGICYT grant to W.B. of hippocampal population spikes, Exp. Brain Res., 13 (1971) 208-221. 3 Artemenko, D.P., Participation of hippocampal neurons in
179 theta-wave generation, Neurophysiology, 4 (1973) 409-415. 4 Bland, B.H., Colom, L.V., Konopacki, J. and Roth, S., Intracellular records of carbachol-induced theta rhythm in hippocampal slices, Brain Research, 447 (1988) 364-368. 5 Bufio, W., Garcia-S~inchez,J.L. and Garcia-Austt, E., Reset of hippocampal rhythmical activities by afferent stimulation, Brain Res. Bull., 3 (1978) 21-28. 6 Dajas, E, Gaztelu, J.M., Rodriguez Zavalla, C., Macadar, O. and Garcia-Austt, E., Effects of intraventricular curarimimetics on hippocampal electrical activity, Exp, Neurol., 79 (1983) 160-167. 7 Fox, S.E., Membrane potential and impedance changes in hippocampal pyramidal ceils during theta rhythm, Exp. Brain Res., 77 (1989) 283-294. 8 Fox, S.E., Wolfson, S. and Ranck, J.B., Investigating the mechanism of hippocampal theta rhythms: approaches and progress. In W. Siefert (Ed.), Neurobiology of the Hippocampus, Academic Press, New York, 1983, pp. 303-319. 9 Fujita, Y., Evidence for the existence of inhibitory postsynaptic potentials in dendrites and their functional significance in hippocampal pyramidal cells of adult rabbits, Brain Research, 175 (1979) 59-69. 0 Fujita, Y. and Sato, T., Intracellular records from hippocampal pyramidal cells in rabbit during theta rhythm activity, J. Neurophysiol., 27 (1964) 1011-1026.
11 Konig, J.ER. and Klippel, R.A., The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Williams and Wilkins, Baltimore, MD, 1963. 12 Lebeda, L.S., Hablitz, J.J. and Johnston, D., Antagonism of GABA-mediated responses by D-tubocurarine in hippocampal neurons, J. Neurophysiol., 48 (1982) 622-632. 13 Leung, L.W.S. and Yim, Ch.Y., Intracellular records of theta rhythm in hippocampal CA1 cells of the rat, Brain Research, 367 (1986) 323-327. 14 MacVicar, B.A. and Tse, EW.Y., Local neuronal circuitry underlying cholinergic rhythmical slow activity in CA3 area of rat hippocampal slices, J. Physiol. (Lond.), 417 (1989)197-212. 15 Mufioz, M.D., Nfifiez, A. and Garcia-Austt, E., In vivo intracellular analysis of rat dentate granule cells, Brain Research, 509 (1990) 91-98. 16 Ntifiez, A., Garcia-Austt, E. and Bufio, W., Intracellular theta rhythm generation in identified hippocampal pyramids, Brain Research, 416 (1987) 289-300. 17 Proctor, W.R., Mynlieff, M. and Dunwiddie, W., Facilitatory action of etomidate and pentobarbital on recurrent inhibition in rat hippocampal pyramidal neurons, J. Neurosci., 6 (1986) 3161-3168. 18 Winson, J., Hippocampal theta rhythm. I. Depth profiles in the curarized rat, Brain Research, 103 (1976) 57-70.
