Departments of 1Pharmacology, 2Pediatrics (Neurology), 3Surgery (Neurosurgery), and 4Neurobiology, Duke University Medical Center, Durham, North Carolina, 27710
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ABSTRACT |
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Mott, David D., Qiang Li, Maxine M. Okazaki, Dennis A. Turner, and Darrell V. Lewis. GABAB-Receptor-Mediated Currents in Interneurons of the Dentate-Hilus Border. J. Neurophysiol. 82: 1438-1450, 1999. GABAB-receptor-mediated inhibition was investigated in anatomically identified inhibitory interneurons located at the border between the dentate gyrus granule cell layer and hilus. Biocytin staining was used to visualize the morphology of recorded cells. A molecular layer stimulus evoked a pharmacologically isolated slow inhibitory postsynaptic current (IPSC), recorded with whole cell patch-clamp techniques, in 55 of 63 interneurons. Application of the GABAB receptor antagonists, CGP 35348 (400 µM) or CGP 55845 (1 µM) to a subset of 25 interneurons suppressed the slow IPSC by an amount ranging from 10 to 100%. In 56% of these cells, the slow IPSC was entirely GABAB-receptor-mediated. However, in the remaining interneurons, a component of the slow IPSC was resistant to GABAB antagonists. Subtraction of this antagonist resistant current from the slow IPSC isolated the GABAB component (IPSCB). This IPSCB had a similar onset and peak latency to that recorded from granule cells but a significantly shorter duration. The GABAB agonist, baclofen (10 µM), produced a CGP 55845-sensitive outward current in 19 of 27 interneurons. In the eight cells that lacked a baclofen current, strong or repetitive ML stimulation also failed to evoke an IPSCB, indicating that these cells lacked functional GABAB receptor-activated potassium currents. In cells that expressed a baclofen current, the amplitude of this current was ~50% smaller in interneurons with axons that projected into the granule cell dendritic layer (22.2 ± 5.3 pA; mean ± SE) than in interneurons with axons that projected into or near the granule cell body layer (46.1 ± 10.0 pA). Similarly, the IPSCB amplitude was smaller in interneurons projecting to dendritic (9.4 ± 2.7 pA) than perisomatic regions (34.3 ± 5.1 pA). These findings suggest that GABAB inhibition more strongly regulates interneurons with axons that project into perisomatic than dendritic regions. To determine the functional role of GABAB inhibition, we examined the effect of IPSPB on action potential firing and synaptic excitation of these interneurons. IPSPB and IPSPA both suppressed depolarization-induced neuronal firing. However, unlike IPSPA, suppression of firing by IPSPB could be easily overcome with strong depolarization. IPSPB markedly suppressed N-methyl-D-aspartate but not AMPA EPSPs, suggesting that GABAB inhibition may play a role in regulating slow synaptic excitation of these interneurons. Heterogeneous expression of GABAB currents in hilar border interneurons therefore may provide a mechanism for the differential regulation of excitation of these cells and thereby exert an important role in shaping neuronal activity in the dentate gyrus.
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INTRODUCTION |
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The excitability of granule cells, the principal
cells of the dentate gyrus, is regulated by a diverse population of
inhibitory interneurons located in the molecular layer (ML), granule
cell layer (GCL), and hilus. One region particularly rich in glutamic acid decarboxylase (GAD) positive, presumably inhibitory, interneurons is the border of the GCL and the hilus (Houser and Esclapez
1994; Kosaka et al. 1985
; Ribak and
Seress 1983
; Seress and Ribak 1983
; Sloviter and Nilaver 1987
), an area referred to by
Amaral (1978)
as the dentate hilus border of Zone 4 or
the D/H border zone. Interneurons located in this area form synaptic
contacts with specific regions of the granule cells they innervate
(Amaral 1978
; Halasy and Somogyi 1993
;
Han et al. 1993
; Lorente de No 1934
; Mott et al. 1997
; Ramon y Cajal 1968
;
Soriano and Frotscher 1993
). Some cells in this area,
such as the pyramidal basket cells, have axons that arborize within or
very close to the granule cell layer. In contrast, other interneurons
are specialized to innervate more distal dendritic regions of granule
cells. Interneurons innervating somatic and dendritic regions of
granule cells may have different inhibitory effects (Miles et
al. 1996
). Although many interneurons in this layer innervate
granule cells, recent studies also have identified a subgroup of
interneurons that are specialized to contact only other inhibitory
cells (Gulyás et al. 1996
, Hájos et
al. 1996
). These connected interneuronal networks may play a
role in rhythm generation in the hippocampal formation. Differential modulation of D/H border zone interneurons thus may provide a mechanism
to alter the functional state of granule cells (Parra et al.
1998
).
The activity of interneurons is regulated by both
GABAA- and
GABAB-receptor-mediated inhibition.
GABAA-receptor-mediated inhibitory postsynaptic
potentials and currents (IPSP/Cs) have been examined in interneurons in
several different regions of the hippocampal formation
(Buckmaster and Schwartzkroin 1995; Hájos
and Mody 1997
; Lacaille 1991
; Lacaille
and Schwartzkroin 1988a
,b
; Lacaille et al. 1987
;
Misgeld and Frotscher 1986
; Morin et al.
1996
; Soltesz and Mody 1994
; Williams et
al. 1994
). These IPSP/Cs are thought to contribute a necessary
timing mechanism for rhythmic oscillation in interneuronal networks and
thereby contribute to the generation of gamma frequency oscillations
(20-100 Hz) in principal cells (Wang and Buzsáki
1996
; Whittington et al. 1995
). In contrast, the
role of GABAB-receptor-mediated inhibition in
interneurons is less clear. GABAB IPSP/Cs have
been reported in hippocampal interneurons (Buckmaster and
Schwartzkroin 1995
; Khazipov et al. 1995
;
Lacaille 1991
); however, they have not been examined in detail. To understand how dentate-hilar border interneurons are regulated. we first must have a clear picture of the currents that are
involved and whether the expression of these currents correlates with
morphological properties of the interneurons. Therefore a primary aim
of this study was to characterize
GABAB-receptor-mediated IPSP/Cs in
morphologically identified D/H border zone interneurons.
A second aim of this study was to determine the role of
GABAB inhibition in regulating the activity of
these interneurons. We previously have shown that activation of
presynaptic GABAB autoreceptors on inhibitory
terminals could suppress the synaptic release of GABA and thereby
disinhibit granule cells (Mott et al. 1990, 1993
).
However, the possibility exists that GABAB
receptors located on the somatodendritic membrane of inhibitory
interneurons may inhibit interneurons and thus contribute to the
disinhibition of granule cells. Because of its slower time course,
GABAB inhibition might be expected to have
markedly different effects on the frequency of interneuron firing than
does GABAA inhibition.
To address these issues, we used the whole cell patch-clamp technique to record pharmacologically isolated slow IPSCs from visually identified interneurons at the dentate-hilus border. The cells were filled with biocytin, allowing us to reconstruct their axonal and dendritic arbors for anatomic identification. We found marked heterogeneity in the magnitude of GABAB responses in these interneurons that largely correlated with their axonal distribution. In addition, we found that IPSPB inhibits interneuronal firing and powerfully suppresses N-methyl-D-aspartate (NMDA) EPSPs. We suggest that GABAB inhibition modulates the activity of D/H border zone interneurons and thereby plays an important role in regulating neuronal activity in the dentate gyrus.
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METHODS |
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Slice preparation and recording
Transverse 300-µm brain slices were prepared from halothane-anesthetized male Sprague-Dawley rats (16- to 30-days old) using a Vibraslicer (Campden 752), and the hippocampal formation was gently dissected free of the remainder of the brain slice. Hippocampal slices were incubated in warmed (32-34°C), artificial cerebrospinal fluid (ACSF) containing (in mM): 120 NaCl, 25 NaHCO3, 10 dextrose, 3.3 KCl, 1.23 NaH2PO4, 1.8 CaCl2, and 1.2 MgSO4, bubbled with a 95% O2-5% CO2 gas mixture at pH 7.4. For recording, slices were placed into a small submersion chamber maintained at 32-34°C and held in place by a bent piece of platinum wire resting on the surface of the slice. Transilluminated slices were viewed with an upright Nikon Optophot microscope with a ×40 water immersion objective, Hoffman Modulation Contrast optics and infrared filtering.
Our whole cell voltage-clamp technique has been described in our
previous publications (Mott et al. 1993; Xie et
al. 1992
). Briefly, recordings were made with microelectrodes
(3-6 M
) pulled from borosilicate glass capillary tubing (1.5 mm OD,
1.05 mm ID, World Precision Instruments, Sarasota, FL) on a
Flaming-Brown microelectrode puller. The intracellular solution
contained (in mM): 130 Kgluconate, 7 KCl, 10 HEPES, 2 MgATP, and 0.3 TrisGTP, and pH adjusted to 7.2 with KOH. Biocytin (Sigma Chemical, St. Louis, MO) 0.2-0.4% was added for later visualization of the neuron morphology. Osmolalities were from 260 to 275 mOsm for pipette solutions and 284 mOsm for the ACSF.
When used as the internal anion, gluconate has been reported to
interact with some types of potassium channels (Zhang et al. 1994) but not the GABAB receptor-coupled
potassium conductance (Lenz et al. 1997
). However, to
confirm this, we compared IPSCBs in a group of 13 interneurons recorded with our gluconate-containing internal solution
to the IPSCBs in a group of 9 interneurons
recorded with an internal solution in which gluconate had been replaced with an equimolar concentration of methylsulfate. We found no difference in the slope conductance of IPSCB
between these groups of cells (independent t-test,
P > 0.87), indicating that gluconate had no specific
effect on the GABAB conductance recorded in this study.
Stimuli were 0.1-ms, monophasic, cathodal, rectangular, constant current pulses (10-1,000 µA) delivered through a monopolar tungsten electrode placed in the outer molecular layer. An Axopatch 1D amplifier with filtering at 3 kHz and series resistance compensation and capacitance compensation set at zero was used. Series resistance was monitored during the recording, and cells were discarded if seal breakdown or sealing over was detected. Responses were digitized and recorded on magnetic disks using a Nicolet digital oscilloscope (model 410). In addition, responses were digitized by a Digidata 1200 A-D board (Axon Instruments, Foster City, CA) in a PC-based computer using Strathclyde Electrophysiology Software Whole Cell Program developed and generously provided by John Dempster.
Cells selected for recording had somata located near the hilar border
of the GCL, were distinctively larger than granule cells, and exhibited
basilar dendrites entering the hilus. On entry into the cells, current
injections were performed to record typical trains of action
potentials. If the cell did not exhibit the typical deep monophasic
spike afterhyperpolarizations seen in interneurons in this area, it was
not used (Mott et al. 1997; Scharfman
1992
).
Fixation, histochemistry, and reconstruction
Our methods have been described previously (Mott et
al. 1997). Briefly, slices were fixed in 4% paraformaldehyde
containing 0.1% gluteraldehyde in 0.1 M phosphate buffer, and 75-µm
sections were cut on a vibratome. Sections were incubated in
Avidin-horseradish peroxidase solution (Vectastain ABC Standard Kit)
for 2-4 h, washed, and incubated in phosphate buffer containing 0.05%
3'-3-diaminobenzidine tetrahydrochloride, CoCl2
(0.025%), and NiNH4SO4
(0.02%) for 15 min and then
H2O2 (0.1% final
concentration) until cells were visible. For neuron reconstruction,
cells were traced across all sections using a ×100 oil immersion lens
and a three-dimensional neuronal reconstruction system consisting of an
automated stage and a high resolution monitor (Neurolucida,
Microbrightfield, Colchester, VT).
Recovery of the stained cell was achieved in 52 of 110 interneurons
from which electrophysiological data were used in this report. The 58 cells in which morphology was not recovered were classified as putative
interneurons based on their visual appearance during recording and
electrophysiological characteristics (see preceding text). Of the 52 cells from which anatomy could be recovered, identification of axon
morphology was possible in 35. In those interneurons the axons of which
were well stained, the distribution of the axons in the dentate could
be classified into four patterns previously described in detail by
Mott et al. (1997), and the reader is referred to that
publication for a more extensive discussion of the interneuron
morphology. Interneurons, such as basket and axo-axonic cells, the
axons of which arborized primarily in the granule cell layer, were
termed GCL cells. Cells with axons that ramified throughout the
molecular layer were termed TML cells. Many of these TML cells may
represent the pyramidal interneurons described by Soriano and
Frotscher (1993)
. Cells with axons primarily in the inner
molecular layer were termed IML cells and most likely correspond to the
hilar commissural-associational pathway (HICAP) cells of Han et
al. (1993)
. Finally, cells with axons preferring the outer
molecular layer are termed OML cells. These cells resemble the hilar
perforant pathway-associated (HIPP) cells of Han et al.
(1993)
.
Electrophysiology
Resting membrane potential for each neuron was determined at the beginning of the experiment by the voltage offset accompanying cell access and rechecked at the end of the experiment by measuring the amplitude of the DC offset produced when the cell access was lost. We have corrected for a junction potential of 10 mV by setting the values reported herein at 10 mV more hyperpolarized than the values read off the amplifier. The input resistance of each neuron was determined in current clamp from the amplitude of the voltage deflection produced by a current injection of small amplitude (10-50 pA).
Interneuron recordings lasted 20-60 min. During these recordings, a
gradual run down in the amplitude of synaptic currents was noted but
not specifically studied. Despite the presence of ATP and GTP in the
pipette solutions, NMDA EPSCs, in particular, ran down with time. In
contrast, GABAA IPSCs were clearly less prone to
this problem and GABAB IPSCs did not show a
consistent decrease. The average change in peak amplitude during the
first 30 min of recording, expressed as percent of the initial peak amplitude of the evoked PSC, was (mean ± SE) 76 ± 20%
for NMDA EPSCs (n = 10 cells),
19 ± 4.9%
(n = 8 cells) for GABAA IPSCs, and
10 ± 7.4% for GABAB IPSCs
(n = 8 cells).
Values of experimental measurements are given in the results as means ± SE followed by sample number (n). Statistical significance was determined using the Student's t-test or one-way ANOVA with post hoc Bonferroni tests.
Decay kinetics of the IPSCs were analyzed using curve fitting functions
in Strathclyde Electrophysiology Software Whole Cell Program (version
1.1) and in Microcal Origin 5.0 (Microcal Software, Northhampton, MA).
For the evoked IPSCs, each trace fitted was an average of three to five
evoked responses. Each trace was fitted to both a monoexponential and
biexponential decay curve. Several criteria were used to conclude
whether a given decay phase was better represented by a biexponential
or a monoexponential function. One criterion was the ratio of the
residual variance of the fit to the background variance as measured by
the software. A second criterion was simple visual inspection and often
it was quite evident which equation was the better fit to the data. In
many cases, the two time constants derived from the biexponential fit differed very little, i.e., by a factor of 2, suggesting to us that
the most parsimonious interpretation was a monoexponential decay.
Reversal potentials for the IPSCs were determined from current voltage
(I-V) curves of the IPSCs elicited at different holding potentials. The expected reversal potential for potassium currents is
97 mV calculated using the Nernst equation with our extracellular potassium concentration of 3.3 mM and intracellular concentration of
137 mM (Hille 1992
).
Drugs
Six,7-dinitroquinoxaline-2,3-dione (DNQX; 20 µM) and
D()-2-amino-5-phosphonovaleric acid (D-APV)
(50 µM) were purchased from Tocris Cookson (Bristol, UK). When
NMDA-receptor-mediated currents were studied, 10 µM DNQX was used.
Bicuculline methiodide (20 µM) and picrotoxin (75 µM) were
purchased from Sigma Chemical . QX 314 was obtained from Astra
Pharmaceutical Products, (Worcester, MA). GABAB
antagonists CGP 35348 and CGP 55845 and the agonist (±)baclofen were
supplied generously by Novartis Pharma AG (Basel). All drugs were bath
applied in the perfusion medium. Drugs were washed on until a
steady-state effect was observed before any measurements were taken.
All drugs were dissolved directly into the ACSF except DNQX, which
first was dissolved in dimethyl sulfoxide (DMSO) and then added to the ACSF.
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RESULTS |
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In this study we recorded from 110 interneurons at the border of
the hilus and the granule cell layer in the dentate gyrus. The input
resistance of these interneurons averaged 242 ± 20 M and the
resting potential averaged
66.0 ± 0.7 mV. Their morphological and passive electrotonic parameters have been described previously (Mott et al. 1997
). In addition, we also recorded from
18 granule cells for comparison with the interneurons. These granule
cells had an average input resistance of 324 ± 38 M
and rested
at
82.2 ± 1.9 mV, significantly more hyperpolarized than the
interneurons (P < 0.001, independent
t-test).
Slow IPSCs
The slow IPSC was examined in 63 interneurons. In the absence of
antagonists a single stimulus to the ML evoked a large fast EPSC
followed in most cells by a slow IPSC. Application of DNQX (20 µM),
D-APV (50 µM), bicuculline methiodide (20 µM), and
picrotoxin (75 µM) blocked the AMPA, NMDA, and
GABAA components of the response, respectively,
leaving a slow IPSC in 55 of the 63 interneurons (Fig.
1). Spontaneous slow IPSCs were not
observed. In 32 of the 55 cells that exhibited a pharmacologically
isolated slow IPSC, we delivered a stimulus which evoked a maximal
response. The maximal amplitude of the slow IPSC at a holding potential
of 70 mV averaged 22.8 ± 2.6 pA but varied markedly over a
range from 1.6 to 56.9 pA. Eight of the 63 interneurons exhibited no
detectable slow IPSC despite strong and/or repetitive stimulation and
the presence of a robust EPSC in the absence of antagonists. These
eight cells appeared electrophysiologically healthy in that they had an
input resistance (268 ± 30 M
) similar to the average of all
interneurons and hyperpolarized resting potentials (
63.0 ± 1.7 mV). In contrast to interneurons, a slow IPSC was present in all 18 granule cells examined. In 10 of these granule cells, we
pharmacologically isolated the slow IPSC and delivered a stimulus that
evoked a maximal response. In these cells, the maximal amplitude of the
slow IPSC averaged 35.7 ± 6.6 pA (range: 8.8-69.9 pA),
significantly larger than that recorded from interneurons
(P < 0.05, independent t-test).
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The GABAB antagonist, CGP 35348 (400 µM) was applied to seven interneurons. It reversibly blocked the slow IPSC in four of these cells (Fig. 2A). However, in the remaining three cells, this antagonist only partially reduced the peak current (Fig. 2C). Similar results were obtained with the more potent GABAB receptor antagonist, CGP 55845. Application of a maximal concentration of CGP 55845 (1 µM) in a separate group of eighteen cells blocked the slow IPSC in 10, but 8 were partially resistant to this antagonist (Fig. 2C). Furthermore increasing the concentration of CGP 55845 to 10 µM in three of these cells produced no further reduction in the slow IPSC. In contrast, 1 µM CGP 55845 blocked the slow IPSC in all 10 granule cells tested (Fig. 2D). Thus in contrast to principal cells where the slow IPSC was entirely GABAB-receptor mediated, the slow IPSC in some D/H border zone interneurons appeared to be mediated by both GABAB receptors and receptors insensitive to GABAB antagonists.
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At a holding potential of 70 mV, the high concentration of glutamate
and GABAA receptor antagonists used in this study
was sufficient to block all inward currents in every granule cell tested (n = 10). In contrast, a small inward current
remained in 36 of 63 interneurons (see Figs. 3B and
8A). Therefore where indicated, this resistant EPSC was
subtracted from the waveform to isolate the
IPSCB.
Baclofen effects
Baclofen (10 µM) produced an outward current in 19 of 27 interneurons and 5 of 5 granule cells on which it was applied (Fig. 3A). In the 19 interneurons in
which baclofen produced a current the amplitude of this current at a
holding potential of 70 mV averaged 39.5 ± 6.8 pA. This was
slightly smaller than the average amplitude of the baclofen current in
granule cells at a holding potential of
70 mV (42.9 ± 14.4 pA).
Subsequent application of the GABAB receptor
antagonist CGP 55845 (1 µM) fully reversed the effect of baclofen in
all cells tested.
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The eight interneurons that did not exhibit a baclofen current had
high-input resistances (306.5 ± 65.2 M) and hyperpolarized membrane potentials (
61.0 ± 2.1 mV), suggesting that they were electrophysiologically healthy. In five of these cells, we were unable
to evoke a slow IPSC (see preceding text), suggesting that these
interneurons did not possess
GABAB-receptor-activated potassium currents. In
contrast, the remaining three cells did exhibit a slow IPSC. Although
this IPSC was suppressed by baclofen, it recovered in the presence of
CGP 55845 (1 µM), suggesting that it was not GABAB-receptor mediated. Thus these findings
suggest the presence of a subgroup of interneurons in the dentate hilar
border region that lack GABAB-receptor-activated
potassium currents.
Application of baclofen to both interneurons and granule cells consistently resulted in the disappearance of IPSCB, presumably due to a reduction of GABA release or to saturation of the GABAB response by baclofen. Interestingly, one interneuron in which the large baclofen current was antagonized completely by CGP 55845 also had a prominent slow IPSC that was only minimally sensitive to CGP 55845 (Fig. 3B). This disparity in the ability of CGP 55845 to antagonize the baclofen current and the IPSC suggests that CGP-55845-sensitive GABAB receptors were present on the postsynaptic membrane of this interneuron but were only minimally activated by synaptically released GABA.
IPSCB heterogeneity
In 13 interneurons that were treated with
GABAB antagonists, we recovered sufficient
biocytin staining to compare the maximal amplitude of
IPSCB with the morphology of the cell. The
amplitude of the GABAB component of the slow IPSC
in each of these cells was determined by subtracting the
antagonist-treated waveform from the control waveform. The maximal
amplitude of the IPSCB did not correlate with
somatic shape or dendritic morphology but did correlate with axonal
distribution. The IPSCB in interneurons with
axons which projected to the inner molecular layer (IML cells) and
granule cell layer (GCL cells) was similar in amplitude to that
recorded from granule cells (Fig.
4A). In contrast, the
IPSCB in interneurons with axons projecting to
the outer molecular layer (OML cells) and the total molecular layer
(TML cells) was significantly smaller. The difference in
IPSCB amplitude between these groups of
interneurons could not be explained by differences in their input
resistance, as the input resistance of TML/OML cells (204.0 ± 25.5 M) was similar to that of GCL/IML cells (171.0 ± 24.2 M
). In contrast to IPSCB, the amplitude of the
antagonist-resistant component of the slow IPSC was not different in
interneurons with axons projecting to perisomatic (10.3 ± 3.4 pA,
n = 3) or dendritic (10.2 ± 5.0 pA,
n = 3) regions of granule cells.
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The differences in IPSCB amplitude could be
caused by differences in our ability to activate GABAergic input to
these cells. Therefore in nine interneurons, we compared the amplitude
of the outward current elicited by baclofen with the axonal projection of that cell. The input resistance (195.3 ± 43.9 M) and
resting membrane potential (
55.0 ± 3.1 mV) of GCL/IML cells
combined (n = 5) was similar to that of TML/OML cells
combined (222.8 ± 50.4 M
;
55.3 ± 1.6 mV,
n = 4). However, like our findings with IPSCB, baclofen produced a twofold larger current
in GCL/IML cells than in TML/OML cells (Fig. 4B). These
findings indicate that interneurons with axons projecting into or near
the granule cell body layer express larger postsynaptic
GABAB receptor currents than do interneurons with
axons projecting to the granule cell dendrites.
Voltage dependence and conductance of IPSCB
In seven cells it was possible to construct complete
current-voltage curves from subtracted IPSCBs. In
each cell we measured the peak amplitude of the isolated
IPSCB over a range of voltages. A regression line
was then fit to each of these curves so that the average slope
conductance and reversal potential could be determined. To average the
curves between cells, peak currents within a given neuron were
normalized to the peak current at 70 mV. The normalized measurements
from each cell then were averaged together to produce the
I-V curves shown in Fig. 5. As
these I-V curves show, the IPSCB in
both interneurons and granule cells exhibited no rectification over the
range of potentials studied (Fig. 5A). The reversal
potential of IPSCB in interneurons was
96.7 ± 1.9 mV, identical to the calculated reversal potential of
97 mV for a potassium conductance. This reversal potential also
compared well with that of
94.6 ± 2.0 mV for the
IPSCB obtained from four granule cells.
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In six interneurons the voltage dependence of the baclofen current was
examined by delivering voltage ramps from 70 to
120 mV in control,
in baclofen and after application of CGP 55845. Subtraction of the ramp
delivered in baclofen from the average of the ramps delivered in
control and CGP 55845 revealed the component of the ramp current
produced by baclofen (Fig. 5B). Unlike
IPSCB, the baclofen current displayed some inward
rectification over the range of membrane potentials tested. The
reversal potential of this current averaged
97.9 ± 3.4 mV,
similar to that of IPSCB. In comparison, the
reversal potential of the baclofen current in granule cells averaged
91.3 ± 1.2 mV (n = 3).
In five of the seven interneurons in which the I-V relationship of IPSCB was examined, a ML stimulus was used that evoked a maximal IPSCB. In these cells, the slope conductance of IPSCB averaged 0.75 ± 0.12 nS (n = 5; range: 0.6-1.35 nS). This value is significantly smaller (P < 0.05, independent t-test) than the IPSCB conductance measured in granule cells (1.31 ± 0.23 nS, n = 3). Similarly, the conductance of the baclofen current, calculated from the amplitude of voltage steps delivered in control, baclofen, and CGP 55845, was smaller in interneurons (1.7 ± 0.3 nS) than in granule cells (2.6 ± 0.6 nS);, however, this difference was not significant.
IPSCB kinetics
IPSCB kinetics were examined in all 25 interneurons to which we applied GABAB
antagonists. For each interneuron, the GABAB component of the slow IPSC was isolated by subtracting the antagonist resistant PSCs. The kinetics of these isolated
IPSCBs were studied at a holding potentials of
70 mV. GABAB currents exhibited an onset
latency ranging from 18 to 50 ms, a peak latency of 114 to 207 ms, and
a duration of 452 to 2,110 ms. Although we found no significant
difference in the onset or peak latency of IPSCB in interneurons and granule cells, we did find a difference in the
decay of the GABAB current (Table
1). The duration of
IPSCB in interneurons was significantly shorter
than that in granule cells (P < 0.05, independent
t-test). In addition, in interneurons IPSCB exhibited a single exponential decay while,
in 9 of the 10 granule cells, IPSCB was better
fit with a biexponential decay curve. The IPSC in the remaining granule
cell was best fit with a single exponential curve with a decay
value of 410 ms. Biphasic current decay in granule cells has been
reported previously (Otis et al. 1993
).
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Summation of IPSCB during high-frequency stimulation
We examined summation of isolated IPSCBs by delivering 100-Hz stimulus trains with up to 10 stimuli (n = 4). The stimulus intensity was set to evoke an IPSCB of maximal amplitude. To isolate the GABAB component of the response, we repeated the series of stimulus trains in each cell in the presence of CGP 55845 (1 µM) and subtracted these responses from the control responses in that cell. On average, the summed IPSCB plateaued after about seven stimuli at an amplitude that was 3.8-fold larger than that of a single maximal IPSCB in the same cells (Fig. 6). Both the peak latency (164 ± 16%) and the duration (147 ± 13%) of the summed response to 10 stimuli were markedly longer than those of a single response.
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The total charge carried by the GABAB current was determined from the net area of IPSCB. This measurement revealed that on the second and third responses of the train the charge carried by the summed IPSCB was actually potentiated relative to the theoretical response expected assuming linear summation of the first maximal IPSCB (Fig. 6C). However, beginning with the fourth stimulus, the summed IPSCB rapidly fell below this theoretical response. Thus by the end of the train, the response to the 10th stimulus added little to the total charge flux of the summed IPSCB beyond that evoked by the 7th stimulus.
Inhibition of interneuron firing by IPSPA and IPSPB
We compared the ability of GABAA and GABAB inhibition to inhibit interneuron firing (n = 6). In current clamp and in the presence of 20 µM DNQX and 50 µM D-APV, a monosynaptic IPSP was evoked during a 2-s-long depolarization-induced train of action potentials (Fig. 7). The monosynaptic IPSP consisted of a fast GABAA IPSP and a slow IPSP. We selected cells in which the slow IPSP kinetics were similar to those of IPSPB to ensure that this response was produced primarily by GABAB receptor activation. With small current injections, the firing rate of the interneuron was slow and both IPSPs were able to completely stop action potential discharge. However, as the amplitude of the current injection increased, the firing rate of the interneuron increased and IPSPB, but not IPSPA, became almost completely ineffective. Thus IPSPA powerfully inhibited interneuron firing at all tested membrane potentials, whereas IPSPB produced a hyperpolarizing inhibition that was effective only during weak to moderate depolarizations.
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IPSPB suppresses NMDA EPSPs
In cells bathed in DNQX (10 µM), BMI (20 µM), and picrotoxin (75 µM), a ML stimulus evoked an NMDA EPSP followed by an IPSPB. D-APV (50 µM) blocked the NMDA EPSP (n = 13) with only a small resistant EPSP remaining in some cells. Application of CGP 35348 (400 µM) blocked the IPSPB and increased the duration of the NMDA EPSP by 5.0 ± 0.8 fold (Fig. 8A). Because of this longer duration, the area of these responses was increased by >2.5-fold (Fig. 8B). Similarly, the latency to peak of the response increased from 48.1 ± 2.8 ms to 68.1 ± 6.0 ms, a significant difference (P < 0.05, paired t -test). Thus when GABAA inhibition is reduced, IPSPB appears to be capable of markedly truncating the NMDA EPSP in these interneurons.
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To further demonstrate the effect of IPSPB on the NMDA EPSPs, a paired-pulse paradigm was used in which a test stimulus was delivered to the ML 150-200 ms after a conditioning stimulus, near the peak of the IPSPB. The effect of IPSPB on the NMDA EPSP was quantitated by measuring the amplitude of the conditioning EPSP from baseline and the amplitude of the test EPSP in a similar manner after subtraction of the conditioning waveform. The amplitude of the test NMDA EPSP was clearly depressed in 19 of 20 interneurons tested (Fig. 8, C-E). In cells in which CGP 35348 (400 µM) blocked the IPSPB, it also significantly reduced depression of the test EPSP (Fig. 8D). However, blockade of IPSPB did not completely eliminate depression of the NMDA EPSP. The inability of this antagonist to completely block the depression, despite its complete blockade of IPSPB, suggests that factors other than CGP 35348-sensitive GABAB receptors also may contribute to the depression.
A paired-pulse paradigm also was used to evaluate the effect of IPSPB on AMPA EPSPs. AMPA EPSPs were isolated by blockade of NMDA and GABAA responses with D-APV (50 µM), BMI (20 µM), and picrotoxin (75 µM). These EPSPs could be blocked by subsequent application of DNQX (10 µM). As opposed to its effect on the NMDA EPSP, IPSPB had no effect on the AMPA EPSP (Fig. 8E), suggesting that presynaptic depression of glutamate release did not contribute to paired-pulse depression of NMDA EPSPs.
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DISCUSSION |
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The principal findings of this study were that in most D/H border zone interneurons, ML stimulation evoked IPSCBs similar to those in granule cells; a subpopulation of these interneurons lacked GABAB currents; the amplitude of GABAB currents was larger in interneurons with perisomatic projections than in those with dendritic projections; IPSPBs inhibited depolarization-induced interneuronal firing during weak, but not strong depolarizations; and IPSPBs effectively blocked NMDA but not AMPA EPSPs.
IPSCBs
IPSCBs have been reported in principal cells
throughout the hippocampal formation as well as in interneurons in CA1
stratum pyramidale (Lacaille 1991; Sik et al.
1995
) and lacunosum-moleculare (Khazipov et al.
1995
). However, this current has not been examined in detail in
any interneuronal population. In this study, we have examined the
kinetics of IPSCBs recorded from D/H border zone interneurons. We found that the onset and peak latency of
IPSCB in these interneurons were similar to those
observed in granule cells both in this study as well as in previous
reports (Hablitz and Thalmann 1987
; Mott et al.
1993
; Otis et al. 1993
; Staley and Mody
1992
; Thalmann and Ayala 1982
; Xie et al.
1992
). However, the decay rate of IPSCB
in interneurons and granule cells differed. In interneurons
IPSCBs decayed significantly more rapidly than in
granule cells and with monoexponential kinetics, whereas in granule
cells they decayed with biexponential kinetics. The mechanism underlying this difference is unknown; however, it may reflect diversity in the subunit composition of either the effector potassium channel or the G protein responsible for the
GABAB-receptor-mediated current. During rhythmic
activity in the dentate network, these differences in the decay of
IPSCB in interneurons and granule cells suggest
corresponding differences in the frequency range over which the
IPSCB will produce inhibition in these two cell types.
As in granule cells (De Koninck and Mody 1997;
Otis et al. 1993
), IPSCB in
interneurons exhibited a nonrectifying current-voltage relation over a
range of membrane potentials from
120 to
60 mV. In contrast, over a
similar voltage range the baclofen current showed some inward
rectification. Inward rectification of the baclofen current also has
been reported in CA3 pyramidal cells (Sodickson and Bean
1996
). The reason for this difference in the I-V
relationship between synaptically evoked currents and baclofen currents
is unclear.
The conductance of IPSCB in D/H border zone
interneurons was quite small (~0.75 nS), consistent with its proposed
function to provide hyperpolarizing inhibition (Misgeld et al.
1989). Given the single channel conductance of 5-11 pS
reported for GABAB-linked potassium channels
(De Koninck and Mody 1997
; Premkumar et al. 1990
), this corresponds to the contribution of 70-150 channels to the peak of a single maximal IPSCB. The
conductance of IPSCB in both interneurons and
granule cells was clearly smaller than that reported in hippocampal
pyramidal cells (range 6-19 nS) (Hablitz and Thalmann
1987
; Oleskevich and Lacaille 1992
;
Rovira et al. 1990
), suggesting that the functional
effects of IPSCB in interneurons may be weaker
than in pyramidal cells.
As in principal cells, repetitive stimulation was more effective than
single stimuli at activating GABAB receptors on
interneurons. This is thought to occur because
GABAB receptors are located extrasynaptically and
therefore require GABA spillover from the synaptic cleft for their
activation (Isaacson et al. 1993; Thompson and
Gähwiler 1992
). During the second and third pulses of the
train, the amplitude of the individual IPSCBs did
not run down but instead was potentiated as might be expected if GABA
spillover is increased. After these initial responses, the amplitude of
individual IPSCBs progressively declined during
the train, such that stimuli after the seventh added little to the
overall response. This progressive decline in the amplitude of
individual responses may reflect GABA depletion, increased GABA uptake,
or the activation of presynaptic GABAB receptors
capable of suppressing further GABA release (Mott et al.
1993
). Taken together, this behavior suggests that
GABAB receptors in these interneurons are
optimally activated by short bursts of inhibitory afferent activation,
such as occurs during rhythmic oscillation in the hippocampal formation
(Ranck 1973
).
IPSCB heterogeneity
GABAB current amplitude in interneurons
exhibited marked heterogeneity. The lack of both a baclofen current and
a slow IPSC in some D/H border zone interneurons suggests the existence
of a subpopulation of these interneurons that lack functional
GABAB-receptor-activated potassium currents. In
those cells that expressed a GABAB current, the
maximal amplitude of this current correlated with the axonal projection
of the interneuron. The heterogeneity in baclofen current amplitude
could not be explained by differences in the dendritic surface area, as
there is little difference between the total dendritic length of
interneurons that project to perisomatic and dendritic regions of
granule cells (Mott et al. 1997). Alternately, because
of the slow kinetics of IPSCB, the difference in
IPSCB amplitude between these groups of
interneurons is unlikely to be caused by differences in dendritic
filtering. Furthermore we have found previously that the electrotonic
length of both TML and OML cells is slightly shorter than
that of GCL and IML cells (Mott et al. 1997
). Therefore
we suggest that interneurons that project to perisomatic regions of
granule cells express greater postsynaptic GABAB
receptor function than do interneurons which project to dendritic regions.
A number of possible mechanisms may account for postsynaptic
differences in GABAB receptor function. First, it
is possible that some interneurons may simply lack or contain fewer
GABAB receptors. A second possibility is that
these interneurons may contain different subtypes of
GABAB receptor (Kaupmann et al. 1997) that couple with differing efficiencies to their effector mechanism. Alternatively, the effector potassium channel may be expressed more abundantly in some interneurons or different subtypes may be expressed. A fourth possibility is that a component of the
GABAB receptor system may be persistently
downregulated in interneurons that project to dendritic regions of
granule cells.
A growing body of evidence indicates that distinct groups of
interneurons express receptors for different neurotransmitters. Receptors that have been reported to be differentially expressed in
subgroups of interneurons include serotonin (Kawa 1994;
Morales and Bloom 1997
), muscarinic (Behrends and
ten Bruggencate 1993
), nicotinic (McQuiston and Madison
1999
), noradrenergic (Bergles et al. 1996
), and
metabotropic glutamate receptors (McBain et al. 1994
).
Similarly, Lambert and Wilson (1993)
have reported heterogeneity in presynaptic GABAB receptor
function in CA3 interneurons. Other studies have reported the
expression of glutamate (McBain and Dingledine 1993
) and
GABAA receptors (Gao and Fritschy
1994
) with different subunit composition in different
subpopulations of interneurons. This diversity provides a mechanism by
which interneurons with distinct roles in hippocampal computation might be differentially regulated. The heterogeneous expression of
GABAB currents in D/H border zone interneurons
may provide another means for the differential modulation of these interneurons.
The animals used in this study were between 16- and 30-days old.
Seress and Ribak (1990) reported that by day 16 the
axonal plexus of basket cells was typically distributed and relatively mature. Therefore it is not clear that the axonal distribution of other
dentate interneurons would be expected to change dramatically after day
16. Although the axonal morphology of D/H border zone interneurons may
be developmentally mature, it is unknown whether the expression pattern
of postsynaptic GABAB receptors in these interneurons is still maturing during the age range of animals used in
this study. In thalamic neurons, functional postsynaptic GABAB receptors are present at birth
(Warren et al. 1997
);however, in hippocampal pyramidal
cells. they are not present before the end of the first postnatal week
(Gaiarsa et al. 1995
). Therefore although we do not feel
that it is likely, we cannot rule out the possibility that differences
in GABAB function between these interneuronal
groups may reflect differences in the developmental time course of
GABAB receptors in some interneuron types.
Resistant PSCs
In addition to the differential expression of
IPSCB in D/H border zone interneurons, we also
saw heterogeneous expression of two unidentified currents. The first of
these currents was a slow IPSC that was resistant to
GABAB antagonists. It was present in ~44% of
D/H border zone interneurons. However, it was not observed in any of
the granule cells tested and has not been reported in principal cells
(Davies et al. 1993; Jarolimek et al.
1993
; Olpe et al. 1993
) or L-M interneurons
(Khazipov et al. 1995
) of the hippocampus. It is not yet
known whether this current is present in other interneurons in the
hippocampal formation. The receptor mediating these resistant slow
IPSCs is not known. It is possible that they are mediated by a subtype
of GABAB receptor not sensitive to the
antagonists used in this study (Bonanno and Raiteri
1993
). Alternatively, they may be produced by activation of a
different receptor system. Dentate inhibitory interneurons are
contacted by serotonergic afferents from the median raphe
(Halasy et al. 1992
), raising the possibility that the
resistant IPSC is serotonergic. Indeed, serotonin applied to pyramidal
cells can evoke a potassium conductance similar to
IPSCB (Andrade et al. 1986
;
Innis et al. 1988
). Alternatively, the resistant IPSC
could be an opioid-mediated potassium conductance (Madison and
Nicoll 1988
; Wimpey and Chavkin 1991
), although
significant release of opioids by a single ML stimulus seems unlikely
(Wagner et al. 1990
).
The second unidentified current in these interneurons was a
synaptically evoked inward current that was not blocked by DNQX and
D-APV and was present in ~57% of the interneurons
examined. It appeared similar to the spontaneous depolarizing synaptic
response recorded from granule cells in the presence of 4-aminopyridine (Forti and Michelson 1997). The presence of each of
these currents in a separate subpopulation of D/H border zone
interneurons suggests that, like GABAB receptors,
they also may provide a means for the differential modulation of
certain subtypes of interneurons.
Functional implications
What may be the functional consequences of
IPSC/PB in these interneurons? The slow time
course and small conductance increase associated with
IPSCB suggest that this form of inhibition will play a more subtle role in regulating interneuron function than does
IPSCA. In neocortical neurons (Connors et
al. 1988), IPSPB has been suggested as a
mechanism to reduce background neuronal firing. As in these neocortical
neurons (Connors et al. 1988
; McCormick
1989
), IPSPB in D/H border zone
interneurons was able to block action potentials evoked by weak but not
strong depolarizations, suggesting that it may serve a similar function
in these cells. Strong membrane depolarization is thought to overcome
GABAB inhibition because
IPSPB relies primarily on its ability to move the
membrane potential away from firing threshold to produce inhibition.
In support of this hyperpolarizing mechanism of inhibition,
IPSCB in interneurons was able to inhibit
excitatory synaptic responses mediated by voltage-dependent NMDA
receptors but not those mediated by AMPA receptors. Inhibition of NMDA
EPSPs by IPSPB has been reported previously in
CA1 pyramidal cells (Morrisett et al. 1991) and in
neurons of the amygdala (Huang and Gean 1994
). In D/H
border zone interneurons, inhibition of NMDA receptors by
IPSP/CB may have important consequences for
rhythmic bursting, synaptic plasticity, and/or excitotoxicity in the
dentate network. Furthermore the ability of IPSCB
to suppress NMDA currents may serve an important compensatory role if
GABAA inhibition is compromised. These findings
indicate that IPSP/CB inhibits the response of these interneurons to slow and/or voltage-dependent excitatory stimuli,
such as that mediated by NMDA receptors, while leaving unaffected the
response to strong fast voltage-independent excitatory stimuli, such as
that mediated by AMPA receptors. Furthermore they suggest that
GABAB inhibition may play an important role in
shaping the excitatory response of these interneurons and thus may
contribute to the GABAB-receptor-mediated
depression of inhibition previously reported in dentate granule cells
(Mott and Lewis 1991
; Mott et al. 1993
).
Conclusions
We conclude that GABAB currents in D/H border zone interneurons modulate interneuron firing rates and inhibit slow excitatory currents. The heterogeneous expression of these currents would produce different levels of GABAB inhibition in subpopulations of interneurons and thus permit these cells to match the computational needs of their environment.
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ACKNOWLEDGMENTS |
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We thank Drs. S. J. Mickel and H.-R. Olpe, Novartis Pharma AG, Basel, for providing CGP 35348 and CGP 55845 and Dr. John Dempster (Strathclyde University) for generously providing data acquisition and analysis software. We also thank Dr. J. Doherty for comments on a draft of this manuscript and K. Mott for expert editorial assistance.
This work was supported by National Institutes of Health Grants DA-06735 (D. V. Lewis) and AG-13165 (D. A. Turner) and a Veterans Affairs Merit Review Award (D. A. Turner).
Present address of D. D. Mott: Dept. of Pharmacology, Emory University School of Medicine, Atlanta, GA, 30322.
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FOOTNOTES |
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Address for reprint requests: D. V. Lewis, Dept. of Pediatrics (Neurology), Box 3430, Duke University Medical Center, Durham, NC 27710.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 21 September 1998; accepted in final form 19 May 1999.
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REFERENCES |
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