1 Neuroscience Program, 2 Department of Anesthesiology, 3 Department of Neurology, and 4 Department of Anatomy, University of Wisconsin, Madison, Wisconsin 53706
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ABSTRACT |
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Kapur, A., R. A. Pearce, W. W. Lytton, and L. B. Haberly.GABAA-mediated IPSCs in piriform cortex have fast and slow components with different properties and locations on pyramidal cells. J. Neurophysiol. 78: 2531-2545, 1997. A recent study in piriform (olfactory) cortex provided evidence that, as in hippocampus and neocortex, -aminobutyric acid-A (GABAA)-mediated inhibition is generated in dendrites of pyramidal cells, not just in the somatic region as previously believed. This study examines selected properties of GABAA inhibitory postsynaptic currents (IPSCs) in dendritic and somatic regions that could provide insight into their functional roles. Pharmacologically isolated GABAA-mediated IPSCs were studied by whole cell patch recording in slices. To compare properties of IPSCs in distal dendritic and somatic regions, local stimulation was carried out with tungsten microelectrodes, and spatially restricted blockade of GABAA-mediated inhibition was achieved by pressure-ejection of bicuculline from micropipettes. The results revealed that largely independent circuits generate GABAA inhibition in distal apical dendritic and somatic regions. With such independence, a selective decrease in dendritic-region inhibition could enhance integrative or plastic processes in dendrites while allowing feedback inhibition in the somatic region to restrain system excitability. This could allow modulatory fiber systems from the basal forebrain or brain stem, for example, to change the functional state of the cortex by altering the excitability of interneurons that mediate dendritic inhibition without increasing the propensity for regenerative bursting in this highly epileptogenic system. As in hippocampus, GABAA-mediated IPSCs were found to have fast and slow components with time constants of decay on the order of 10 and 40 ms, respectively, at 29°C. Modeling analysis supported physiological evidence that the slow time constant represents a true IPSC component rather than an artifactual slowing of the fast component from voltage clamp of a dendritic current. The results indicated that, whereas both dendritic and somatic-region IPSCs have both fast and slow GABAA components, there is a greater proportion of the slow component in dendrites. In a companion paper, the hypothesis is explored that the resulting slower time course of the dendritic IPSC increases its capacity to regulate the N-methyl-D-aspartate component of EPSPs. Finally, evidence is presented that the slow GABAA-mediated IPSC component is regulated by presynaptic GABAB inhibition whereas the fast is not. Based on the requirement for presynaptic GABAB-mediated block of inhibition for expression of long-term potentiation, this finding is consistent with participation of the slow GABAA component in regulation of synaptic plasticity. The lack of susceptibility of the fast GABAA component to the long-lasting, activity-induced suppression mediated by presynaptic GABAB receptors is consistent with a protective role for this process in preventing seizure activity.
For several decades after its discovery, the study of inhibition mediated by Slices were prepared from the piriform cortex of 3- to 4-wk-old male Sprague Dawley rats. Decapitation was under ether anesthesia. Slices were cut perpendicular to the cortical surface by making a small adjustment in block-face orientation relative to the coronal plane to preserve as much of the dendritic trees of pyramidal cells as possible. Sectioning was carried out with a Vibratome (Lancer) at 500 µm thickness in carbogen-saturated artificial cerebrospinal fluid at ~4°C. This medium contained (in mM) 126 NaCl, 3 KCl, 2 CaCl2, 1 MgSO4, 1.25 NaH2PO4, 26 NaHCO3, and 10 D-glucose and was equilibrated with 95% O2-5% CO2. Osmolarity was 320 mOsm.
Pharmacologically isolated monosynaptic GABAA-mediated IPSCs were evoked in layer II pyramidal cells by stimulation of inhibitory cells and their axons at a distance of ~250 µm from the recording site. Glutamatergic excitatory postsynaptic currents and polysynaptic IPSCs were blocked by bath-applied DNQX (20 µM) and APV (15 µM D-APV or 30 µM D,L-APV) and GABAB-mediated IPSCs blocked by Cs+ (135 mM) and QX-314 (5 mM) in the recording pipette. IPSCs were evoked from a "superficial" tungsten microelectrode in layer Ia and from a "deep" tungsten microelectrode in layer Ib, layer II, or the superficial part of layer III (Fig. 1). No differences were detected in results obtained with stimulation in layers Ib, II, or III. Bath application of 10 µM bicuculline reversibly blocked responses to both superficial and deep stimulation (Fig. 2), confirming that the pharmacological isolation of the GABAA-mediated IPSC was complete.
Laminar specificity of inhibitory circuitry
To test the prediction that separate inhibitory circuits provide input to distal dendritic and somatic regions, the effects of focally applied bicuculline on IPSCs evoked from different layers were examined. Bicuculline methiodide was pressure ejected from micropipettes positioned either in the distal apical dendritic region or in the vicinity of the cell body of the neuron under study. Results from a typical experiment are illustrated in Fig. 3. When bicuculline was applied in layer Ia, the IPSC evoked by superficial stimulation was blocked by 60% before there was any effect on the IPSC evoked by deep stimulation (Fig. 3A, 1 and 2). A small decrease in deep response was observed but required 30 s to develop, indicating that few inhibitory axons activated by deep stimulation terminated at the site of bicuculline application. Conversely, when bicuculline was applied near the patched cell body in layer II, the deep-evoked IPSC was blocked by 80% when the superficial-evoked IPSC was blocked by 10% (Fig. 3B, 1 and 2). In this case, the latency of action on both deep and superficial responses was equally brief, suggesting that a small proportion of inhibitory axons activated from the superficial site terminated in the vicinity of bicuculline application in layer II.
Kinetics of GABAA-mediated IPSCs
To determine if GABAA-mediated IPSCs in piriform cortex consist of fast and slow components as in the hippocampus (Pearce 1993
Test for effects of imperfect space clamp
Because the time course of synaptic currents in dendrites can be slowed when measured by voltage clamp at the cell body (Mainen et al. 1996
Laminar separation of fast and slow GABAA components
The finding that IPSCs evoked by superficial and deep stimulation have different proportions of fast and slow components (Fig. 4), together with the evidence for laminar segregation of synaptic responses (Fig. 3), suggests that fast and slow components are generated in different parts of pyramidal cells as postulated for hippocampus. If this is true, then the observed mixture of fast and slow components in responses to both superficial and deep stimulation could be due to a limited exchange of axons between superficial and deep zones or to the spread of current over depth from stimulating electrodes. Alternatively, there could be a well-segregated innervation of two discrete groups of synapses, each of which evokes both fast and slow components but in different proportions as a consequence of a varying proportion of fast and slow receptors (or a single receptor with varying biexponential kinetics).
Reversal potentials
Reversal potentials for the fast and slow components of IPSCs evoked from all layers were similar (Fig. 7, Table 2). The overall mean of
Use-dependent depression of GABAA-mediated IPSCs
The hypothesis that the slow GABAA component is selectively susceptible to presynaptic GABAB inhibition was tested using paired pulse depression (PPD) and pharmacological manipulation. The strength of presynaptic GABAB regulation was assessed by measuring PPD of the IPSC at an intershock interval of 150-200 ms where presynaptic GABAB inhibition is near maximal. PPD also was measured at a shorter interval (10-20 ms).
GABAA-mediated IPSCs are generated in apical dendrites of pyramidal cells in piriform cortex
The results provide the first direct evidence for the generation of GABAA-mediated IPSCs in the dendrites of pyramidal cells in piriform cortex: IPSCs evoked by superficial stimulation (layer Ia) were blocked at brief delays after the application of bicuculline to distal segments of apical dendrites in contrast to a long delay and lesser extent with application near cell bodies. A previous study had provided indirect evidence for dendritic GABAA-mediated inhibition through the demonstration that dendritic, but not somatic, application of bicuculline facilitates the NMDA component of EPSPs in dendrites (Kanter et al. 1996 Inhibition in piriform cortex is segregated in depth and mediated by feedforward and feedback pathways
The results obtained by stimulation in different layers and application of bicuculline at different locations on pyramidal cells (Fig. 3) indicate that different populations of GABAergic interneurons provide input to distal dendritic and somatic regions of pyramidal cells. If axonal processes from superficial and deep populations were to overlap extensively, the observed laminar specificity in the action of bicuculline could not have been obtained. Preliminary results obtained by intracellular dye injection of interneurons in piriform cortex support this conclusion (Ekstrand and Haberly 1995 GABAA-mediated IPSCs in piriform cortex have fast and slow components
The results suggest that two kinetically distinct GABAA-mediated IPSC components are present in pyramidal neurons in piriform cortex: a fast component that decays with a time constant of 6-15 ms and a slow component with a decay time constant of 30-80 ms. Several lines of evidence suggest that the slow component represents a longer lasting conductance rather than a space-clamp artifact. First, the delayed voltage-step experiment (Fig. 5) indicates that capacitative current is not responsible for the slow decay. Second, the evidence that both fast and slow components are generated in distal dendrites and in the vicinity of cell bodies (Figs. 4 and 6) is incompatible with an artifactual origin of the slow component from an inadequate space clamp. Third, reduction in the extent of PPD of the slow but not the fast IPSC component by CGP 35348 and a greater block of the slow component by baclofen suggests that the two are generated by different populations of synaptic terminals. A dissociation in the effect of baclofen on fast and slow components also has been reported in the hippocampal CA3 (Lambert and Wilson 1993 Fast and slow GABAA conductances are differentially distributed in somatic and distal dendritic regions
The results obtained by focal application of bicuculline suggest that both fast and slow GABAA components are generated in somatic and dendritic regions of pyramidal cells but that the distribution of the slow component is skewed toward the distal dendritic region and the fast skewed toward the somatic region. However, the simulation studies presented in APPENDIX B reveal that the present evidence cannot exclude the possibility that cell bodies express only the fast component and that distal-most apical dendrites express only the slow.
Use-dependent depression of fast and slow components of GABAA-mediated IPSCs
The susceptibility of GABAA mediated IPSCs to depression during repetitive activation was assessed by examining the extent of PPD. These studies revealed that GABAA-mediated IPSCs are depressed at both long (150-200 ms) and short (10-20 ms) intervals. At long intervals, the slow component was depressed preferentially, whereas at short intervals only the fast component was depressed. A similar result was obtained in the hippocampal CA1 region (Pearce et al. 1995 Functional implications
The results are consistent with a role of dendritic GABAA-mediated inhibition in the regulation of NMDA-mediated processes in piriform cortex. Such regulation would have an impact on the integration of repetitive inputs that can activate the NMDA component as well as on NMDA-dependent LTP (Artola et al. 1990
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
-aminobutyric acid-A (GABAA) receptors largely consisted of confirmation in many systems that it is generated in or near cell bodies by feedback pathways. In recent years, however, it has become apparent that GABAA-mediated inhibition in the cerebral cortex is a complex process that is generated in dendrites as well as cell bodies (Buhl et al. 1994a
; Lambert et al. 1991
; Miles et al. 1996
; Pearce 1993
), has fast and slow components (Pearce 1993
), and is regulated by diverse mechanisms. A thorough knowledge of GABAergic processes clearly will be required for understanding a wide variety of neuronal functions including the integration of spatially and temporally patterned inputs, the regulation of relationships between inputs and output, the coupling of activity to learning-related changes in synaptic efficacy, and maintenance of the intricate balances between excitation and inhibition that are required for optimal function while minimizing the risk of seizure activity.
; Haberly 1973
; Satou et al. 1983
). More recently, evidence from a study of modulation of the N-methyl-D-aspartate (NMDA) component of excitatory postsynaptic potentials (EPSPs) indicated that GABAA inhibition also is generated in apical dendrites of piriform pyramidal cells (Kanter et al. 1996
) as in other types of cerebral cortex. The results of that study indicated that even though the dendritic GABAA inhibitory postsynaptic potential (IPSP) is small in somatic recordings, it can strongly regulate expression of the NMDA component. Because NMDA-dependent long-term potentiation (LTP) in piriform cortex is regulated by GABAA inhibition (del Cerro et al. 1992
; Kanter and Haberly 1993
) as in other types of cerebral cortex, this finding suggests that dendritic inhibition may play a role in controlling synaptic plasticity.
), and evidence that feedback inhibition in the vicinity of cell bodies is largely responsible for prevention of such bursting through control of cell firing (Miles et al. 1996
; Traub et al. 1987
). If dendritic and somatic region inhibitory systems are separate in piriform cortex as in the hippocampus (Freund and Buzaki 1996
), selective block of the dendritic component for purposes of enabling dendritic processes such as NMDA-dependent LTP would be possible during normal function. Separation also could allow feedback adjustments in system excitability through changes in the strength of somatic-region inhibition with minimal effect on integrative processes (see Kapur et al. 1997
).
), stems from the hypothesis that a slower dendritic inhibitory postsynaptic current (IPSC) would exert a stronger controlling action on the NMDA component by virtue of a better match in time course (see Kapur et al. 1997
).
; Mott and Lewis 1991
), the induction of NMDA-dependent LTP in piriform cortex requires activity-dependent blockade of GABAA inhibition. Selective susceptibility of the slow GABAA component to the long-lasting suppression mediated by presynaptic GABAB receptors as demonstrated in hippocampus (Pearce et al. 1995
) could serve to protect the system from regenerative bursting that might develop if the fast component also were subject to prolonged interruption.
METHODS
Abstract
Introduction
Methods
Results
Discussion
References
. Darkfield illumination through a transparent base allowed visualization of all layers. Recordings were made in layer II using the blind whole cell patch technique (Blanton et al. 1989
). Pipette resistances were 7-12 M
in the extracellular space; tips were coated with Sigmacote (Sigma) to lower capacitance. Adequate voltage clamp could be attained when series resistance was <30 M
. Recordings were made with an Axoclamp-2A amplifier in discontinuous voltage clamp mode. Headstage output was monitored with a separate oscilloscope to assure adequate settling time. Switching frequency was 5 kHz in most experiments. Recording pipettes contained (in mM) 135 Cs-gluconate, 2 MgCl2, 0.5 CaCl2, 10 N-[2-hydroxyethyl]piperazine-N
-[2-ethanesulfonic acid] (sodium salt), 5 ethylene glycol-bis(
-aminoethylether) N,N,N
,N
-tetraacetic acid, 2 ATP (ATP, Mg salt), and 5 lidocaine N-ethyl bromide (QX-314). The solution was buffered with CsOH to pH 7.3; final osmolarity was 290-330 mOsm. With Cs+-containing pipettes, resting potential was approximately
45 mV. Cells were typically held at
35 mV to increase the driving force on IPSCs. Responses were recorded and analyzed with pClamp v 5.5 (Axon Instruments). Time constants of IPSCs were determined with the simplex routine provided in pClamp (Axon Instruments). The junction potential at the pipette tip (12 mV), measured as described by Neher (1992)
, was subtracted from responses.
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FIG. 1.
Lamination of piriform cortex, excitatory inputs to pyramidal cells, postulated inhibitory circuitry, and placement of electrodes. Cell bodies of superficial pyramidal cells (P) are in layer II; their apical dendrites extend into layer I and basal dendrites into layer III. Afferent fibers from the olfactory bulb are confined to layer Ia; association fibers (recurrent collaterals of pyramidal cell axons) are in layers Ib and III and, to a lesser extent, layer II. Afferent fibers synapse on distal segments of pyramidal cell apical dendrites; association fibers synapse on proximal segments of pyramidal cell apical dendrites in layer Ib and basal dendrites in layer III (not shown). Postulated superficial (SG) and deep (DG) GABAergic interneurons synapse on apical dendritic and somatic regions of pyramidal cells, respectively. Recording was with a patch pipette (REC) from cell bodies of layer II pyramidal cells. One stimulating electrode was placed in superficial Ia (Sup Stim) to activate inhibitory circuitry in the distal dendritic region, and 1 in the association fiber layers in Ib, II, or superficial III (Deep Stim) to activate inhibitory circuitry in the somatic region. -aminobutyric acid-A (GABAA)-mediated inhibition was blocked locally by pressure injection from bicuculline-containing pipettes in the distal dendritic (BIC in Ia) or somatic region (BIC in II) of the patched neuron.
, A-M Systems) placed under direct vision. One electrode was placed in the layer to be stimulated, the other nearby to create a bipolar stimulus.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
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FIG. 2.
Isolated GABAA-mediated inhibitory postsynaptic currents (IPSCs) can be evoked by superficial (afferent layer) stimulation and deep (association fiber layer) stimulation after pharmacological blockade of glutamatergic excitatory postsynaptic currents (EPSCs). Responses in A and B are voltage-clamp, whole cell patch recordings from the soma of a layer II pyramidal cell. In this and all subsequent figures, EPSCs were blocked by bath applied 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 2-amino-5-phosphonovaleric acid (APV). A: response to shock stimulation in the superficial part of layer Ia in DNQX and APV (control), after addition of 10 µM bicuculline (BIC), to confirm that the response in DNQX and APV was GABAA-mediated and after washout of BIC (wash). B: same as A, but for deep stimulation (layer Ib). Holding potential in this and subsequent figures was depolarized with respect to the GABAA reversal, except as noted.
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FIG. 3.
The IPSC evoked by superficial stimulation is concentrated in the distal apical dendrites of pyramidal cells, whereas the IPSC from deep stimulation is concentrated near cell bodies. GABAA-mediated IPSCs were recorded from cell bodies as described for Fig. 2. A: effect of local application of bicuculline (BIC) in layer Ia on the GABAA-mediated IPSC evoked by deep and superficial (sup) stimulation. B: effect of bicuculline applied near the cell body in layer II. A1 and B1: superimposed responses to superficial (layer Ia) and deep (layer III) stimulation, before and after application of bicuculline. With distal dendritic application (A1), the IPSC evoked by superficial stimulation was reduced by 60% at a time when the IPSC evoked by deep stimulation was unaffected. With somatic application (B1), the deep layer-evoked IPSC was blocked by 75% when the IPSC from superficial stimulation was reduced by 10%. A2 and B2: amplitudes of IPSCs evoked by deep and superficial stimulation as a function of time after application of bicuculline. Control amplitudes are normalized to 100%. , time of bicuculline application. A3 and B3: mean amplitude of IPSCs evoked by deep and superficial stimulation in all experiments at the latency of maximal differentiation of the bicuculline effect (10-30 s postapplication). Error bars are SEs. Significance was P < 0.001 (n = 7) in A3; and P < 0.0001 (n = 6) in B3.
), the falling phase was fitted to exponential functions. The time course of decay of GABAA-mediated IPSCs evoked from all layers was usually best fit by a sum of two exponentials: one with a "fast" time constant on the order of 10 ms, and one with a "slow" time constant on the order of 40 ms at 29°C (Fig. 4A). Fast and slow time constants for IPSCs evoked from different layers are summarized in Table 1. Fast and slow time constants remainedwell separated over a wide range of relative amplitudes(Fig. 4C).
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FIG. 4.
GABAA-mediated IPSCs decay with 2 time constants. A: isolated monosynaptic GABAA-mediated IPSC evoked by superficial stimulation (layer Ia). Line through data points, best-fit biexponential function; the other solid lines are plots of the fast and slow components alone. Faster decaying component ( = 10.6 ms) contributed 63% of the peak amplitude and the slower component (
= 50.2 ms) contributed 37%. B: isolated monosynaptic GABAA-mediated IPSC evoked by deep stimulation (deep part of layer Ib). Peak amplitude consisted of 82% fast (
= 7.2 ms) and 18% slow (
= 42.3 ms). Time scale in B also applies to A. Holding potential was hyperpolarized (
90 mV) with respect to GABAA reversal so that currents in A and B are inward. C: time constants of fast and slow components were well separated over a wide range of relative amplitudes. Values of
slow/
fast for IPSCs recorded in 57 cells are plotted against the percent contribution of the fast component.
, IPSCs evoked by superficial stimulation (layer Ia);
, evoked by deep stimulation (Ib, II, or superficial III).
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TABLE 1.
Time constants of decay and relative amplitudes of fast and slow components of GABAA-receptor mediated IPSCs evoked from different layers
).
; Major 1993
; Rall and Segev 1985
; Spruston et al. 1993
), it could be argued that the slow IPSC component is artifactual. To determine if the slow component is kinetically distinct from the fast, the delayed voltage-clamp method of Pearce (1993)
was applied. The objective was to test for the presence of an active synaptic conductance at a latency when the fast IPSC component would be minimal. Recorded cells were held at or near the reversal potential of the IPSC during shock stimulation of inhibitory axons to minimize current flow. The membrane potential was stepped to a more positive level at a series of latencies after the stimulus so that any synaptic conductance still active at that time would appear as an inward current. The brief capacitative transients resulting from the voltage step were subtracted digitally.
52 mV) to
32 mV at latencies
75 ms after superficial stimulation. In contrast, no synaptic current was evoked by a voltage step at 75 ms in response to deep stimulation (Fig. 5B). Because little current could flow before the voltage step, the presence of current at 75 ms poststimulus suggests that an active synaptic conductance was present at that latency. A similar result was observed in 3/3 cells.
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FIG. 5.
Slow time constant of decay of the GABAA-mediated IPSC represents a slowly decaying component of conductance. Membrane potential was held initially near the reversal potential of the IPSC ( 52 mV) and subsequently stepped to a depolarized level (
32 mV) at a series of latencies after a shock in either layer Ia (A) or in layer III (B). Synaptic currents observed on stepping the membrane potential away from the IPSC reversal at the various latencies after the stimulus have been overlaid. Capacitative currents resulting from the voltage step alone (no stimulus) were subtracted. Note that outward current is elicited by the voltage step at intervals of
75 ms for the response evoked from layer Ia, consistent with the presence of currents of synaptic origin at this latency.
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FIG. 6.
Slow component of the IPSC evoked by superficial stimulation is blocked preferentially by application of bicuculline to the distal apical dendritic region. A1: sample responses from 1 experiment. Coefficients of the fast and slow components before (top) and after bicuculline (bottom) reveal changes in relative amplitude. A2: mean amplitudes (relative to control) of fast and slow components of IPSCs evoked by superficial (sup) stimulation after application of bicuculline to distal apical dendrites in layer Ia. Slow component was blocked to a greater extent than the fast (slow blocked by 67 ± 6%, fast by 33 ± 8%;n = 7, P < 0.01). Application of bicuculline near cell bodies in layer II (B) or to mid-apical dendrites in layer Ib (C) did not differentially block fast and slow components of IPSCs evoked by superficial or deep stimulation. Error bars are SE.
57 mV for IPSC reversal was ~5 mV more depolarized than the value obtained in adult rats with sharp intracellular pipettes (mean
62 mV, Table 3) (E. D. Kanter and A. Kapur, unpublished results). The younger age of the rats in the present studies (Luhmann and Prince 1991
) might have contributed to a more depolarized reversal potential. The substantial deviation from the value that would be predicted from the Cl
concentrations in the bathing medium and pipette solution suggests that equilibration between the contents of the relatively high resistance patch pipettes and cell bodies was incomplete. Alternatively, HCO
3 or other anions may contribute to current through GABAA channels in piriform cortex as concluded for hippocampus (Bonnet and Bingmann 1995
; Fatima-Shad and Barry 1993
; Staley et al. 1995
).
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FIG. 7.
Reversal potentials of fast and slow IPSC components evoked by deep and superficial stimulation were similar. A: IPSCs evoked by deep (layer III) and superficial (layer Ia) stimulation in a pyramidal cell at a series of holding potentials from 32 to
82 mV. B: reversal potentials of the fast and slow components of the superficial- and deep-evoked IPSCs were all approximately
50 mV.
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TABLE 2.
Reversal potentials of GABAA-mediated IPSCs for slices from 3-4 wk old rats and whole cell patch pipettes
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TABLE 3.
Reversal potentials of GABAA-mediated IPSCs for slices from adult rats and sharp micropipettes
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FIG. 8.
Paired pulse depression of fast and slow components at long (150-200 ms) and short (10-20 ms) intershock intervals. A: when paired pulses were separated by 150-200 ms, the slow component of the second response was depressed more than the fast. A1: responses to a pair of stimuli in layer Ib separated by 200 ms. Holding potential was hyperpolarized ( 67 mV) with respect to GABAA reversal so that current is inward. Coefficient of the slow component in the second response was 59% of that in the first; the coefficient of the fast in the second was 89% of the first. A2: pooled results for 24 cells. Mean amplitudes of the fast and slow components in the second response are expressed as a percentage of their amplitudes in the first response. Stimuli were separated by 150-200 ms. B: fast component was selectively depressed when paired pulses were separated by 10-20 ms. B1: responses to a pair of identical stimuli in layer Ib separated by 20 ms from a different cell than illustrated in A1. Holding potential was
30 mV. Top: response to the shock pair (S1,S2). Middle: response to first shock alone (S1). Bottom: after subtraction of the response to the first shock alone. Coefficient of the fast component in the second response was 59% of that in the first; the coefficient of the slow in the second was 97% of the first. B2: pooled data as in A2, but for intervals of 10-20 ms (n = 9). Error bars in A2 and B2 are SEs. * Significantly different from 100% (P < 0.0001, 1 group t-test). ** Depression of slow component significantly greater than depression of fast (P < 0.0001, paired t-test).
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FIG. 9.
The GABAB antagonist 3-amino-propyl (diethoxymethyl) phosphinic acid 35348 (CGP) reduced paired pulse depression (PPD) at long, but not short intervals. A: paired pulses at long intervals. A1: sample responses to paired stimulation in layer Ia before and after bath application of 1 mM CGP, 200-ms interval. Peaks of the first responses are normalized. A2: pooled data for long interval. Mean amplitudes of fast and slow components in the second response are expressed as a percentage of the first response amplitudes before (ctrl) and after CGP. Note that CGP partially reversed the depression of the slow component. * Difference between control and CGP conditions significant at P < 0.005 (paired t-test). B: paired pulses at brief intervals. B1: sample responses to paired stimulation in layer Ib before and after CGP, same cell as in A1 but pulses separated by 20 ms. Peaks of first responses are normalized. B2: pooled data as in A2, except for 10- to 20-ms intervals. Error bars in A2 and B2 are SEs.
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FIG. 10.
R(+)-baclofen preferentially reduced the slow component of the GABAA-mediated IPSC. A: GABAA-mediated IPSC evoked from layer Ib before (control) and after bath application of 0.5 µM R(+)-baclofen. Peaks have been normalized to illustrate differences in their time courses. Coefficient of the slow component in baclofen was 45% of that in the control; coefficient of the fast component in baclofen was 84% of control. B: pooled data. Mean amplitudes of fast and slow components in baclofen expressed as a percentage of their control amplitudes. Error bars are SEs.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
).
; Miles et al. 1996
), current source-density analysis (Lambert et al. 1991
) and local application of bicuculline (Pearce 1993
), indicate that GABAA-mediated IPSCs are generated in both apical and basal dendrites of pyramidal cells. In cat motor cortex, evidence has been provided that inhibitory neurons in all layers give rise to horizontal axons that generate GABAA-mediated IPSPs in apical dendrites of pyramidal cells (Kang et al. 1994
). It therefore appears that dendritic GABAA-mediated inhibition is a general feature of pyramidal cells in cerebral cortex.
; Ekstrand et al. 1996
). For the hippocampus, there is an extensive literature supporting such laminar specificity in inhibitory circuitry that includes studies combining anatomic and physiological methods (Buhl et al. 1994a
,b
, 1995
; Freund and Buzaki 1996
; Halasy and Somogyi 1993
; Lacaille et al. 1989
; Miles et al. 1996
).
), the present evidence for GABAergic neurons and axons in this layer suggests that afferents mediate a feedforward inhibition onto the apical dendrites of pyramidal cells (see Fig. 1). Studies in progress have shown that interneurons in layer Ia are excited by afferent fibers, consistent with this hypothesis (Ekstrand and Haberly 1995
).
; Gellman and Aghajanian 1993
; Haberly and Bower 1984
; Kubota and Jones 1992
; Satou et al. 1983
). The present studies provide additional evidence that the action of this circuitry is concentrated in somatic and proximal dendritic regions of pyramidal cells.
) and CA1 (Pearce et al. 1995
) regions. Finally, in a computer simulation analysis (APPENDIX A), the observed slow component could not be replicated with fast-only GABAA conductances on dendrites. In contrast, the observed IPSCs could be simulated readily with a mixture of fast and slow GABAA conductances (APPENDIX B).
). In the present study, the fast time constant ranged from 6 to 15 ms and the slow from 30 to 80 ms, consistent with the lower recording temperature (29 vs. 36°C). GABAA-mediated IPSCs with dual decay time constants of 7 ± 1.6 ms and 59 ± 16 ms at 22-24°C also have been reported in cerebellar granule cells (Puia et al. 1994
).
) where paired-pulses with intervals of 150-200 ms depressed the slow component by ~50% but did not reduce the fast component, whereas paired-pulses with intervals of 20-40 ms depressed the fast component by ~30% but did not affect the slow component. It is interesting to note that a commonly used LTP-inducing protocol, theta-burst stimulation (Larson and Lynch 1988
), uses both long and short intervals as tested above and therefore would result in the depression of both the slow and fast GABAA-mediated IPSCs, but depression of the slow would develop more slowly and be much longer lasting.
; Kanter and Haberly 1993
; Steward et al. 1990
; Wigström and Gustafsson 1983
). The laminar specificity in inhibitory circuitry that has been demonstrated could allow a relatively selective block of the dendriticGABAA-mediated IPSC by action of centrifugal inputs or local processes at the interneuronal level. In the companion paper, evidence will be presented that the greater proportion of the slow GABAA component in distal apical dendrites on which afferent fibers synapse could increase the efficacy of this control: computer simulation studies confirmed that the slow component is more effective that the fast for control of current through NMDA channels and the resulting depolarization (Kapur et al. 1997
). Regulation of the slow component by presynaptic GABAB receptors further supports a role of this process in the control of NMDA-dependent synaptic plasticity, because, in the hippocampus, a reduction in strength of GABAA-mediated inhibition by presynapticGABAB regulation is required for the induction of LTP (Davies et al. 1991
; Mott and Lewis 1991
). This requirement, together with a lack of presynaptic GABAB inhibition of the fast GABAA component, indicates that regulation of NMDA-dependent LTP by GABAergic inhibition in hippocampus is exclusively by way of the slow component (Pearce et al. 1995
).
; Lipowsky et al. 1996
; Magee and Johnson 1995; Schwindt and Crill 1995
), one action of which is presumably a modulation of the NMDA component. Regulation of voltage-gated Ca2+ channels by dendritic GABAA-mediated inhibition also might play a role in control of NMDA-independent synaptic plasticity (Johnston et al. 1992
). The ability of dendritic GABAA-mediated inhibition to block burst firing mediated by dendritic Ca2+ channels in pyramidal cells in the CA3 region of hippocampus supports this role (Miles et al. 1996
; Traub et al. 1994
). Dendritic GABAergic inhibition also would modulate the back-propagation of Na+ spikes into dendrites from cell bodies (Magee and Johnston 1997
; Svoboda et al. 1997
; Tsubokawa and Ross 1996
). Because NMDA-dependent LTP of weak EPSPs can be enabled by a pairing with back-propagating action potentials (Magee and Johnston 1997
; Markram et al. 1997
), this could provide an additional means for GABAergic regulation of synaptic plasticity.
).
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ACKNOWLEDGEMENTS |
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We thank M. Hines for technical assistance with Neuron.
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-19865 to L. B. Haberly.
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APPENDIX A |
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Can the biexponential decay of GABAA-mediated IPSCs be reproduced with monoexponentially decaying dendritic inputs?
Because the true time course of synaptic processes in dendrites is faster than the time course recorded in voltage-clamp mode from the cell body, the possibility must be considered that the observed biexponential decay in GABAA-mediated IPSCs is artifactual. To explore this possibility, simulations were carried out with the model cell described in the companion paper (Kapur et al. 1997). This model was developed from a serially reconstructed layer II pyramidal cell in piriform cortex with physiological data derived by whole cell patch recording. The morphologically complete version of the model (345 compartments) was used for all simulations. The conductance change associated with the activation of GABAA synaptic inputs (GABAA conductance) was simulated with exponential rising and falling phases as follows
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35 mV (level in most of the present experiments) during application of the GABAA conductance. Runs were performed with two values of Ri (100 and 200
cm) and two values of Rm (26,845 and 67,114
cm2) that yielded membrane time constants of 40 and 100 ms, respectively, with Cm = 1.49 µF/cm2 (the computed value for the modeled cell). The response was a simulated somatic voltage-clamp recording (current required at the cell body to maintain a constant potential at this location during activation of the dendritic conductances). The results revealed a substantial slowing of the decay phase of the somatic current relative to the time course of the applied GABAA conductance, as expected (Fig. A1A). However, convergence could not be obtained with a biexponential fit with any combination of parameters, and the time constants for monoexponential fits were 23-30 ms in contrast to experimentally derived values for the slowGABAA component of 30-80 ms.
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FIG. A1.
Simulated somatic voltage-clamp recordings of dendriticGABAA-mediated IPSCs. Simulations were carried out with the compartmental model of a layer II pyramidal cell (40C2) developed in the companion paper (Kapur et al. 1997 ). A: with fast-only GABAA conductances confined to distal apical dendrites, response decays could not be well-fitted with either mono- or biexponential functions. Illustrated trace was obtained with 200 conductances with a decay time constant (
d) = 10 ms and maximum conductance (
) = 0.5 nS at distances
325 µm from the cell body. Closest fit was the illustrated single exponential with a time constant of 23 ms. B: when fast-only GABAA conductances were distributed over middle and distal apical segments, decay of the somatic response could be biexponential. Illustrated result (best match to actual response) was obtained with 100 conductances with
d = 10 ms and
= l50 nS on distal segments at 300-600 µm from the cell body, and 100 conductances with
d = 10 ms and
= 2 nS on middle apical segments at 75-300 µm from the cell body. Slow time constant for the biexponential fit (39 ms) was at the lower limit of actual responses (48 ± 3 ms), as was the contribution of the fast component to peak amplitude (34% vs. 57 ± 4%). C: fast-only conductances on middle and distal segments also could generate a monoexponential decay. Illustrated result was obtained with 100 conductances with
d = 10 ms and
= 17.5 nS at 300-600 µm from the cell body and l00 with
d = 10 ms and
= 1.75 nS at distances of 75-300 µm. In all simulations (A-C), reversal potential of the leak conductance was
50 mV (approximate value with Cs+-containing patch pipettes); holding potential at the cell body was
35 mV. Passive membrane constants were: Rm = 26,845
cm2 and Cm = 1.49 µF/cm2 for A-C; Ri = 200
cm in A and C and 300
cm in B.
d) for the GABAA conductance was varied from 7 to 10 ms in steps of 1 ms. Tests showed that simulated somatic IPSC recordings with a slow component in the experimentally observed range (>30 ms) could not be reproduced when
d was <7 ms.
cm in steps of 100
cm.
m) in the range observed experimentally with Cs+-containing patch pipettes. Cm = 2 µF/cm2 andRm = 26,845
cm2 yielded a
m = 40 ms; Cm = 1.5 µF/cm2 and Rm = 50,000
cm2 yielded
m = 100 ms. The reversal potential of the leak conductance (represented by Rm) was set to
50 mV, again to reproduce findings with Cs+-containing pipettes in slices from immature animals.
20% of the peak amplitude.
s:
f) >3.5. Experimentally,
s:
f = 4.61 ± 0.28 (Table 1).
Simulation of biexponential IPSCs with fast and slow GABAA conductances
Based on the failure of simulations with a single time constant of decay to adequately reproduce the observed IPSCs, studies were carried out with the assumption that there are two GABAA conductance components with fast and slow time constants of decay that are distributed differentially on the apical dendrite. These simulations were carried out with the same model cell used for the studies in APPENDIX A, with the cable parameters that provided the best fit to voltage transients for the modeled cell [Ri = 137
Extent of artifactual PPD of the IPSC from imperfect voltage clamp
In response to the second of a pair of stimulus pulses separated by a brief (10-20 ms) interval, the fast-decaying component of the GABAA-mediated response was depressed significantly. A potential problem in interpretation is that an imperfect voltage clamp could produce a similar decrease in amplitude. This is because the driving force for the IPSC would be decreased during the response to the second stimulus if voltage is not well controlled during the response to the first. Because the extent of this error would decrease over time, it would be greatest for the fast component at short intershock intervals. To evaluate the extent of this error, simulations were carried out with the same parameters used to replicate the deep stimulus-evoked IPSC in APPENDIX B. The simulated response to paired activation of identical conductances separated by 10 ms is illustrated in Fig. C1A. Comparison of the response to the first stimulus alone (Fig. C1B) with the subtraction-isolated response to the second stimulus of the pair (Fig. C1C) reveals a decrease in amplitude as a result of the incomplete dendritic voltage clamp. The fast component was decreased by 22%, which compares with a typical 50% decrease observed experimentally (Fig. 8). It is concluded that there is a PPD of the fast GABAA component at short intervals, but the magnitude of depression is approximately one-half that observed experimentally in somatic voltage-clamp recordings.
Present address of A. Kapur: Div. of Neuroscience, Baylor College of Medicine, Houston, TX 77030.
Address for reprint requests: L. B. Haberly, Dept. of Anatomy, University of Wisconsin, 1300 University Ave., Madison, WI 53706. Received 19 March 1997; accepted in final form 3 July 1997.
in Fig. A2), the proportion of the fast component was, at best, at the lower limit of the experimentally observed range (maximum of 35% vs. 57 ± 4% for actual responses, Table 1).
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FIG. A2.
Simulated somatic voltage-clamp recordings of dendriticGABAA IPSCs with fast-only decays do not match experimentally recorded IPSCs. Each point is a result from a systematic parameter exploration for which the decay phase could be well fit by a biexponential function with a fast time constant ( f) between 6 and 15 ms and a slow time constant (
s) between 30 and 80 ms. Results of 2 explorations are presented:
, results for which
s:
f was within the experimental range;
, results for which the percent contribution of the fast component to peak amplitude was within the experimental range. As described in the text, when
s:
f was in-range, percent fast was not, and when percent fast was in-range,
s:
f was not.
in Fig. A2). However, maximum
s:
f was 3.4 versus 4.61 ± 0.28, as observed experimentally (Table 1).
s:
f exceeded 3.5, the simulations were repeated with the proximal population of GABAA conductances starting at 25 µm from the cell body rather than 75 µm, in an attempt to increase the proportion of the fast component. This resulted in a maximum contribution of the fast component of 42%
still at the lower limit of the experimentally observedvalues.
s:
f ratio, could not be duplicated. In cases where a monoexponential fit was better than a biexponential, the slowest time constant was 30 ms, which was substantially faster than the time constant of the actual slow component evoked by superficial stimulation (Table 1). It therefore is concluded that the experimentally observed biexponential decay results from the presence of fast and slow GABAA conductance components.
APPENDIX B
cm, Rm = 14,005
cm2, and Cm = 1.49 µF/cm2; cell 40C2 in Table 1 from Kapur et al. (1997)
] and a resting potential (reversal for Rm) of
50 mV.
that was validated by direct counts in serial sections. Synapses with symmetrical contacts and associated pleomorphic vesicles were assumed to be GABAergic. Calculations were made of the number per unit volume as a function of depth within layer I. Because this layer contains few dendrites of nonpyramidal cells (Haberly 1983
; Haberly and Feig 1983
), these numbers are thought to be reasonable estimates for synaptic inputs to pyramidal cell dendrites. The same methods were used to estimate the number of synapses with round vesicles and asymmetrical contacts (presumed glutamatergic synapses). Because dendritic spines on pyramidal cells in piriform cortex typically receive a single synapse of this form, these estimates are approximately equal to the volume density of spines. From the ratio of the volume density of spines to the volume density of putative GABAergic synapses for the opossum and from the present estimates of spines per unit membrane area for the rat (Kapur et al. 1997
), estimates of the density of GABAergic synapses per unit membrane area were derived for different dendritic segments (Tables 2 and 3 in Kapur et al. 1997
). Dendritic compartments of the model cell were chosen for the assignment ofGABAergic synapses by random sampling using the estimates in Table 2 from Kapur et al. (1977) and the locations and dimensions of compartments in the model cell.
d = 7.25 ms and
= 1.15 nS were distributed randomly in apical dendritic compartments at 100-400 µm from the cell body. Slow conductances with
d = 37 ms and
= 0.25 nS were placed on compartments at 200-600 µm from the cell body. These distributions were loosely based on the experimental observations that both fast and slow components of the IPSC evoked by superficial stimulation were blocked by bicuculline in the mid-apical dendritic region (Fig. 6C) and that the slow component was blocked to a greater extent in the distal apical region (Fig. 6A). No GABAergic inputs were placed on the soma or basal dendrites because local application of bicuculline at the depths of these structures had little effect on the response to superficial stimulation. This distribution of conductances produced simulated IPSCs that adequately matched actual IPSCs evoked by superficial stimulation: decay time constants were 11 and 51.6 ms, and the fast component constituted 60% of the peak amplitude (Fig. B1A).
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Box 1.
IPSCs simulated by a combination of fast and slow GABAA components in apical dendrites can match experimentally recorded IPSCs. A: experimental IPSC evoked by superficial stimulation ( ) and simulated IPSC (
). Both time constants and relative proportions of the 2 components were well reproduced by the model. B: same as A but for deep stimulation.
d = 37 ms,
= 0.2 nS) were distributed on apical and basal dendrites at distances of 100-300 µm from the cell body. Ninety out of a total of 100 fast GABAA conductances (
d = 7.25 ms,
= 1.0 nS) were placed on apical and basal dendrites at distances of 0-300 µm from the soma; the remaining 10 were placed on the cell body. This distribution of fast and slow conductances produced a realistic IPSC at the soma that decayed biexponentially with time constants of 10 and 45.6 ms, with the fast component comprising 73% of the peak amplitude (Fig. B1B).
APPENDIX C
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FIG. C1.
Evaluation of the extent of artifactual paired pulse depression of dendritic GABAA-mediated IPSCs when voltage clamp is applied at the cell body. A: simulated somatic recording of identical IPSCs separated by 10 ms. B: simulated recording of first IPSC alone. C: simulated recording of the second IPSC in the pair, isolated by subtracting the response in B from that in A. Peak amplitude of the isolated second response was 81% of the first. Fast component was depressed by 22% and the slow by 11%. Passive membrane properties and synaptic distributions used for the simulation were the same as those for Fig. B1B.
FOOTNOTES
REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References
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0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society