Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110
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ABSTRACT |
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Shao, Zhengwei and
Andreas Burkhalter.
Role of GABAB receptor-mediated inhibition in
reciprocal interareal pathways of rat visual cortex. In
neocortex, synaptic inhibition is mediated by -aminobutyric acid-A
(GABAA) and GABAB receptors. By using
intracellular and patch-clamp recordings in slices of rat visual cortex
we studied the balance of excitation and inhibition in different
intracortical pathways. The study was focused on the strength of fast
GABAA- and slow GABAB-mediated inhibition in
interareal forward and feedback connections between area 17 and the
secondary, latero-medial visual area (LM). Our results demonstrate that
in most layer 2/3 neurons forward inputs elicited excitatory
postsynaptic potentials (EPSPs) that were followed by fast
GABAA- and slow GABAB-mediated hyperpolarizing inhibitory postsynaptic potentials (IPSPs). These responses resembled those elicited by horizontal connections within area 17 and those evoked by stimulation of the layer 6/white matter border. In contrast, in the feedback pathway hyperpolarizing fast and slow IPSPs were rare.
However weak fast and slow IPSPs were unmasked by bath application of
GABAB receptor antagonists. Because in the feedback pathway disynaptic fast and slow IPSPs were rare, polysynaptic EPSPs were more
frequent than in forward, horizontal, and interlaminar circuits and
were activated over a broader stimulus range. In addition, in the
feedback pathway large-amplitude polysynaptic EPSPs were longer lasting
and showed a late component whose onset coincided with that of slow
IPSPs. In the forward pathway these late EPSPs were only seen with
stimulus intensities that were below the activation threshold of slow
IPSPs. Unlike strong forward inputs, feedback stimuli of a wide range
of intensities increased the rate of ongoing neuronal firing. Thus,
when forward and feedback inputs are simultaneously active, feedback
inputs may provide late polysynaptic excitation that can offset slow
IPSPs evoked by forward inputs and in turn may promote recurrent
excitation through local intracolumnar circuits. This may provide a
mechanism by which feedback inputs from higher cortical areas can
amplify afferent signals in lower areas.
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INTRODUCTION |
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Neurons in mammalian primary visual cortex respond
with increased firing when a stimulus falls into the receptive field.
Although retinal inputs provide the primary excitatory drive, responses can be modulated by the context in which a stimulus appears, by attention, and by signals that are unrelated to the current sensory stimulus (Lamme 1995; Motter 1993
;
Press et al. 1997
; Zipser et al. 1996
).
Unlike neuronal responses to stimuli in the classical receptive field
that encode orientation, spatial position, stereoscopic depth, illusory
contours, and faces which all show selective tuning a few milliseconds
after onset of firing (Celebrini et al. 1993
; Oram and Perrett 1992
; Rolls et al 1991
;
Thorpe et al.; von der Heyt and Peterhans 1989
),
modulatory responses evoked from outside the classical receptive field
and by attention are delayed by tens to hundreds of milliseconds
(Knierim and Van Essen 1992
; Motter 1993
;
Roelfsema et al. 1998
; Zipser et al.
1996
). To account for this delay it was suggested that visual
inputs are first sent to the top of the cortical hierarchy and after
some latency return to V1 via interareal feedback connections
(Hupé et al. 1998
; Zipser et al.
1996
). However, latency measurements in different cortical
areas have shown that many neurons at hierarchically different levels
can be activated simultaneously (Nowak et al. 1995
;
Raiguel et al. 1989
). This argues against large
differences in the arrival of forward and feedback activity in
different areas. An alternative to this "delay-line" hypothesis is
that responses to forward and feedback inputs are simultaneous but that
each pathway elicits a different temporal pattern of polysynaptic excitation.
In the rat, forward and feedback projections between primary and
secondary visual cortex (Coogan and Burkhalter 1993)
arise from pyramidal cells (Jiang et al. 1993
) that
provide monosynaptic excitation to postsynaptic neurons
(Domenici et al. 1995
; Shao and Burkhalter
1996
). Most of these neurons are pyramidal cells, and only
~10% of axons form synapses with GABAergic nonpyramidal cells
(Johnson and Burkhalter 1996
, 1997
) which
in turn innervate pyramidal neurons that receive input from interareal
connections (Shao and Burkhalter 1996
). In the forward
pathway this disynaptic inhibition produces powerful hyperpolarizing
fast inhibitory postsynaptic potentials (IPSPs) (Shao and
Burkhalter 1996
). By contrast, in the feedback pathway
hyperpolarizing fast IPSPs are rare (Shao and Burkhalter
1996
).
Inhibition in the cortex is mediated by -aminobutyric acid-A
(GABAA) and GABAB receptors
(Connors et al. 1988
). Together these receptor
mechanisms contribute to the balance of excitation and inhibition that
influences the magnitude and temporal pattern of spike discharge
(Berman et al. 1992
; Connors et al. 1988
;
McCormick 1989
). Excitatory responses consist of
monosynaptic and polysynaptic excitatory postsynaptic potentials
(EPSPs). Although monosynaptic EPSPs result from direct connections
between pairs of pyramidal cells, polysynaptic EPSPs arise in chains of
pyramidal cells that are linked by local axon collaterals
(Douglas et al. 1995
). The recurrent excitatory
interactions produced in this network are important for the
amplification of afferent inputs (Douglas and Martin
1991
; Douglas et al. 1995
). It is known from
developmental studies in rat somatosensory cortex that polysynaptic
excitation is regulated by slow inhibition (Fukuda et al.
1993
; Luhmann and Prince 1990a
,b
). This suggests
that GABAB receptor-mediated IPSPs play a role in the
modulation of signal amplification. Excitation from feedback
connections that is unopposed by slow inhibition might be important in
this process. We therefore studied the effects of GABAB
receptor-mediated inhibition on polysynaptic excitation in reciprocal
interareal circuits of rat primary and secondary visual cortex.
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METHODS |
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Slice preparation
Slices were prepared from 3- to 6-wk-old male Long-Evans rats.
For this purpose animals were anesthetized with pentobarbital (40 mg/kg
body weight ip) and decapitated. The left occipital pole of the
forebrain was removed and placed in ice-cold, oxygenated artificial
cerebrospinal fluid (ACSF) in which NaCl was substituted by sucrose
(Aghajanian and Rasmussen 1989) [sucrose ACSF (in mM): 252 sucrose, 3.3 KCl, 2.5 CaCl2, 1.2 MgSO4,
25.5 NaHCO3, 1.2 KHPO4, 15 D-glucose, pH 7.4]. Tissue blocks were glued with the cut
anterior surface down onto the stage of a Vibratome, submersed in
sucrose ACSF and sectioned at 400 µm in the coronal plane. To ensure
the integrity of reciprocal connections between primary visual cortex (area 17) and the secondary visual area latero-medial visual area (LM)
we collected slices at 1.6-2 mm in front of the posterior pole
(Domenici et al. 1995
). The area 17/LM border was
identified as a sharp transition between the opaque, heavily myelinated
area 17 and the sparsely myelinated more transparent area LM adjoining laterally. This border was marked with a cut in the subiculum below the
visual cortex. Slices were transferred to a recording chamber (Fine
Science Tools, Foster City, CA) and were maintained at the liquid/air
interface in a 33°C, humidified atmosphere saturated with 95%
O2-5% CO2. Slices were perfused (1 ml/min)
first with a 1:1 mixture of sucrose ACSF and normal ACSF (in mM: 124 NaCl, 3.3 KCl, 2.5 CaCl2, 1.2 MgSO4, 25.5 NaHCO3, 1.2 KH2PO4, 15 D-glucose). After 30 min, the mixture was replaced with
normal ACSF, and slices were allowed to recover for ~2 h before recording.
Stimulation
For activation of specific pathways microstimulation with
bipolar platinum/iridium electrodes (200-µm tip separation; F. Haer, New Brunswick, ME) was used. Shocks (0.1-2 mA, 100 µs, 0.1-10 Hz)
were delivered by a stimulator. We have shown previously that stimuli
of comparable intensities do not spread beyond a radius of 350 µm
(Domenici et al. 1995). Stimulus intensity was scaled to
the minimal strength necessary to evoke postsynaptic potentials (PSPs).
This strength was considered threshold intensity (T). To control for
activation of fibers of passage, in some experiments, glutamate was
used to stimulate pathways. Glutamate (0.5-1 mM; ~5-20 pl) was
applied to the slice by delivering pulses (10-50 ms; 40 psi) of
pressurized air to the back of glass pipets (tip diameter 2-5 µm)
with a picospritzer (General Valve, Fairfield, NJ).
Recording and stimulating electrodes/glutamate puffer pipettes were
positioned in the slice under a dissecting microscope. To study
synaptic potentials/currents elicited by forward connections stimulating electrodes/glutamate puffer pipettes were placed in layer
2/3 of area 17. Intracellular/patch-clamp recordings were obtained in
layer 2/3 of area LM. For studying synaptic inputs of feedback
connections to cells in area 17, stimulating and recording sites were
reversed. Extracellular recordings of field potentials (maximal
response at minimal stimulation strength) were used to find the
topographically optimal configuration of stimulating and recording
electrodes (Domenici et al. 1995). The minimal allowed separation between stimulating and recording electrodes was 1.2 mm, and
the minimal allowed distance from the 17/border was 0.3 mm. Stimulation
from the layer 6/white matter (L6/WM) border was used for activating
intralaminar/thalamocortical inputs to layer 2/3 neurons
(Burkhalter 1989
; Miller et al. 1993
).
Horizontal long-range inputs from within area 17 to layer 2/3 neurons
(Burkhalter 1989
; Burkhalter and Charles
1990
; Johnson and Burkhalter 1994
) were
activated by stimulating layer 2/3
1-mm medial to the recording site.
Recording and analysis
Intracellular and whole cell patch-clamp recordings were
obtained from randomly selected cells in layers 2/3 of area 17 and LM.
Sharp electrodes were pulled from thin-walled glass capillaries (0.75 mm ID, 1 mm OD; World Precision Instruments, Sarasota, FL). When filled
with 4 M K-acetate (pH 7.4) electrode impedances were 70-120 M.
Patch electrodes were pulled from soda lime glass capillary tubing (ID
1.1, OD 1.5; Chase Instrument Company Norcross, GA) and had impedances
of 6-8 M
when filled with intracellular solution containing (in mM)
120 K-gluconate, 5 NaCl, 1 CaCl2, 1 MgCl2, 5 EGTA, 10 HEPES, 0.2 GTP, 2 ATP, pH 7.4; 285 mosmol/l, adjusted with
sucrose). For recording of inhibitory postsynaptic currents (IPSCs)
pipettes were filled with a solution in which K+ was
replaced by Cs+ and 5 mM QX-314 was added to block
GABAB-receptor activated K+ currents and
delayed Na+ currents (Gähwiler and Brown
1985
; Nathan et al. 1990
; Otis et al.
1993
). Field potentials were recorded with extracellular electrodes pulled from glass capillaries filled with 3 M NaCl (impedance: 2-5 M
).
Voltage signals from intracellular recordings were amplified with an
Axoprobe-1 amplifier (Axon Instruments, Foster City, CA). An active
bridge balance allowed simultaneous passage of current and measurements
of voltage with a single electrode. Signals were displayed
simultaneously on an oscilloscope and after digitization (at 25 kHz)
with a 1401 plus interface (Cambridge Electronic Design, Science Park,
Cambridge, UK) and Spike-2 software (Cambridge Electronic Design)
on-line on a monitor. Data were stored on optical disk or digital tape
for off-line analysis. Only cells with resting membrane potentials of
less than or equal to 68 mV and overshooting action potentials were
used for analyses. Peak amplitudes of EPSPs and IPSPs/IPSCs (average of
3-6 sweeps) were measured as maximal positive or negative deflections
from baseline. The amplitude of glutamate stimulated IPSCs was measured
as peak within 150 ms of response onset. Peak latencies reflect the
time elapsed between stimulus and the maximal amplitude of the
response. Mean PSPs of a population of cells were obtained by averaging
responses of different neurons recorded under comparable conditions and pooling three sweeps per cell.
Voltage-clamp recordings were made with standard patch-clamp methods
(Blanton et al. 1989). The liquid junction potential (<5 mV) was measured immediately after making contact between electrode and slice and was offset in the amplifier output so that the
voltage and current measurements remained unaffected. Recordings were
filtered at 10 kHz and digitized at 25-50 kHz with an Axoclamp-2B
amplifier (Axon Instruments) in continuous mode and a Digidata 1200 A/D
converter (Axon Instruments) with p-Clamp 6.0 software (Axon
Instruments). Only cells with access resistance of <15 M
were used
for analyses.
Pharmacology
The effects of GABAB receptor blockade on PSPs were studied by bath application of 200 µM 2-OH-saclofen (Tocris Cookson, Ballwin, MO) or CGP55845 (1-4 µM, gift from Drs. Bittinger and Olpe, Novartis, Switzerland).
INTRACELLULAR INJECTION OF BIOCYTIN.
To allow subsequent identification of the cells recorded with patch
pipettes, in selected experiments 0.2-0.5% biocytin was added to the
intracellular solution. Slices with injected cells were fixed for 1-3
days in a mixture of 4% paraformaldehyde and 0.5% glutaraldehyde. The
tissue was cryoprotected in 30% sucrose and sectioned at 80 µm.
Sections were incubated with 10% methanol and 1%
H2O2 for 10 min, washed in 0.1 M phosphate
buffer, and processed with the avidin-biotin-peroxidase method, and
staining was intensified with AgNO3 and HAuCl4
as described by Jiang et al. (1993). Stained sections were mounted on
slides, treated with ethanol followed by xylene, and coverslipped with
DPX mountant. Biocytin-filled cells were analyzed under the microscope
with a ×60 oil immersion lens with high numerical aperture.
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RESULTS |
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Cell types recorded
Intracellular recordings with sharp electrodes were performed from
92 randomly selected layer 2/3 neurons in areas 17 and LM. In the
majority of cells (83/92) injection of depolarizing current pulses
showed relatively broad action potentials (half-width at half-maximal
amplitude: 0.8 ± 0.1 ms; n = 83) and marked
accommodation of firing (Fig. 7). These neurons were classified as
regular spiking and presumed to represent pyramidal cells
(Huettner and Baughman 1988; Kawaguchi
1995
; McCormick et al. 1985
). Resting membrane potential (Vm) and input resistance
(Rm) of cells recorded in areas 17 (Vm:
75 ± 5 mV;
Rm: 35 ± 6 M
; n = 66) and LM
(Vm:
76 ± 4 mV; Rm: 35 ± 5; n = 17) were similar. In a small group of nine
cells action potentials were narrow (half width at half maximal amplitude: 0.4 ± 0.05 ms), and firing frequency remained constant during the injection of a 200-ms pulse of positive current. These cells
were classified as fast spiking (Huettner and Baughman
1988
; Kawaguchi 1995
; McCormick et al.
1985
) and were not included in this study.
Whole cell patch-clamp recordings were made from 16 randomly selected
layer 2/3 neurons in areas 17 and LM. Blockade of K+
channels by intracellular Cs+ precluded the physiological
determination of cell types. However all of the eight biocytin-filled
neurons (5 in area 17, 3 in LM) were pyramidal cells, suggesting that
this type of neuron represents the majority of cells tested. The
average resting membrane potential and input resistance of cells in
areas 17 (Vm: 63.6 ± 6 mV;
Rm: 114.6 ± 44 M
) and LM
(Vm:
64.1 ± 5 mV; Rm:
120 ± 38 M
) were similar.
Synaptic responses to forward and feedback inputs
We compared intracellularly recorded PSPs evoked by forward
and feedback inputs of different strengths. The stimuli used ranged from threshold intensity for EPSP activation to more than double the
intensity necessary to elicit the maximal EPSP amplitude. Forward and
feedback responses of layer 2/3 neurons showed very different
waveforms, especially when recorded at membrane potentials 10-15 mV
depolarized from rest (Fig. 1,
A and B). Threshold stimulation (1T) of forward
inputs evoked small EPSPs. Stronger stimuli (1.2T) elicited larger
responses, which reached the peak more rapidly. Over 90% of responses
had mono- and early polysynaptic components (Fig. 1C), which
resembled those recorded in the frontal cortex (Sutor and
Hablitz 1989a). The different EPSPs were easily distinguished by stimulating at different frequencies. Early polysynaptic components had variable amplitudes and decayed to baseline within <100 ms. Raising the stimulus intensity to 1.4T revealed effects of fast IPSPs
that let to an accelerated decay of monosynaptic EPSPs and abolished
early polysynaptic components. In addition, in >90% of trials 1.4T
stimuli evoked late polysynaptic EPSPs with variable peaks at latencies
of ~100-400 ms. Early and late polysynaptic EPSPs were confined to a
narrow window of stimulation intensities and were not seen with
stronger stimuli (
1.5T). The disappearance of polysynaptic EPSPs
correlated with the appearance of hyperpolarizing fast and slow IPSPs
whose amplitudes grew larger with increasing stimulus strength (Fig.
1A). Slow IPSPs were always preceded by a small depolarizing
wave that temporally overlapped with the decay of fast IPSPs. Because
polysynaptic responses were variable we averaged recordings from seven
cells, six trials each, to obtain the mean forward response ±SD (Fig.
1D). This average response showed the same dependence on
stimulus intensity as the individual responses (Fig. 1A).
However, the late depolarizing peak of the average response was smaller
than the individual response caused by trial-by-trial variability of
polysynaptic EPSPs.
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Unlike polysynaptic EPSPs in the forward pathway, which were seen only after weak stimulation, polysynaptic EPSPs in the feedback pathway were elicited over a broad range of stimulus intensities (Fig. 1B). Most (>90%) responses showed multiple peaks whose amplitude and time course varied from trial to trial but often lasted >500 ms (Figs. 1B and 5). Fast IPSPs were weak, and their presence was only evident at higher stimulus strengths by an accelerated decay of the early depolarization. In the majority of cases fast IPSPs were too weak to hyperpolarize the membrane. The most interesting difference, however, was that at all stimulus intensities tested feedback inputs failed to evoke slow hyperpolarizing IPSPs (Figs. 1B and 5).
Although the responses shown in Fig. 1 were seen in the majority of neurons, recordings with sharp electrodes revealed cells with a range of different properties. Of 17 cells that gave responses to activation (1.8T) of forward inputs, 13 (76%) exhibited both fast and slow IPSPs when tested at membrane potentials 10-15 mV positive to rest (Fig. 2, Aa and Ad). Three cells (3/17, 18%) showed early IPSPs only (Fig. 2, Ab and Ad). In a single cell the response was entirely depolarizing (Fig. 2, Ac and Ad). In every case slow IPSPs, if present, were preceded by fast IPSPs. As shown in Table 1 the distribution of responses evoked by stimulation of the L6/WM border and horizontal connections was similar to that seen in the forward pathway, and 78-86% of the cells tested showed both early and late IPSPs. In marked contrast, in the feedback pathway hyperpolarizing fast and slow IPSPs were rare, and most responses (18/27, 67%; Fig. 2Bd) resembled that shown in Fig. 2Bc. Few cells (5/27, 18%) showed fast IPSPs (Fig. 2, Bb and Bd), and only 15% (4/27) exhibited fast and slow IPSPs (Fig. 2, Ba and Bd). In none of the pathways tested did we encounter cells that showed late IPSPs in isolation.
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Pathway-specificity of PSPs
ISOLATION OF PATHWAYS.
To assess whether the responses shown in Fig. 1 are pathway specific it
was necessary to control for stimulation of fibers of passage. For this
purpose we voltage clamped layer 2/3 neurons in areas 17 and LM at the
reversal potential of EPSCs (~0 mV) and compared IPSCs elicited by
electrical stimulation or drop application of glutamate at the same
site. On the basis of previous observations that disynaptic inhibition
in forward pathways is stronger than in feedback pathways (Shao
and Burkhalter 1996), the expectation was that IPSC amplitudes
show similar pathway differences and that these differences can also be
demonstrated after stimulation with glutamate. As expected, in the
forward pathway IPSCs elicited by medium- to high-intensity (1.5-1.8T) electrical shocks were larger (538 ± 90 pA) and decayed more
slowly than in the feedback pathway (263 ± 50 pA; Fig.
3, A and B). For eight cells tested in each pathway the average IPSC amplitude in the
feedback pathway was ~49% smaller (P < 0.01).
Stimulation with glutamate evoked smaller and longer-lasting responses
than electrical stimulation. However the relative difference (40%, P < 0.05) in IPSC amplitude between forward (120 ± 31 pA) and feedback pathways (48 ± 22 pA) was similar to that
in tests with electric shocks (Fig. 3, C and
D).
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PATHWAY-SPECIFIC ACTIVATION OF SLOW IPSPS. To directly test whether activation of slow IPSPs is pathway specific we compared responses of individual area 17 neurons with converging inputs from feedback (FB), intralaminar (WM), and horizontal (HC) pathways. The results show that the incidence of undershooting fast and slow IPSPs evoked by the different inputs are similar to those of experiments in which neurons were activated from a single pathway (Table 1).
After stimulating the L6/WM border with intensities of 1.5-1.8T most cells (13/15, 86%) responded with short-latency EPSPs followed by fast and slow IPSPs (Fig. 4, A and C). Slow IPSPs peaked at 146 ± 13 ms and had a mean amplitude of 4.7 ± 1.5 mV. Similar responses were seen in the majority of cells (11/15, 74%) stimulated by inputs from horizontal connections (Fig. 4, A andC), although slow IPSPs were slightly smaller (3.5 ± 1.2 mV). Responses to feedback inputs differed in that repolarization of the early depolarizing wave was slower than after stimulation of both the L6/WM border or forward inputs. This indicates that early IPSPs in the feedback pathway were weak. Interestingly, in the feedback pathway only 20% (3/15) of the cells that after WM and HC stimulation showed the usual fast and slow IPSPs responded with hyperpolarizing slow IPSPs (Fig. 4, B and C). The presence of cells with slow IPSPs, similar to those seen after HC stimulation (peak amplitude: 3.1 ± 1.4 mV; peak latency 134 ± 14 ms), resulted in a small, slow hyperpolarization of the average feedback response at high stimulation intensities (Fig. 5). In spite of this, the mean slow IPSP evoked by high-intensity feedback input was much smaller than the response elicited by high-intensity stimulation of the L6/WM border or horizontal inputs (Fig. 5). In fact the amplitude time integral of the average slow hyperpolarization after strong stimulation of the feedback pathway was a mere 3% of that evoked by L6/WM stimulation and was only 7% of that seen after HC stimulation. This indicates that the net hyperpolarizing effect of the slow IPSP is largely independent of stimulus strength and that its weakness is a characteristic feature of the feedback circuit.
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Identification of slow IPSP
Because slow IPSPs were rare in the feedback pathway detailed analyses of slow hyperpolarizing responses were focused on the forward pathway.
VOLTAGE DEPENDENCE.
To test the voltage dependence of slow IPSPs current injection was used
to alter the membrane potential. During the 500 ms of current
injection, forward inputs were activated by stimulating at 1.8T in
upper layers of area 17. In all 13 cells tested monosynaptic EPSPs were
followed by fast and slow IPSPs. Membrane depolarization away from the
equilibrium potential of Cl and K+
progressively increased amplitudes of fast and slow IPSPs. On average,
fast IPSPs reversed polarity at approximately
68 mV, whereas slow
IPSPs reversed at approximately
95 mV. Similar reversal potentials
were obtained in two cells tested in the feedback pathway. Although
because of the temporal overlap of EPSPs and IPSPs these measurements
represent approximations and suggest that fast and slow IPSPs reverse
polarity near the equilibrium potential for Cl
and
K+, respectively.
BLOCKADE OF GABAB RECEPTORS.
The apparent reversal of polarity at membrane potentials below 90 mV
strongly suggested the involvement of a GABAB
receptor-mediated K+ conductance in the generation of slow
IPSPs (Connors et al. 1988
; Deisz and Prince
1989
; McCormick 1989
). To test this possibility directly we used bath application of 2-OH-saclofen to block
GABAB receptors. Five neurons that showed fast and slow
IPSPs after activation of forward inputs were tested. At slightly
depolarized membrane potentials control responses showed prominent fast
and slow IPSPs (Fig. 6A). In
three cells, perfusion of 200 µM saclofen markedly reduced slow IPSPs
(Fig. 6B). This partial blockade released a powerful wave of
late polysynaptic EPSPs whose onset coincided with the peak of slow
IPSPs. Similar results were obtained with the GABAB
antagonist CGP55845 (Brugger et al. 1993
). In every cell
tested (5/5) the drug greatly suppressed late IPSPs evoked by
activation of forward inputs. In many instances the excitation released
by this blockade was strong enough to trigger a barrage of action
potentials (Fig. 6, C and D).
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Effects of slow IPSP on neuronal firing
To study whether late IPSPs can influence neuronal firing we compared the effects of feedback inputs with responses to stimulation of the L6/WM border. Recordings were performed in area 17 neurons that received convergent input from both the feedback pathway and connections from the L6/WM border. The magnitude of neuronal discharge was varied by injecting 500-ms pulses of positive current of different amplitudes. Synaptic responses were evoked by delivering stimuli of constant intensity (100 µs, 1.8T) 200 ms after the onset of intracellular current injection to either area LM or the L6/WM border. Each of five neurons tested behaved similarly to the cell shown in Fig. 7 and exhibited radically different responses to inputs from each stimulation site. At the resting membrane potential (no current injection) feedback input evoked a series of EPSPs (Fig. 7Aa). When the membrane depolarization reached firing threshold but was still too weak to sustain firing, feedback input triggered a train of action potentials that was maintained for the duration of the DC pulse and showed pronounced spike frequency adaptation (Fig. 7Ad). As the firing rate increased with stronger depolarization, feedback input produced a further increase in firing, but the relative change in rate imposed by the synaptic inputs was less than at firing threshold (Fig. 7Ae). At the highest firing frequency tested feedback input had no detectable effect on discharge rate (Fig. 7Af).
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Unlike feedback inputs, subthreshold activation of inputs from the L6/WM border evoked a sequence of EPSPs followed by fast and slow IPSPs (Fig. 7, Ba-Be). Near spike threshold synaptic inputs produced a transient increase in firing by evoking a single additional spike. This was followed by powerful fast and slow IPSPs that inhibited firing for most of the duration of the depolarizing current injection. Additional depolarization slightly increased the firing rate and made the transient suppression of repetitive firing by synaptic input less effective than at more negative potentials (Fig. 7Bf). This shortening of the delay of firing was accompanied by a reduction in the amplitude of the slow IPSP.
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DISCUSSION |
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Our results show that intracortical feedback inputs from the
secondary visual area, LM, rarely evoke slow GABAB
receptor-mediated hyperpolarizing IPSPs in regular spiking layer 2/3
pyramidal cells (Kawaguchi 1995; McCormick et al.
1985
) of area 17. By contrast, forward inputs from area 17 evoke slow IPSPs in most upper layer pyramidal neurons of area LM. A
similarly high incidence of GABAB responses was found in
superficial layers of area 17 after local activation of interlaminar
and horizontal inputs. Because in the same neuron feedback inputs evoke
weaker slow inhibition than horizontal and intralaminar inputs, these
differences reflect pathway-specific synaptic properties and are not
due to recordings from different cell types.
A low incidence of slow IPSPs in the feedback pathway could result from
an antagonism by polysynaptic EPSPs (Scharfman 1992). However, we found no evidence that polysynaptic EPSPs are larger in the
feedback pathway, suggesting that in feedback-recipient neurons slow
IPSPs are less effective.
Unlike in interareal forward, horizontal, and interlaminar circuits in
which polysynaptic excitation is curbed by powerful fast and slow
IPSPs, in the feedback circuit polysynaptic EPSPs are more common
because slow IPSPs are weak. Douglas et al. (1995) have shown that
polysynaptic excitation is important for the amplification of afferent
visual inputs. Our results suggest that feedback inputs are capable of
generating polysynaptic EPSPs that in turn influence the gain of
neuronal responses in primary visual cortex.
Source of synaptic potentials
A central assumption of this study is that distinct intracortical
pathways were activated and that neurons at the stimulation site were
the source of synaptic input to the cells recorded. Although definitive
proof is lacking, observations indicate that stimulation at <2T is too
weak for activating fibers of passage. Stimuli in this intensity range
evoke synaptic activity that corresponds to the laminar distribution of
forward and feedback terminals (Domenici et al. 1995).
Furthermore, antidromic spikes at these intensities are rare
(Asanuma and Sakata 1967
; Nowak and Bullier 1996
; Shao and Burkhalter 1996
; Stoney et
al. 1968
), which rules out contamination by local axon
collaterals. Finally, stimulation of forward and feedback-projecting
neurons with electric shocks or glutamate yields a qualitatively
similar asymmetry of disynaptic inhibition. From these results we
conclude that stimulation of the L6/WM border preferentially activates
ascending projections of layer 6 neurons (Burkhalter
1989
; Hirsch 1995
; Katz 1987
) and that activation of thalamocortical (Miller et al.
1993
) and cortico-cortical axons (Coogan and
Burkhalter 1993
; Olavarria and Van Sluyters 1985
) was negligible. For similar reasons we believe that
stimulation in upper layers of area 17 activates inputs from
topographically distant points (Burkhalter and Charles
1990
) and that layer 2/3 neurons in areas 17 and LM are the
source of forward and feedback inputs, respectively.
Differential expression and effectiveness of slow IPSP
Recordings in many different species demonstrated fast and slow
inhibitory responses in upper layers of cerebral cortex
(Chagnac-Amitai and Connors 1989; Chagnac-Amitai
et al. 1990
; Connors et al. 1982
, 1988
;
Deisz and Prince 1989
; Deisz et al. 1993
;
Douglas and Martin 1991
; Hirsch 1995
;
Hirsch and Gilbert 1991
; Kang et al.
1994
; Kawaguchi 1992
; Lohmann and
Rörig 1994
; McCormick 1989
; Silva et al. 1991
; van Brederode and Spain 1995
).
Several of these studies described variability in the efficacy of fast
and slow IPSPs, suggesting cell type- or lamina-specific synaptic
effects (Chagnac-Amitai and Connors 1989
; Douglas
and Martin 1991
; Silva et al. 1991
; van
Brederode and Spain 1995
). Our study provides the first
demonstration that distinct intracortical pathways produce different
amounts of slow inhibition.
Although the results show that fast and slow hyperpolarizing IPSPs are
smaller in the feedback pathway, it is possible that an amount of
inhibition equivalent to that seen in the other pathways studied was
attenuated by powerful excitation. This seems unlikely, however,
because in the feedback pathway the size of polysynaptic EPSPs was
similar to that in the forward pathway (Figs. 1C and 5). In
addition, polysynaptic EPSPs released by blocking
GABAB receptors at feedback synapses were often
smaller than those released at forward synapses (Fig. 6, A,
B, E, and F). This argues against a
drug-induced increase in glutamate release from presynaptic terminals
(Deisz et al. 1993) and indicates that in the feedback pathway slow inhibition is weak and that strong synaptic activation of
GABAergic neurons is necessary to attenuate recurrent polysynaptic EPSPs (Fig. 5). This interpretation is consistent with observations in
cat motor cortex where the demonstration of slow IPSPs requires elevation of GABA release from presynaptic terminals (van
Bredenrode and Spain 1995
). Thus it seems that the small IPSCs
evoked by feedback inputs are insufficient to activate
GABAB receptor-mediated slow IPSPs.
It is important to stress that the same neuron that responds to feedback inputs with small IPSPs gives much larger IPSPs to "forward-type" inputs (Fig. 5). Therefore the weakness of fast and slow IPSPs in the feedback pathway is not due to different intrinsic membrane properties of feedback-and forward-recipient neurons; rather it reflects a distinct synaptic organization of feedback and forward circuits.
Determinants of inhibitory strength
Inhibitory responses in forward and feedback pathways may differ
because the density of inhibitory synapses on forward-recipient pyramidal neurons is higher than on feedback-recipient cells. This
seems unlikely because White et al. (1994) found no positive correlation between the density of GABAergic inputs and IPSP amplitude. Alternatively, the distribution of GABAergic synapses on target neurons
may differ. In both pathways these synapses derive mainly from
parvalbumin (PV) immunoreactive neurons (Gonchar and Burkhalter 1999
). PV neurons include basket and axo-axonic cells
(DeFelipe et al. 1989
; Kawaguchi and Kubota
1997
), which form synapses with pyramidal cell bodies,
dendrites, or axon initial segments, respectively (DeFelipe et
al. 1986
; Peters et al. 1982
). Inhibition by
axo-axonic cells is thought to be most effective. Thus the presence of
a larger contingent of axo-axonic cells in forward than in feedback circuits may underlie the stronger inhibition in forward pathways. Whether this cellular diversity can account for the pathway difference in GABAB responses rests on the demonstration that
different GABAergic cells have different affinities for
GABAB receptors (Benardo 1994
; Otis
and Mody 1992
; Sugita et al. 1992
;
Thomson and West 1997
).
Recent findings that forward synapses on PV neurons are larger than
feedback synapses and are located more proximally on the dendritic tree
suggest that the pathway selectivity of inhibitory effects may be
linked to the inputs to PV neurons (Gonchar and Burkhalter
1999). This organization may cause stronger activation of
inhibitory neurons in the forward pathway (Deuchars et al. 1994
; Stuart and Sakmann 1995
) and increase the
probability for activating GABAB receptors
(Howe et al. 1987
; Kim et al. 1997
; Sodickson and Bean 1996
).
Polysynaptic circuit
Unlike interareal forward, interlaminar, and horizontal pathways
in which polysynaptic EPSPs were confined to weak inputs (Sutor
and Hablitz 1989a; this study), strong feedback inputs elicited
polysynaptic EPSPs that lasted
500 ms. Similar responses were
recorded after stimulation of layer 1 (Cauller and Connors 1994
), which receives strong feedback input (Coogan and
Burkhalter 1993
). Because antidromic activity in layers 2/3 of
area 17 was rare, it is unlikely that polysynaptic EPSPs arise from
reverberations within the reciprocal interareal loop between areas 17 and LM. The two more likely sources of these secondary depolarizations are inputs from feedback-recipient pyramidal cells in the same layer
and/or inputs from deep layers of the same column (Burkhalter 1989
; Czeiger and White 1993
; Elhanany
and White 1990
; Mason et al. 1991
). In layer 2/3
the convergence of inputs to individual pyramidal cells is large
(Deuchars et al. 1994
; Gabbott et al. 1987
; Kisvárday et al. 1986
;
Markram et al. 1997
; Peters 1987
). Thus
it is thought that summation of unitary EPSPs from a pool of
neighboring neurons could easily evoke polysynaptic EPSPs
(Douglas and Martin 1991
; Douglas et al.
1995
). In the feedback pathway this pool consists of
interconnected forward projecting neurons with few contacts to
inhibitory cells (Johnson and Burkhalter 1997
). As a
result, recurrent excitation is only weakly opposed by inhibition
(Markram et al. 1997
; Mason et al. 1991
;
Thomson and Deuchars 1994
). The local connectivity of
forward-recipient pyramidal cells is not known. However, the strong
inhibitory component in the forward response suggests that they are
more intimately connected to inhibitory cells that curb recurrent excitation.
Functional implications
Our study demonstrates that GABAB receptor
antagonists block slow IPSPs elicited by interareal forward inputs and
release polysynaptic EPSPs from inhibition. The role of
GABAB receptors in the suppression of polysynaptic EPSPs is
consistent with the low probability of late EPSPs and an increased
spike threshold (Berman et al. 1992; Connors et
al. 1988
; McCormick 1989
) that renders responses
to moderately strong interareal forward inputs more transient (Fig. 7).
In contrast, in the feedback pathway late IPSPs are weak, and
polysynaptic EPSPs are present even at high stimulation strengths and
are capable of increasing and prolonging neuronal firing.
Activation of polysynaptic excitation by feedback connections over a
broad range of stimulus intensities is advantageous for enhancing
striate cortical responses to a variety of afferent inputs. If strong
enough, this depolarization can overcome the GABAB-mediated slow hyperpolarization generated by
forward inputs, increase spike frequency, and make responses more
sustained (Fig. 7) (Connors et al. 1988). Therefore the
proposed role of the feedback pathway is to modulate late inhibition
elicited by forward inputs.
To understand the significance of two types of circuits (i.e., forward
type, feedback) with unique balances of excitation and inhibition and
partially overlapping but different operating ranges, consider how
convergent inputs are synaptically integrated. Because of the lower
activation threshold for inhibition in forward pathways, two coincident
forward-type inputs may lead to strong amplification of IPSPs and to a
complete suppression of recurrent excitation (Hirsch
1995). By contrast, because inhibition in feedback circuits is
weak, coactivation of forward and feedback inputs may have a much
smaller effect on the summation of inhibition. Indeed, slow inhibition
provided by forward inputs might be suppressed, and late, polysynaptic,
N-methyl-D-aspartate (NMDA) receptor-dependent excitation (Sutor and Hablitz 1989b
) might be enhanced
so that responses to forward inputs are more robust and more sustained. Interestingly, single-unit recordings in cat visual cortex have shown
that the response gain depends on NMDA receptors that act to amplify
small synaptic signals over the entire range of effective inputs
(Fox et al. 1990
). Thus the capacity of feedback inputs to turn on recurrent excitation in an intensity range in which these
circuits are suppressed by forward input-dependent slow IPSPs may
provide a mechanism by which higher cortical areas can amplify
responses in lower areas.
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ACKNOWLEDGMENTS |
---|
We thank M. Ariel, N. Kogo, and H. Xu for expert advice on whole cell patch-clamp recording in slices. Many thanks also to L. Peng for help with the histology and to L. Domenici, Y. Gonchar, R. R. Johnson, and J. M. Nerbonne for helpful discussions and comments on the manuscript.
This work was supported by National Eye Institute Grant EY-05935.
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FOOTNOTES |
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Address for reprint requests: A. Burkhalter, Dept. of Anatomy and Neurobiology, Box 8108, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110.
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 soley to indicate this fact.
Received 6 May 1998; accepted in final form 4 November 1998.
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REFERENCES |
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