Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201
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ABSTRACT |
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Morishita, Wade and
Bradley E. Alger.
Direct Depolarization and Antidromic Action Potentials
Transiently Suppress Dendritic IPSPs in Hippocampal CA1 Pyramidal
Cells.
J. Neurophysiol. 85: 480-484, 2001.
Whole-cell current-clamp recordings were made from distal dendrites of
rat hippocampal CA1 pyramidal cells. Following depolarization of the
dendritic membrane by direct injection of current pulses or by
back-propagating action potentials elicited by antidromic stimulation,
evoked -aminobutyric acid-A (GABAA) receptor-mediated inhibitory postsynaptic potentials (IPSPs) were transiently suppressed. This suppression had properties similar to depolarization-induced suppression of inhibition (DSI): it was enhanced by carbachol, blocked
by dendritic hyperpolarization sufficient to prevent action potential
invasion, and reduced by 4-aminopyridine (4-AP) application. Thus DSI
or a DSI-like process can be recorded in CA1 distal dendrites. Moreover, localized application of TTX to stratum pyramidale blocked somatic action potentials and somatic IPSPs, but not dendritic IPSPs or
DSI induced by direct dendritic depolarization, suggesting DSI is
expressed in part in the dendrites. These data extend the potential
physiological roles of DSI.
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INTRODUCTION |
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The retrograde signaling
process called "depolarization-induced suppression of inhibition"
(DSI) can regulate inhibitory postsynaptic potentials (IPSPs) in CA1
pyramidal cells and cerebellar Purkinje cells (Alger and Pitler
1995). DSI is induced by a rise in
[Ca2+] in pyramidal cells, but is
expressed as a decrease in the release of GABA from
interneurons. Experimentally, step depolarizations of the soma have
generally been used to induce DSI. However, pyramidal cell
depolarization is mainly initiated normally by synaptically induced
depolarization of the dendrites, and so it will also be important to
know if dendritic depolarization is an effective stimulus for DSI
induction. Moreover, dendritic inhibitory synapses exist, and
regulation of dendritic inhibition could be an important function of DSI.
CA1 DSI is blocked by voltage-gated Ca channel antagonists (Lenz
et al. 1998), and L-type and N-type Ca channels are distributed heavily on somata and proximal apical dendrites of CA1 pyramidal cells
(Westenbroek et al. 1990
, 1995
), and dendritic
Ca2+ influx has been measured (Jaffe et
al. 1992
, 1994
; Spruston et al. 1995
;
Tsubokawa and Ross 1996
). Thus we hypothesized that direct depolarization of the dendrites might be an especially effective
way of initiating DSI, and that DSI might have pronounced effects on
dendritic IPSPs. Because of their potential importance for
understanding the physiological roles of DSI, we have begun testing
these hypotheses by recording from distal dendrites of CA1 pyramidal cells.
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METHODS |
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Hippocampal slice preparation
Following deep halothane anesthesia and cardiac perfusion with ice-cold saline, brains were removed from male Sprague-Dawley rats (4- to 8-wk-old); the hippocampi were removed and transversely sectioned at 400-µm intervals. Recordings were made from slices in a chamber on a fixed stage of a Nikon E600FN microscope and perfused (1-1.5 ml/min) with oxygenated saline at 20-24°C.
Solutions
The extracellular saline comprised the following (in mM): 120 NaCl, 3 KCl, 1 NaH2PO4, 25 NaHCO3, 2.5 CaCl2, 2 MgSO4, and 20 D-glucose (pH 7.4 when
bubbled with 95% O2-5%
CO2). 6-cyano7-nitroquinoxaline-2,3-dione (CNQX, 20 µM, RBI), MK-801
[(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten5,10-imine hydrogen maleate, 50 µM, RBI], D,
L-2-amino-5-phosphonovaleric acid (APV, 50 µM, Sigma), and CGP
35348 (150 µM) were present to block ionotropic glutamate
receptor-mediated responses and -aminobutyric acid-B
(GABAB) receptors. Carbachol (5-10 µM, Sigma) and 4-aminopyridine (4-AP, 50 µM, Sigma) were bath applied. Patch pipettes (15-35 M
, in saline) were filled with the following (in
mM): 150 KCH3SO3, 10 HEPES,
0.2 or 0.4 bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA, 2 MgCl2), 2 MgATP, and 10 sodium
phosphocreatine (pH adjusted to 7.2 with KOH). In some experiments,
biocytin (0.4%, Molecular Probes) was in the patch solution, and these
slices were fixed and stained. For the experiments of Fig. 4, a
whole-cell pipette was filled with 0.1 mM TTX and placed
extracellularly in stratum pyramidale near the recorded cell. TTX was
pressure-ejected via a Picospritzer (General Valve) using pulses of 20 psi lasting 1 s.
Whole-cell recording and data analysis
Series resistances were between 60 and 110 M and were
compensated by bridge balance (Axoclamp 2B amplifier, Axon
Instruments). Only cells with stable resting membrane potentials (>
65 mV) and series resistances were accepted. Antidromic action
potentials were elicited with a bipolar concentric stimulating
electrode (Rhodes Electronics) positioned near the alveus toward the
subiculum. IPSPs were evoked at 0.33 Hz with a stimulating electrode in
stratum oriens. Voltage signals were filtered at 2 kHz with an
eight-pole Bessel filter (Frequency Devices) and digitized at 10 kHz
(Digidata 1200). Data were stored on a PC and VHS video tape.
IPSP suppression was quantified by %Reduction = (1 IPSPt/IPSPc) × 100%, where IPSPc is the mean amplitude of
seven consecutive IPSPs recorded during the pre-DSI period (the time
immediately before the antidromic train or postsynaptic depolarization), and IPSPt is the mean of five
consecutive IPSPs evoked during the test period (time following the
train or depolarization). The first IPSP was generally not used to
calculate IPSPt (Pitler and Alger
1994
). Input resistances were obtained from the least-squares
linear regression fit of the plots of voltage response versus current
steps from
100 to +100 pA. Data were analyzed with PClamp7 (Axon
Instruments) and SigmaPlot 4 (SPSS Inc.) software. Student's
t-tests, paired and unpaired as appropriate
(P < 0.05), were used to determine statistical significance of the data. All values given in text and figures are
mean ± SE.
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RESULTS |
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Slices were visualized with infrared videomicroscopy
(Stuart et al. 1993), and whole-cell current-clamp
recordings were made from the distal portion of 22 apical dendrites
250-400 µm from the CA1 pyramidal cell layer. Dendritic recordings
were verified by: 1) appearance under videomicroscopy,
2) declining peaks of back-propagating antidromic action
potentials (9 of 9 dendrites) (Andreasen and Lambert
1995
; Spruston et al. 1995
), and 3)
location of biocytin-labeled cell somata in the CA1 stratum pyramidale (n = 5 of 5 cells) (Fig.
1, A and B)
following filling of the cell through the dendritic electrode. Figure
1, C1 and C2, show typical dendritic and somatic
data. Note the marked inward rectification with strong
hyperpolarizations of the dendrite.
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Input resistances were 83.1 ± 6.06 M (n = 7)
in the dendrites and 115.4 ± 9.62 M
(n = 8, significant, P
0.02) in the somata (different
cells). The inward rectification ratio was calculated from the
steady-state response (usually taken at 800 ms after the
hyperpolarizing step onset) divided by the peak response (usually ~100 ms after step onset). The mean rectification ratio in the dendrites was 0.34 ± 0.04, n = 7, whereas in the
somata it was 0.50 ± 0.03, n = 7 (P
0.01), providing further evidence of the dendritic recording site (cf. Andreasen and Lambert
1995
; Magee 1998
). Both dendritically and
somatically recorded IPSPs have a linear conductance (Fig.
1C2), although the dendritic current-voltage plot is shifted
slightly to hyperpolarized membrane potentials. The peak amplitudes of
the IPSPs in dendrites and somata were similar (P = 0.08) when measured at a membrane potential near
70 mV (
7.8 ± 0.28 mV, n = 8, dendrites;
6.55 ± 0.62 mV,
n = 8, somata).
We first asked if depolarizing the dendritic membrane to elicit a train of action potentials would induce DSI of the evoked IPSP. In fact, IPSPs evoked after the 0.65-nA, 500-ms-long depolarizing current step were consistently smaller than IPSPs in control. The suppression was reversible; however, it was modest in magnitude, 15 ± 4% peak, n = 5 (Fig. 1, D1 and D2).
CA1 DSI is markedly enhanced by bath application of a cholinergic
agonist, such as carbachol, acting at a muscarinic receptor (Martin and Alger 1999), and muscarinic agonists can
enhance Ca2+ influx into dendrites. Application
of 5 µM carbachol caused a large Ca2+ spike to
appear following the dendritic depolarization (Fig. 1D1),
and there was a significant increase in IPSP suppression (to 36 ± 5%, P < 0.05) (Fig. 1, D1 and
D2).
Action potentials can back-propagate into CA1 pyramidal cells and
increase intracellular [Ca2+] (Andreasen
and Lambert 1995; Jaffe et al. 1992
, 1994
;
Spruston et al. 1995
; Tsubokawa and Ross
1996
). A 20-Hz train of back-propagating action potentials
lasting 1 s was followed by a transient reduction of evoked IPSPs
(6 of 8 dendrites) by 25.9 ± 4.11% (Fig.
2, A2 and A4).
Extending the antidromic stimulus train in the same dendrites to 2 s of 20-Hz stimuli significantly increased the suppression to 38 ± 6% (5 of 5 cells tested) (Fig. 2, A3 and A4;
P < 0.05).
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We attributed the suppression of IPSCs following the alveus stimulation
to a DSI-like process because: 1) During the IPSC suppression there were no changes in passive cell properties; e.g.,
Fig. 2A2 (5 of 5 cells). 2) Limiting
back-propagating action potential invasion by a strong
hyperpolarization of the dendrites abolished the IPSC suppression (Fig.
2, B1 and B2; n = 3). Because of
the small size of the reversed IPSP (Fig. 2B1) in three
cases, we blocked back-propagating spikes by hyperpolarizing the
dendrite only during the antidromic train, so that the larger IPSPs
were recorded at the resting membrane potential. This also prevented DSI (data not shown). 3) Bath application of 50 µM 4-AP, a
treatment that blocks DSI (Alger et al. 1996),
significantly reduced the IPSC suppression caused by back-propagating
action potentials (from 30.8 to 17.1%, P < 0.03, paired t-test, n = 7).
When carbachol, 5 µM, was bath-applied to the same dendritic recording, the 20-Hz/1-s antidromic train caused a significantly greater suppression of the IPSP (control suppression 20 ± 2%; carbachol suppression 30 ± 4%, n = 5, Fig. 3, A1 and A2; P < 0.05), which was reversible (Fig. 3A2). Interestingly, carbachol failed to enhance IPSP suppression by a 20-Hz/2-s train (Fig. 3, B1 and B2). Presumably, DSI was saturated by the 2-s train.
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Both dendritic depolarization and antidromic stimulation produced somatic action potentials; it was not clear if DSI depended on somatic action potentials, nor if dendritic IPSPs were reduced (somatic IPSP reduction might be recorded distally). To address these issues, we placed an extracellular pipette containing 0.1 mM TTX into the slice in s. pyr. and pressure-applied a small bolus of TTX (see Fig. 4A1). When DSI was induced by antidromic action potentials, TTX reduced the IPSP and blocked both the antidromic spikes and DSI (Fig. 4A2). Note the remaining IPSP evoked by stratum radiatum stimulation must have been produced by dendritic synapses in this case. However, when DSI was induced in the same cells by direct dendritic depolarization, somatic TTX application did not block DSI, even though the somatic spikes were clearly blocked (Fig. 4A3). Because the residual IPSP was dendritic in origin and was reduced by DSI, we infer that DSI is expressed at least partly in the dendrites. Figure 4A4 summarizes data from all six dendrites tested as in Fig. 4, A2 and A3.
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DISCUSSION |
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This work suggests that direct dendritic depolarization can induce
a DSI-like phenomenon in CA1 pyramidal cells. The present results
extend our previous observations (Pitler and Alger 1994) by showing that antidromic action potentials can induce DSI when they
invade the soma-dendritic regions, and thus demonstrating another
physiological mode of inducing DSI. However, as nearby cells that are
antidromically activated probably also undergo DSI (Fig.
4A2), the influence of DSI induced in these cells evidently does not spread effectively to the recorded cell when it is prevented from firing. Thus CA1 DSI appears to be a local form of communication, unlike cerebellar DSI (Vincent and Marty 1993
). This
demonstration, which has not previously been reported, would have
significant functional implications. A caveat is that we do not have
direct evidence that all pyramidal cells were equally well activated by
the antidromic stimulation.
Our results show for the first time that DSI can be recorded in distal
dendrites and can be expressed on dendritic IPSPs. Nevertheless, the
relatively small magnitude of DSI induced by direct dendritic
depolarization in the absence of carbachol did not support our
prediction that DSI would be especially prominent when induced in this
way. This is partly attributable to our recent finding that the
"slow" component of the dendritic GABAA IPSP (Pearce 1993) is not affected by DSI (L. A. Martin,
D.-S. Wei, and B. E. Alger, unpublished observations);
i.e., the greater the proportion of IPSP that is not susceptible to
DSI, the smaller is the percentage reduction in the total IPSP by DSI.
In any case, dendritic IPSPs were significantly reduced by DSI, and
thus DSI may influence dendritic electro-responsiveness. Further
experiments will be needed to test this hypothesis about the functional
roles of DSI.
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ACKNOWLEDGMENTS |
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We thank N. Spruston for advice on recording from pyramidal cell dendrites and E. Elizabeth for expert typing and editorial assistance.
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-30219 to B. E. Alger.
Present address of W. Morishita: Dept. of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Lab Surge Building, 1201 Welch Rd., P152, Palo Alto, CA 94304.
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FOOTNOTES |
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Address for reprint requests: B. E. Alger, Dept. of Physiology, University of Maryland School of Medicine, 655 W. Baltimore St., Baltimore, MD 21201 (E-mail: balger{at}umaryland.edu).
Received 7 December 1999; accepted in final form 3 October 2000.
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REFERENCES |
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