Inhibition Evoked From Primary Afferents in the Electrosensory
Lateral Line Lobe of the Weakly Electric Fish
(Apteronotus leptorhynchus)
NEIL J. BERMAN AND LEONARD MALER
Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1H
8M5, Canada
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.
INTRODUCTION
The importance of synaptic inhibition in processing sensory
input is acknowledged, yet the functional role played by inhibition is still under debate (Berman et al. 1992; Martin 1988;
Somers et al. 1995). The major inhibitory neurotransmitter, gaminobutyric acid (GABA) has been shown to shape the responses of neurons in sensory systems (Sillito 1984), yet it has
proved difficult to identify the inhibitory neurons and pathways
responsible for the various response properties of sensory neurons. This is largely due to the complexity and number of inhibitory circuits present in the most commonly studied sensory structures, e.g., visual cortex. The electrosensory system of weakly
electric fish is a far simpler sensory structure, which has been
extensively studied from both a morphological (Carr and Maler
1986) and functional point of view (Bastian 1986a; Heiligenberg
1991). In the electrosensory lateral line lobe (ELL) of the
weakly electric fish, we have identified GABAergic neurons and
pathways that are likely to be involved in electroreception (Maler
and Mugnaini 1994). We anticipate that attempts to correlate
inhibitory pathways with particular sensory processing functions
will be more successful in this system and will help to focus
investigations in other sensory systems.
The gymnotiform species, Apteronotus leptorhynchus, has
a neurogenic electric organ in its tail that generates a highfrequency electric organ discharge (EOD) (reviewed by
Bass 1986). The EOD of this species resembles a sine wave
ranging from 500 to 1,200 Hz (at 287C) and is constant
except when used intermittently for communication (Bullock 1969; Zupanc and Maler 1997). The fish has specialized
electroreceptors distributed on its body surface that detect
changes in the electric field surrounding the fish. There are
two types of electroreceptors (see Carr and Maler 1986;
Zakon 1986 for review): ampullary receptors ( Ç360) (Carr
et al. 1982) detect slowly varying exogenous electric fields,
whereas tuberous receptors ( Ç6,500–8,500) (Carr et al.
1982) are tuned to the EOD of the individual fish. Tuberous
receptors are divided into two types: phase coders (T-units)
fire 1:1 with each cycle of the fish’s EOD, whereas probability coders (P-units) scale their firing rate according to the
local amplitude of the EOD. A. leptorhynchus has few phase
coders, and so these are not discussed here (see Mathieson
et al. 1987). Probability coders are essential for both electrolocation and electrocommunication (Bastian 1986a; Heiligenberg 1991).
The sole recipient of electroreceptor afferents is the ELL
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Berman, Neil J. and Leonard Maler. Inhibition evoked from primary afferents in the electrosensory lateral line lobe of the weakly
electric fish (Apteronotus leptorhynchus). J. Neurophysiol. 80: 3173–
3196, 1998. The responses of two types of projection neurons of the
electrosensory lateral line lobe, basilar (BP) and nonbasilar (NBP)
pyramidal cells, to stimulation of primary electrosensory afferents
were determined in the weakly electric fish, Apteronotus leptorhynchus. Using dyes to identify cell type, the response of NBP cells to
stimulation of primary afferents was inhibitory, whereas the response
of BP cells was excitation followed by inhibition. g-Aminobutyric
acid (GABA) applications produced biphasic (depolarization then
hyperpolarization) responses in most cells. GABAA antagonists
blocked the depolarizing effect of GABA and reduced the hyperpolarizing effect. The GABAB antagonists weakly antagonized the hyperpolarizing effect. The early depolarization had a larger increase in cell
conductance than the late hyperpolarization. The conductance changes
were voltage dependent, increasing with depolarization. In both cell
types, baclofen produced a slow small hyperpolarization and reduced
the inhibitory postsynaptic potentials (IPSPs) evoked by primary afferent stimulation. Tetanic stimulation of primary afferents at physiological rates (100–200 Hz) produced strongly summating compound
IPSPs ( Ç500-ms duration) in NBP cells, which were usually sensitive
to GABAA but not GABAB antagonists; in some cells there remained
a slow IPSP that was unaffected by GABAB antagonists. BP cells
responded with excitatory or mixed excitatory / inhibitory responses.
The inhibitory response had both a fast ( Ç30 ms, GABAA ) and longlasting slow phase ( Ç800 ms, mostly blocked by GABAA antagonists). In some cells there was a GABAA antagonist-insensitive slow
IPSP ( Ç500 ms) that was sensitive to GABAB antagonists. Application of glutamate ionotropic receptor antagonists blocked the inhibitory response of NBP cells to primary afferent stimulation and the
excitatory response of BP cells but enhanced the BP cell slow IPSP;
this remaining slow IPSP was reduced by GABAB antagonists. Unit
recordings in the granule cell layer and computer simulations of pyramidal cell inhibition suggested that the duration of the slow GABAA
inhibition reflects the prolonged firing of GABAergic granule cell
interneurons to primary afferent input. Correlation of the results with
known GABAergic circuitry in the electrosensory lobe suggests that
the GABAergic type 2 granule cell input to both pyramidal cell types
is via GABAA receptors. The properties of the GC2 GABAA input
are well suited to their putative role in gain control, regulation of
phasicness, and coincidence detection. The slow GABAB IPSP
evoked in BP cells is likely due to ovoid cell input to their basal
dendrites.
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N. J. BERMAN AND L. MALER
( see atlas of Maler et al. 1991 for location ) . The ELL cal synaptic contacts with the basal bush of basilar pyramiconsists of four independent segments, each containing a dal neurons, and mixed ( chemical and gap junction ) syntopographic electrosensory map ( Carr et al. 1982; Heili- apses onto dendrites of several types of putative inhibitory
genberg and Dye 1982 ) . One segment ( medial segment: interneurons: type 1 granular cells ( GC1; transmitter not
MS ) receives only ampullary input, whereas the other seg- identified ) , type 2 granular cells ( GC2; GABAergic ) ,
ments ( centromedial: CMS, centrolateral: CLS, lateral: LS ) ovoid cells ( GABAergic ) and polymorphic cells ( GAreceive tuberous input ( mostly P units ) . There are physio- BAergic ) ( Maler 1979; Maler and Mugnaini 1994; Maler
logical and morphological differences among the segments et al. 1981; Yamamoto et al. 1989 ) .
( Metzner and Juranek 1997; Shumway 1989a,b; Turner et
The principal output neurons of the ELL are the pyramidal
al. 1996 ) but all three tuberous segments contain similar cells (Fig. 1). There are two morphologically and physiologcanonical circuits of excitatory and inhibitory neurons and ically distinct types of ELL pyramidal neurons: basilar (BP)
pathways ( Fig. 1 ) ( Fig. 2 in Maler and Mugnaini 1994 ) . and nonbasilar pyramidal cells (NBP). BP cells (E units)
This and the companion studies ( Berman and Maler receive direct glutamatergic synaptic input from tuberous
1998a,b ) were conducted exclusively in the CMS; hence afferents onto their basal dendrite and are excited by inall results are interpreted for this segment only, as intrinsic creases in EOD amplitude (Maler 1979; Maler et al. 1981;
cell properties and proportions of cell types vary across the Saunders and Bastian 1984; Wang and Maler 1994); this
segments. All four segments of the ELL have a similar cell type also receives an input from the GABAergic ovoid
laminar structure consisting of: a deep fiber layer ( DFL ) cell, which climbs on its basal dendrite as well as input from
consisting mainly of electrosensory afferents, a granular GC1 and GC2 cells onto its soma (Maler and Mugnaini
cell layer ( GCL ) of interneurons, a pyramidal cell layer 1994). NBP (I units) somata and somatic dendrites receive
( PCL ) , and dorsal and ventral molecular layers ( DML, input from GABAergic and non-GABAergic granular cells
VML ) containing apical dendrites of pyramidal cells, inter- (GC2 and GC1) and are excited by decreases in EOD amplineurons, and feedback input from higher brain centers. The tude (Maler 1979; Maler and Mugnaini 1994; Maler et al.
electroreceptor afferents terminate in a deep neuropil region 1981; Saunders and Bastian 1984). The descending excit( ventral to the granular cell layer ) where they form chemi- atory and inhibitory inputs to pyramidal cells, which termi-
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FIG . 1. Topography and relevant inhibitory circuitry of the electrosensory lateral line lobe (ELL). A: coronal section
(cresyl violet stain) from the atlas of Maler et al. (1991; level T-7). ELL layers are approximately indicated for the segment
investigated in this study: the centromedial segment. CCb, cerebellum; mol, molecular layer; StF, stratum fibrosum; PCL,
pyramidal cell layer; GCL, granule cell layer; DNL, deep neuropil layer; DFL, deep fiber layer. B: circuit diagram of
pyramidal cell types and the relevant inputs that could be driven by stimulation of the DFL. Nonbasilar and basilar cells:
both receive GABAergic input from type 2 granule cells (GC2) (Maler and Mugnaini 1994) and inhibitory (based on
synapse ultrastructure) (Maler et al. 1981) input of unknown transmitter type from type 1 granule cells (GC1). Basilar
pyramidal cells differ only in that their basilar dendrites receive GABAergic input along its length from ovoid cells and
direct excitatory (glutamatergic) input from the primary afferents.
PRIMARY AFFERENT-EVOKED INHIBITION
METHODS
Slice preparation
Slices of ELL were prepared according to a modified method
of Mathieson and Maler (1988) and Turner et al. (1994). Briefly,
fish of either sex were anesthetized by immersion in water containing 0.2% 3-aminobenzoic acid ethyl ester (MS-222; Sigma
provided all chemicals unless otherwise indicated). All surgical
procedures were approved by the University of Ottawa Animal
Care Committee. The fish then were transferred to a foam-lined
clamp, and a glass tube was introduced into their mouth that supplied aerated water containing the same anesthetic. The brain was
exposed by dissection of the skin and overlying skull while the
surgery site was irrigated continuously with chilled artificial cerebrospinal fluid [ACSF, which contained (in mM) 124 NaCl, 3
KCl, 0.75 KH2PO4 , 2 CaCl, 2 MgSO4 , 24 NaHCO3, and 10 Dglucose]. Care was taken to avoid touching the ELL while underlying nerves were cut. The brain was blocked to ensure a true transverse section of the ELL by cutting the rostral face of the block
at Ç457 angle at the level of the optic tectum. The brain block was
removed from the skull after severing the spinal cord, glued to a
chilled aluminum chuck with cyanoacrylate adhesive, and covered
with warm ( Ç257C) low gelling temperature agarose (5% in
ACSF; FMC, Rockland, ME). The chuck was placed immediately
in a Vibratome (modified to provide high-amplitude blade vibrations, Technical Products International, St. Louis, MO), immersed
in chilled carbogenated (95% O2-5% CO2 ) ACSF and cut into 350mm sections. As each section was cut, it was sucked up into a
wide-mouthed plastic pipette and ejected into a holding chamber
containing carbogenated and chilled ACSF. Slices then were transferred to an interface type slice chamber where they were maintained at room temperature (21–237C) and perfused with carbo-
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genated ACSF (0.5–1 ml/min). The total time from removal of
the ELL from its oxygenated blood supply to immersion of the
first slice in the holding chamber was Ç5–7 min. Recordings were
started after a 1- to 2-h recovery period.
Stimulation and recording
Electrical stimulation of the primary afferents of the ELL was
achieved by placing a lacquer-coated sharpened tungsten electrode
(50 mm exposed tip) into the DFL region of the CMS, lateral to the
recording site in the PCL (see Fig. 1). Square wave pulses (50–
100 ms, 1–50 V or 20–600 mA cathodal, NL102 or SIU unit,
Digitimer, UK) were delivered via the stimulating electrode. Stimulus intensity was adjusted to evoke Ç70% of maximal response
amplitude. Excessively high stimulation parameters caused direct or
antidromic activation of the cells. Intracellular recordings were made
in the CMS PCL with 2 M potassium acetate-filled electrodes bevelled to 55–120 MV DC resistance. For recovery of cells, some
recordings were made with either biocytin (5% in potassium acetate,
bevelled to 70 MV ) or Lucifer yellow (tips were backfilled by
capillary action with a 10% solution in distilled water, then filled
with 2 M potassium acetate and bevelled to 120 MV ) pipettes. All
experiments were software controlled (PCLAMP, Axon Instruments, Foster City, CA or A/Dvance, Rob Douglas, University of
British Columbia, Vancouver BC, Canada). Electrical activity was
amplified (Axoclamp, Axon Instruments), filtered (10 kHz fc ), digitized (1.25–50 kHz) and analyzed off-line (Igor Pro 3.0, Wavemetrics, OR; Macintosh PowerPC, Apple, CA).
Drug applications
For iontophoretic applications, drugs were dissolved in 100 mM
NaCl at the following concentrations: GABA and baclofen
(GABAB agonist) 0.1 M [Research Biochemicals International
(RBI), MA], 5 mM bicuculline (GABAA antagonist: Tocris Cookson, UK; RBI, MA; Sigma, MO). For application to the slice by
microdroplet, antagonists were dissolved in ACSF at the following
concentrations: 70 mM bicuculline, (Tocris Cookson; RBI; Sigma),
100 mM SR(-95531) (GABAA antagonist: RBI), 0.05–1 mM [6cyano-7-nitroquinoxaline-2,3-dione (CNQX) a -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-receptor antagonist: RBI], 2 mM DL-2-amino-5-phosphonovaleric acid (APV)
[N-methyl-D-aspartate (NMDA)-receptor antagonist 1 CPP ( { )3-2(2-carboxypiperazin-4-gl)-propyl-1-phosphonic acid, NMDA
antagonist, RBI, MA; Tocris Cookson], and 1–3 mM phaclofen
and saclofen (GABAB antagonists: RBI; Tocris Cookson]. Microdroplets were applied by pressure via a broken-back pipette (10–
20 mm) so that a single droplet covered an area of the slice surface
Ç200–400 mm in diameter. This ensured that most of the input to
the recorded cell, from the GCL to the PCL, would be exposed to
the drug. Iontophoretic pipettes were inserted into the PCL as close
as possible to the estimated recording site both in lateral distance
and recording depth. Depth was adjusted to gain the best response
to a test GABA pulse.
Histology and reconstruction
After recording, slices were allowed to incubate for ¢1 h before
fixation. Slices, which lay on individual pieces of lens tissue paper
(Kodak, Rochester, NY), were removed from the chamber, placed
on a glass slide, and had a droplet of fixative applied [4% paraformaldehyde 0.5% glutaraldehyde in 0.1 M phosphate buffered saline
(PBS)]. Slices were postfixed at room temperature for 1–3 h and
then stored in PBS overnight. The slices were resectioned on a
vibratome at 80 mm, placed on glass slides, dried, and processed
according to a modified Horikawa and Armstrong (1988) protocol.
Background peroxidase activity was removed by pretreatment of
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nate in the dorsal laminae of the ELL (Carr and Maler 1986;
Maler and Mugnaini 1993, 1994) are discussed in the accompanying papers (Berman and Maler 1998a,b).
The responses of pyramidal cells to sensory input bear
remarkable resemblance to those of mammalian sensory systems, such as the retina. ELL pyramidal cells have receptive
fields that have center-surround antagonism (Shumway
1989a) and flexible gain control/contrast adaptation (Bastian
1986b, 1995, 1996). Maler and Mugnaini (1994) have attempted to relate ELL inhibitory circuitry to its sensory physiology. The inhibitory input to pyramidal cells from GABAergic GC2 was proposed to control the size and strength of
receptive field centers (Maler and Mugnaini 1994). They
further hypothesized that non-GABAergic input from GC1
contributes to surround regulation. Ovoid cell input to BP cells
is thought to mediate common mode rejection (see Bastian et
al. 1993; Maler and Mugnaini 1994). However, little is known
of the mechanisms and dynamics of inhibition in the ELL.
Such data are needed to assess the validity of these hypotheses. In this paper we have investigated the mechanisms of
inhibition that may underlie the first stage of electrosensory
processing in the ELL. This study identifies the biophysical
and pharmacological nature of inhibitory responses recorded
in pyramidal cells in response to stimulation of the DFL. Our
results show that BP and NBP cells can be identified by
their response to DFL stimulation and that both GABAA and
GABAB receptors are involved in their inhibitory responses.
GC2 are likely to generate mostly GABAA-receptor-mediated
inhibition, whereas ovoid cell input is mediated partly by
GABAB input to BP cells.
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TABLE
N. J. BERMAN AND L. MALER
1.
Summary of biophysical parameters of cells recorded in the ELL slice preparation
BP
Passive parameters
Vrest, mV
Rin, MV
t, ms
Spike parameters
Amplitude, mV
Rise,* ms
Decay,* ms
Half width,† ms
067.9
17.6
15.7
80.9
0.299
0.515
0.454
NBP
{ 6.2 (47)
{ 4.4 (45)
{ 6.3 (42)
{
{
{
{
067.3
16.3
18.2
10.3 (41)
0.278 (40)
0.3892 (40)
0.159 (40)
77.7
0.275
0.435
0.415
All Cells
{ 4.5 (31)
{ 4.7 (33)
{ 6.4 (32)
{
{
{
{
9.7 (31)
0.107 (31)
0.355 (31)
0.170 (31)
067.8
17.2
16.6
79.7
0.296
0.453
0.432
{ 5.7 (80)
{ 4.8 (78)
{ 6.4 (74)
{
{
{
{
10.0 (74)
0.231 (73)
3.709 (73)
0.162 (73)
Number of observations in parentheses. All values are means { SD. Nonbasilar (NBP) and basilar (BP) pyramidal cell means were not significantly
different (Student’s t-test). ELL, electrosensory lateral line lobe; Vrest, resting membrane potential; Rin, input resistance; t, cell time constant. * 10–90%
rise or decay time. † Measured at half-height.
RESULTS
Biophysical parameters
Intracellular recordings were obtained from cells impaled
in the PCL of ELL slices. This study concentrates on the
inhibitory responses that are evoked by DFL stimulation;
this inhibition must arise from activation of three inhibitory
interneurons: GC1, GC2, and ovoid cells (Maler and Mugnaini 1994) are the only known interneurons that receive
direct primary afferent input via glutamatergic and gap junction synapses (Maler 1979; Maler et al. 1981; Mathieson et
al. 1987) and project to pyramidal cells.
Of the 101 cells impaled in the PCL, 15 cells were histologically identified as BP (n Å 9) or NBP (n Å 6). Based
on their electrophysiological response to DFL stimulation
(see next section), a further 78 cells were classified as either
BP (n Å 49) or NBP (n Å 29). The remaining cells could
not be assigned unambiguously to either group or were suspected to be polymorphic type cells (see following text) and
were not included in the sample. The summary statistics of
the pyramidal cells’ passive and active biophysical characteristics are presented in Table 1. There were no significant
differences between the means of the BP and NBP cell
groups. The passive parameters are unremarkable and are
similar to those reported for ELL pyramidal cells (Berman
et al. 1997; Mathieson and Maler 1988; Turner et al. 1994)
and for pyramidal cells in the benchmark rat hippocampal
slice preparation (Schwartzkroin 1975).
ELL BP and NBP spikes had a half-width (0.4 ms) nar-
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rower than typical cortical or hippocampal pyramidal cells
( Ç0.5–0.6 ms) (Mason and Larkman 1990). Two cells, not
included in Table 1, had very narrow spikes (half-width
õ0.18 ms), a large afterhyperpolarization (AHP; Ç10 mV),
and rapid accommodation of spike rate to injected current.
These spike characteristics have been observed in identified
polymorphic cells (Maler 1979; R. Turner, personal communication). Twelve cells probably were impaled in their dendrites because their current-evoked spikes decayed slowly
[1.00 { 0.47 (SE) ms], were wide (half-width 0.617 {
0.147 ms), and lacked a fast AHP (see Turner et al. 1994).
Basilar and nonbasilar pyramidal cells can be identified
by their response to DFL stimulation
MORPHOLOGY. Histological recovery of Lucifer yellow
(LY)- or biocytin-filled cells were used to correlate their
responses to DFL stimulation with their classification as BP
or NBP cells. The cellular morphology and intracellular responses of a typical LY-filled BP cell is shown in Figs. 2
and 3. The soma of the BP cell straddled the pyramidal and
plexiform layers. In this case the sparse somatic dendrites
arose from the apical dendrite trunk within the pyramidal cell
layer (the site of termination of granular cell axons) (Maler
1979; Maler and Mugnaini 1994). The axon can be seen
leaving the soma and coursing horizontally through the plexiform layer (Fig. 2A). The thick apical dendrite courses
through the stratum fibrosum and ramifies in the molecular
layer. The thick basal dendrite courses ventrally through the
plexiform and granular cell layers to form a compact but
dense bush in the deep neuropil layer (Fig. 3C), where it
receives excitatory input from primary afferents (Maler et al.
1981). Our fills demonstrated that the basal bush was far
denser than seen in typical golgi impregnations (Maler 1979).
The NBP cell soma was situated within the pyramidal cell
layer (Fig. 2A) and had a more extensive somatic dendritic
arbor than the BP cell (cf. Fig. 3, A with B); this was typical
of our sample and corresponds with our previous Golgi description of these cells [although the previous studies (Maler
1979; Saunders and Bastian 1984) did not allow full resolution of these fine processes]. Because these processes receive rich GABAergic input (Maler and Mugnaini 1994;
Maler et al. 1981), this implies that NBP cells might have
more potent inhibitory responses than BPs. The functional
implications of placement of GABAergic inputs on these
extensive and fine somatic arbors were further explored by
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slides with 0.3% H2O2 in PBS. Horseradish peroxidase was revealed by using a cobalt-nickel-intensified (Adams 1981) diaminobenzidine (0.2 mg/ml in 0.1 M sodium acetate buffer, pH 6.0)
procedure. Treatment with 0.1% (in PBS, 30–90 s) Osmium tetroxide further darkened the reaction product. Sections then were
dried, defatted, and cover-slipped. For Lucifer yellow visualization,
slices were fixed with paraformaldehyde (4% in PBS). Sections
were cleared using dimethyl sulfoxide to minimize shrinkage distortions (Grace and Llinas 1985). Sections were viewed on a fluorescence microscope (BH-2; Olympus America, NY). Pyramidal
cells were identified as basilar versus nonbasilar on the basis of
the presence of a basal dendrite (Maler 1979). For illustration,
sections were scanned on a confocal microscope in 5- or 10-mm
optical slices ( 116 averaging). Final montages were made by
merging all optical slices (Photoshop Version 5.0, Adobe Systems,
CA) using the ‘‘lighten’’ algorithm (each pixel has the highest
value sampled from each optical slice).
PRIMARY AFFERENT-EVOKED INHIBITION
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computer simulation (see APPENDI X ). The apical dendritic
morphology of NBP and BP cells was indistinguishable
(Maler 1979; Saunders and Bastian 1984).
PHYSIOLOGY. We recorded the intracellular responses of the
same cells to single (Fig. 2B) and tetanic (200 Hz; Fig. 2C)
stimulation of the primary afferents via an electrode in the
DFL. This BP cell responded to a single stimulus with a
short-lasting excitatory postsynaptic potential (EPSP) ( õ10
ms half-width, Fig. 2B, left) followed by a longer lasting
IPSP; when it appeared, the IPSP was always associated
with a preceding EPSP. The NBP cell responded with an
IPSP alone (2 ms onset, 14 ms to peak, Fig. 2B, right).
Postsynaptic potentials were classified as EPSPs if they were
depolarizing at resting membrane potential (Vm ) and, when
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the cell was depolarized with current injection, increased the
probability of spike firing. The polarity of IPSPs depended
on resting Vm ; cells with large (approximately less than 075
mV) resting Vm had depolarizing IPSPs that reversed with
depolarizing current injection and reduced the probability of
spike firing (see following text).
Tetanic stimulation (Fig. 2C) produced a summating series of EPSPs or compound EPSP in the BP cell and a
summating series of IPSPs or compound IPSP in the NBP
cell. The cells in Fig. 2 were tested both at rest and during
injection of current ( /0.2 nA).
The BP cell did not fire at rest, and tetanic stimulation
(Fig. 2C, bottom) could evoke spikes only on the last few
EPSPs in the tetanus; tetanic stimulation was effective in
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FIG . 2. Morphology and physiology of
pyramidal cells. A: montage of fluorescent
confocal images of a basilar (BP, left) and
nonbasilar pyramidal cell (NBP, right) recorded in different transverse ELL slices.
Approximate lamina boundaries for both
slices are indicated ( – – – ). GCL, granule
cell layer; pl, plexiform layer; VML, ventral
molecular layer; DML, dorsal molecular
layer. Labels near the somas (a and b) and
the basilar dendritic bush (c) indicate the
regions scanned at high magnification in Fig.
3. B: intracellular responses (average of 3
sweeps) at rest (BP: 068 mV; NBP: 066
mV) of each cell in A to a single stimulus
(20 us, 70 mA) applied to the DNL Ç300
mm ventral to the granule cell layer. Stimulus
artifacts ( m ) have been removed in this and
all subsequent figures for clarity. C: intracellular responses of same cells to tetanic stimulation (200 Hz, triangles) at rest ( bottom)
and during current injection ( /0.2 nA, top).
Both cells were quiescent at rest. Responses
of BP cell are to single trials; NBP traces
are averages of 3 trials. Spike heights truncated by cropping and/or averaging in this
and subsequent figures.
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N. J. BERMAN AND L. MALER
producing six spikes in the depolarized condition (Fig. 2C,
left, top). Higher stimulation intensities were capable of synaptically evoking spikes at rest both to single stimuli and to
every stimulus in the tetanus (not shown). The depolarized
condition (Fig. 2C, left, top) revealed a slow IPSP following
the compound EPSP. Depolarizing current injection ( °0.6
nA) induced discharge in this cell that was blocked by the
slow IPSP. The NBP also fired during current injection, and
this discharge was completely suppressed both during the
tetanus and during the evoked slow IPSP ( ú300 ms).
The preceding results were representative for nine BP and
six NBP cells filled with either HRP or LY. BP cells always
displayed a clear short-latency EPSP (0.96 { 0.48 ms, n Å
8) or compound EPSP to tetanic stimulation (3 of 3 cells
tested), whereas filled NBP cells displayed a short-latency
IPSP (2.8 { 0.4 ms to peak, n Å 4) or compound IPSP to
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tetanic stimulation (3 of 3 cells tested). In one filled NBP
cell, we observed a short-latency depolarizing potential (0.4
ms to peak) that was presumably due to gap junction input
from granular cells (Maler et al. 1981). The shape and amplitude of this potential remained constant at all membrane
polarizations, unlike the EPSPs in BP cells the amplitudes
of which were voltage sensitive (not shown).
Pyramidal cell response to exogenous GABA and baclofen
GABA was ionophoresed (1–10 s,
10–200 nA) within the PCL close to the intracellular recording site for 13 physiologically identified pyramidal cells
while membrane potential and input resistance (Rin ) changes
were monitored. Both BP and NBPs displayed similar responses to GABA at the resting Vm (Fig. 4); a rapid initial
RESPONSE TO GABA.
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FIG . 3. High magnification views of regions of BP
and NBP cells indicated in Fig. 2. A: BP cell soma.
r, site of axon hillock and axon emerging from the
soma. Axon can be followed for several hundred micrometers in Fig. 2A (left). There is a plexus of fine
(0.2–2 mm diam) varicose dendrites that arise from
the most dorsal region of the soma and the proximal
apical dendrites. These are confined to the PCL. B:
NBP cell soma. r, axon emerging from ventral tip
of soma (see Fig. 2A, right). Axon continue in the
plexiform layer. NBP cell soma has a denser but otherwise similar plexus of fine varicose dendrites. In this
case, these fine dendrites arise from the soma only.
C: basilar dendrite bush. Basilar dendrite terminates
in a dense varicose plexus with process diameters
ranging from 8 to 0.2 mm.
PRIMARY AFFERENT-EVOKED INHIBITION
3179
hyper- or depolarization followed by a lower amplitude sustained hyperpolarization. The initial response was brief
(0.5–4 s), and its polarity dependent on resting Vm (Figs.
4 and 5). It was depolarizing in 8 of the 13 cells.
Polarizing the cell with injected current revealed that this
initial response may be composed of more than one phase
with reversal potentials near rest (Fig. 4B). The small hyperpolarization seen at resting potential reversed at 065 mV.
A second phase appeared as a later depolarizing peak (Fig.
4B; hyperpolarized trace, ● ) with a slightly more depolarized reversal potential ( 062.5 mV). The peak of the second
component coincided with the greatest decrease in Rin (Fig.
A2). After the iontophoretic current was turned off, a small
hyperpolarization (0.5–1 mV) persisted for °15 s. The slow
response, which outlasted the GABA application, was of
longer duration ( ú4 s) and did not reverse polarity even in
the most hyperpolarized traces ( õ-86 mV; Fig. 5).
GABA produced a large decrease in Rin throughout the
applications irrespective of whether the initial response was
hyper- or depolarizing ( Figs. 4 – 6 ) ; the postapplication hyperpolarization was accompanied by a much smaller decrease in Rin ( Figs. 4 and 5 ) . The conductance change
underlying the GABA response was strongly voltage dependent ( e.g., Fig. 4. A2 ) ; in this experiment, Rin decreased
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by °2.5 MV when the cell was hyperpolarized and GABA
applied, whereas when the cell was depolarized to 055
mV, Rin decreased by as much as 7 MV during the GABA
application. Although all pyramidal cells showed some
voltage dependency ( rectification ) of their steady-state Rin ,
this could not account for the voltage dependence of the
GABA response. Indeed, the hyperpolarizing response to
GABA ( most depolarized case ) would offset the intrinsic
voltage dependency of Rin , therefore voltage dependence
of the GABA response may be underestimated. The cell
in Fig. 4 was typical in that control Rin decreased with
depolarization and vice versa ( compare control period voltage deflections at the 3 membrane potential levels ) . This
rectification was probably due to the voltage dependence
of intrinsic currents ( e.g., IA and INap ) ( see Mathieson and
Maler 1988 ) and is not discussed further. The change in
Rin produced by GABA scaled with iontophoretic current,
ejection time, and proximity of the drug electrode to the
impalement site; Rin became unmeasurably low with large
GABA ejection parameters.
EFFECT OF GABAA ANTAGONISTS ON THE GABA RESPONSE.
To determine whether the responses to GABA were due to
activation of GABAA receptors, bicuculline (70 mM) or SR
(50 mM) were applied by microdroplet to the pyramidal cell
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FIG . 4. Response of BP cell to g-aminobutyric acid (GABA) application. A1: response to GABA at 3 membrane potentials
(resting Vm Å 061 mV). Input resistance (Rin ; control Å 22 MV ) was monitored by injecting 00.5-nA current pulses (50
ms, 1 Hz) and measuring the resulting voltage deflections. – – – , control membrane potential. DC currents were injected
into the cell to depolarize (top) and hyperpolarize (bottom) the cell from rest (middle). This and subsequent figures: short
depolarizing voltage deflections were caused by 0.5-nA pulses used to monitor cell excitability (firing response). These
deflections have been truncated digitally. A2: normalized Rin measurements derived from responses to hyperpolarizing current
pulses in A1. B1: same data as in A, but with voltage deflections digitally removed and all 3 traces plotted on same voltage
axis. This plot indicates ¢2 components to the GABA response. Middle: hyperpolarizing peak at 1 s after the onset of
GABA. Bottom: depolarizing peak at 5 s. Markers indicate latency from onset of GABA application where measurements
(plotted in B2) of the GABA response were made. B2: measurements of the GABA response magnitude vs. the control
holding potential indicate the approximate reversal potential (membrane potential where the interpolated GABA response
would be 0 mV) of the early ( h; 065 mV) and late ( ●; 062.5 mV) components of the GABA response.
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N. J. BERMAN AND L. MALER
layer prior to iontophoresis of GABA (Fig. 5). The efficacy
of the antagonists was apparent after several seconds by the
appearance of membrane potential oscillations (Turner et al.
1991). Both GABAA antagonists blocked or substantially
decreased the initial (hyper- or depolarizing) part of the
GABA response (n Å 8). In the example in Fig. 5, the
GABA response was initially depolarizing followed by a
longer lasting hyperpolarization. Note that the cell’s resting
potential was 074 mV and was not polarized (with injected
current) beyond the GABA depolarizing response’s reversal
potential ( 056 mV by extrapolation). SR blocked the depolarizing response but did not affect the longer hyperpolarization (Fig. 5B1) the extrapolated reversal potential of which
was 0100 mV. This hyperpolarization, which under SR
block started at the onset of the GABA response, probably
was masked by the depolarization under control conditions.
The Rin changes underlying the GABA responses (Fig. 5,
A2 and B2) showed typical strong voltage dependency. The
SR block of the depolarizing response was associated with
a block of the early peak conductance shift seen in control
conditions (1st r in Fig. 5, A2 and B2), while the second
peak in the conductance shift was relatively unaffected by
SR (2nd r, Fig. 5, A2 and B2).
GABAB-MEDIATED RESPONSES TO GABA AND BACLOFEN. Because the slow GABA-evoked hyperpolarization was insensitive to bicuculline or SR, GABAB antagonists (saclofen or
phaclofen) and an agonist (baclofen) were tested on the
pyramidal cells. Figure 6 shows one such experiment; in
this cell (which displayed a small oscillation of membrane
potential under control conditions) both components of the
GABA response were hyperpolarizing at rest. Treatment
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with saclofen (2 mM) curtailed mainly the duration of the
hyperpolarization. Conductance measurements showed a decrease in the magnitude of the GABA-induced conductance
increase (Fig. 6B, rrr). Treatment with both SR and saclofen before the GABA application blocked both the voltage
and conductance shifts. Thus at least part of the GABA
response was sensitive to the GABAB antagonist. GABAB
antagonists clearly affected part of the GABA response in
only 5 of 12 cases.
The GABAB agonist baclofen consistently (n Å 13) produced a small (1–2 mV) hyperpolarization and increase in
spike current threshold (not shown). The conductance
change underlying the response was either small ( °1 MV )
or undetectable. This is expected because the effect of rectification with hyperpolarization (which would tend to increase Rin ) would cancel the small conductance increase
associated with the response to baclofen. Treatment of the
slice with microdroplets of saclofen (n Å 3, 2 mM) was
unable to antagonize the response to baclofen. In the example
shown in Fig. 6, the cell depolarized slightly (by 4 mV) after
saclofen treatment; this actually increased the magnitude of
the response to baclofen.
These experiments demonstrated that both types of pyramidal cells respond with both GABAA and GABAB-like potential and conductance changes and pharmacology, as seen
in mammalian pyramidal cells in vitro (hippocampus: Alger
and Nicholl 1979, 1982; Scharfman and Sarvey 1988b; neocortex: Scharfman and Sarvey 1988a). Thus although a molecular characterization is presently lacking, we conclude
that ELL pyramidal cells (BP and NBP) have both GABAA
and GABAB receptors; traditional GABAA antagonists are
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FIG . 5. Effect of GABAA antagonist on
the response of a pyramidal cell to GABA.
A1: response of pyramidal cell to 5 s (middle: rest Vm ; top: depolarized) and 10 s
(bottom: hyperpolarized) GABA iontophoresis (50 nA). Rin and excitability monitored as in Fig. 5. Control membrane potential is indicated ( – – – ). A2: absolute
Rin change during the GABA responses at
the 3 polarization levels in A1. r, 2 peaks
(2 and 6 s) in the time course of the decrease of Rin . B1: response of the same cell
to GABA applications after microdroplet
application of 50 mM SR. Early depolarizing component of the response is blocked,
whereas the slower hyperpolarizing component is unaffected by SR. B2: changes
in Rin during the responses in B1. First peak
arrowed in A2 is reduced by SR. Extrapolated reversal potentials: GABA depolarizing response in A1 Å 040 mV, GABA
hyperpolarizing response (measured at
GABA offset) was 094 mV in before and
0100 mV during the SR treatment.
PRIMARY AFFERENT-EVOKED INHIBITION
3181
highly effective in the ELL, whereas the GABAB antagonists
(saclofen and phaclofen) appear to have a relatively low
potency.
EFFECT OF BACLOFEN ON INHIBITION. GABAB receptors
have been shown to decrease presynaptic release of GABA
in other systems (Thompson and Gähwiler 1992). This was
tested in the ELL by observing the effect of baclofen treatment on the inhibition evoked by DFL stimulation (NBP
cells, n Å 3). Under control conditions, tetanic stimulation
produced strong hyperpolarizing inhibition (Fig. 7A, r ).
The cell was current-clamped at various potentials to monitor
the efficacy of the inhibition. After baclofen treatment, DFL
stimulation did not evoke strong inhibition. To test whether
the baclofen-induced postsynaptic decrease in Rin could account for the disappearance of the stimulus-evoked inhibition, the cell was depolarized to levels that previously had
produced strong repetitive firing (Fig. 7, B and C). Under
control conditions, the stimulus-evoked inhibition potently
reduced the firing rate of the cell (Fig. 7C, r ), however,
after baclofen treatment, it had only a small inhibitory effect
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on spike rate (Fig. 7, B and C, right). We conclude that
GABAB receptors are likely to be present on GABAergic
nerve terminals in the PCL.
Electrophysiological and pharmacological characteristics
of synaptically evoked inhibition in NBP cells
ELECTROPHYSIOLOGY OF NBP CELL INHIBITION. As shown in
Fig. 2, the response of NBP cells to DFL stimulation was
inhibitory. IPSPs could be classified into at least two categories, fast and slow. Fast IPSPs peaked at between 1.8 and
25 ms (mean 8.4 { 8.7 to peak, n Å 28), whereas slow
IPSPs peaked at between 150 and 600 ms (mean 501.3 {
371.7 ms to peak, n Å 20) and endured for °2 s. The slow
IPSP was absent in 26% of NBP cells. The fast IPSP reversed
at –71.1 { 7.1 mV (n Å 8); this is more negative than the
reversal potential of the early phase of the response to GABA
application ( 065 mV; recall that the early phase of the response to exogenous GABA lasts ú500 ms and may be a
combination of ¢2 components). The slow IPSP reversed
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FIG . 6. Response of pyramidal cell to GABA and baclofen in the presence of SR314 and saclofen. A: GABA (76 nA,
10 s) applied during control conditions produced a strong hyperpolarization of the cell. After a microdroplet application of
saclofen (2 mM), the response to GABA was diminished (middle). When SR314 also was applied to the pyramidal cell
layer, the GABA response was blocked almost completely. Note the presence of spontaneous slow membrane potential
oscillations during control and saclofen conditions. SR314 enhanced the oscillations causing the cell to fire action potentials
on the depolarizing phase. Action potentials are truncated in the figure. B: Rin measurements derived from the data in A.
Saclofen reduced the Rin change during GABA applications. Under saclofen and SR314 conditions GABA did not change
Rin appreciably. Oscillations caused fluctuations in control Rin that were accentuated during the SR314-induced large amplitude
oscillations. Saclofen alone had no effect on the oscillations. C: under control conditions, 20-s iontophoresis (150 nA) of
baclofen caused a slow hyperpolarization from rest ( – – – ). Prior application of saclofen did not antagonize the baclofen
response (bottom). D: change in Rin during the baclofen responses in A. Baclofen caused a small decrease in Rin ( Ç0.5 MV )
that, if anything, was enhanced by the presence of saclofen.
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N. J. BERMAN AND L. MALER
at –83.9 { 6.5 mV, which is very negative to the GABAA
(antagonist sensitive) hyperpolarization caused by exogenous GABA. The probably mixed origin of the slow IPSP
is discussed later (see GABAA ANTAGONISM ).
In vivo, the probability coding receptors (P units) of highfrequency wave species have high spontaneous firing rates
( Ç200–400 spikes/s) and stimulus-evoked firing rate increases of several hundred spikes per second (Bastian 1981a;
Xu et al.1996). Although single stimulus pulses could evoke
IPSPs in the slice preparation, more physiological tetanic
stimulation (100–200 Hz) was far more reliable in evoking
both fast and slow IPSPs. An example of a NBP cell that
responded well to both single and tetanic stimulation is
shown in Fig. 8. Single-pulse stimulation at rest evoked a
hyperpolarization that appeared to consist of at least two
components, a fast and a slow IPSP (Fig. 8, A and C). The
fast IPSP was strongly inhibitory; it prevented spike firing
during depolarization, albeit for only a few milliseconds. The
slow IPSP was only weakly inhibitory. Tetanic stimulation,
however, produced a summating IPSP that was strongly inhibitory; it prevented firing in the face of strong depolarizing
currents ( °0.6 nA).
The slow IPSPs were variable in duration and effect. Figure 9 shows a NBP that was spontaneously active. Singlepulse stimulation (Fig. 9A) evoked a slow IPSP that was
moderately inhibitory (some spikes ‘‘broke’’ through after
a few hundred milliseconds). Tetanic stimulation (Fig. 9 B)
evoked a large, strong summating IPSP that prevented spiking for °1.5 s. Note the tight clamping of the membrane
potential during the early part of the summating IPSP.
PHARMACOLOGY OF NBP CELL INHIBITION. NBP cells receive inhibitory input from at least two cell populations,
GC1 and GC2, of which the latter are GABAergic (Maler
and Mugnaini 1994). GABAA (bicuculline, picrotoxin, or
FIG . 8. NBP cell response to single-pulse and tetanic PA stimulation during current injection. A: response to single stimulus pulse. At rest ( 062 mV),
a single stimulus evokes a fast (peak at 3 ms) and
slow inhibitory postsynaptic potential (IPSP; peak
at 120 ms). Current injection (0.15-nA increments)
reversed the fast IPSP at about 066 mV. Top 2
traces: (in this and subsequent figures, traces with
spikes will be displaced vertically and spikes truncated for clarity) only the fast IPSP has an appreciable effect on spike firing probability. B: response of
same cell to tetanic (200 Hz, m and shaded area)
stimulation. Summating IPSP during the tetanus and
the slow IPSP after stimulation flatten but do not
fully reverse during the hyperpolarizing current injection. Top: evoked summed IPSP is strongly inhibitory; spikes cannot be evoked during the summed
IPSP. Note the voltage-activated conductances on the
depolarized traces (see Mathieson and Maler 1988).
C: expanded time-base trial shows 2 distinct phases
to the IPSP(s) evoked by single-pulse stimulation.
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FIG . 7. Effect of baclofen on tetanic
stimulus evoked inhibition of a nonbasilar
pyramidal cell. Left: data collected under
control conditions. Right: data collected
after microdroplet of baclofen (50 mM). A:
response of the NBP cell to tetanic stimulation (100 Hz, shaded area) of the DFL during current clamp ( 00.75–0.25 nA, 720
ms) above and below resting Vm ( 062
mV). Under control conditions (left), the
depolarizing current evoked 1 spike before
the tetanic stimulation produced a large inhibitory hyperpolarization (r ). After
baclofen application, the tetanic stimulation
failed to evoke the strong inhibition. Some
inhibition persisted as evidenced by the
pause in firing after cessation of the tetanic
stimulus (top). B: response to the same
tetanic stimulation during injection of
strong depolarizing current (0.7 nA) that
evoked repetitive firing of action potentials.
Effect of the tetanic stimulus on the firing
rate is plotted in C. C: firing rate derived
from data in B. Under control condition, r,
drop in firing rate coinciding with the peak
inhibition seen in A. Firing rate during the
baclofen condition (right) is compared
with the control response replotted (– – –).
PRIMARY AFFERENT-EVOKED INHIBITION
SR) and GABAB (phaclofen and saclofen) antagonists were
applied to the slice to identify the receptor subtypes responsible for the IPSPs evoked by DFL stimulation.
GABAA ANTAGONISM. In all cases, applications of the
GABAA antagonists either induced slow oscillations (Turner
et al. 1991) or increased the oscillation amplitude of spontaneously oscillating cells. The oscillation cycles consist of a
slowly depolarizing ramp, which leads to spike firing; this
then is terminated by a rapid hyperpolarization followed by
the depolarizing ramp of the next cycle (Fig. 6A) (see
Turner et al. 1996 for details). As GABAA antagonists did
not block the rapid hyperpolarization of the oscillation cycle,
it is unlikely to be due to GABAA receptors.
Both bicuculline (n Å 7) and picrotoxin (n Å 1) blocked
the fast IPSPs evoked by single stimuli or tetani. The slow
IPSP was reduced in four of the eight cells by GABAA
antagonists (e.g., Fig. 10, A and C), and the remaining hyperpolarization was unaffected by the GABAB antagonist
saclofen (n Å 2, not shown). Hence a portion of the slow
IPSP is likely to be attributable to non-GABAergic inhibition
from GC1s. Representative examples are shown in Fig. 10.
In one cell, tetanic stimulation produced a compound IPSP
(Fig. 10A, left, r ) that inhibited cell firing (Fig. 10, A, top
2 traces, and B,
). After bicuculline was applied, the
IPSPs were blocked almost completely and the tetanic stimulus caused a much smaller reduction of the firing rate evoked
by current injection. In another NBP cell (Fig. 10C), bicuculline substantially reduced the size of the IPSPs, but there
remained a significant slow IPSP that resisted further attempts at antagonism, either with additional applications of
bicuculline or with saclofen (data not shown). The reversal
potential of this slow IPSP shifted from 078 to less than
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085 mV after bicuculline, suggesting it was K / -mediated.
This explains the generally very negative reversal potentials
of the drug-naive slow IPSP: it is a mix of Cl 0-mediated
responses (GABAA : reverses at 062 with exogenous GABA
and 070 mV for the fast IPSP) and K / -mediated (unknown
transmitter, likely GC1 in origin) responses.
In some NBP cells (n Å 3), not only were the IPSPs antagonized, but a depolarizing or excitatory component was evoked
by DFL stimulation after bicuculline application. This took the
form of a slow depolarization (Fig. 11; n Å 2) or spikes
activated at very short latency (formed part of the stimulus
artifact, n Å 1). The cell shown in Fig. 11 responded to DFL
stimulation with IPSPs alone. The resting membrane potential
was very negative ( –74 mV), therefore the short-latency IPSP
evoked by a single DFL stimulus was depolarizing (Fig. 11,
inset, control trace). This depolarizing IPSP and the compound
IPSP evoked by tetanic stimulation (Fig. 11A, control) were
abolished completely by bicuculline (Fig. 11B, bicuculline).
Under these conditions, the tetanic stimulus evoked a slowly
depolarizing potential; this potential was clearly voltage sensitive. We attribute these responses to the electrotonic synapses
made by dendrites of GC1 and GC2 cells onto NBP somata
and somatic dendrites (Maler et al. 1981) and the voltagesensitive persistent Na / currents present in these cells (Mathieson and Maler 1988; Turner et al. 1994). The rarity of this
response is presumably also due to the low density of these
connections (Maler 1979) and the difficulty of activating them
in the slice preparation. This interpretation must however still
be experimentally confirmed.1
It has been suggested that gap junction input from granule
cells and disinhibition may be responsible for NBP cell ON
responses (Maler et al. 1981; Shumway and Maler 1989);
however, the response of NBP cells to DFL stimulation when
inhibition was blocked was at best only weakly excitatory
in our experiments. Our data predict that other excitatory
inputs, perhaps descending feedback, must be contributing
to the excitatory responses of NBP cells seen in vivo.
GABAB ANTAGONISM. The effects of GABAB antagonists
(saclofen and phaclofen) were tested on IPSPs evoked in
NBP cells (n Å 7). Only one cell showed any sensitivity to
the antagonists, and even then the effect was marginal (Fig.
11, C and D). The tetanic stimulus produced a large compound IPSP. Several trials are shown to indicate the variability in the response. After 2 mM saclofen application, the
IPSP showed a small reduction in duration. In all other NBP
cells tested, the antagonists failed to significantly alter the
time course or amplitude of IPSPs evoked by tetanic or
single stimuli. We conclude that there is no significant
GABAB component to the NBP cell IPSPs evoked via primary afferent stimulation.
EFFECT OF EXCITATORY ANTAGONISTS ON NBP CELL INHIBITION. The effect of blockade of excitatory-amino-acid
(EAA) transmission was tested on NBP cell tetanus-evoked
1
Note that in his in vivo intracellular study of NBP cells, Bastian (1993)
showed that the cells can be excited moderately by stimulation of commissural avoid cell fibers, presumably via disinhibition of granule cells. This
is consistent with morphological evidence for a projection from ovoid cells
to granule cell interneurons (Maler and Mugnaini 1994). Under conditions
shown to isolate ovoid cell activity, we failed to observe such an excitatory
effect (e.g., Fig. 12). Presumably granule cells are not (or less) spontaneously active in vitro and hence would not be susceptible to disinhibition.
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FIG . 9. Response of NBP cell to single and tetanic stimulation. A: responses to single stimulus (5 trials superimposed). Cell exhibited spontaneous voltage oscillations. Stimulus triggered a slow IPSP (peak at Ç120
ms), and spikes could ‘‘break through’’ the inhibition after Ç300 ms. B:
response of same cell to tetanic stimulation (200 Hz; 7 trials superimposed).
Stimulus evokes a large, rapidly summating IPSP that takes ú1.3 s to fully
recover. Note that the cell is inhibited for longer than in A; spikes do not
occur during the decaying IPSP.
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N. J. BERMAN AND L. MALER
FIG . 11. Effect of GABA antagonists on NBP cell
IPSPs. A and B: GABAA antagonist bicuculline. A: cell
responded to single-pulse stimulation with a reversed (depolarizing) IPSP (see inset, control trace) at rest ( 074
mV). Current injection during control conditions shows
the compound IPSP to reverse a few millivolts above
resting potential (main plot, – – – , control traces on
left). B: application of bicuculline not only completely
abolished the compound IPSP but revealed a slow depolarizing potential. Note that bicuculline blocks the depolarizing IPSP in the single-stimulus pulse experiment (A,
inset). C and D: GABAB antagonist saclofen. Cell displayed spontaneous oscillations. C: tetanic stimulation
(200 Hz, ø ) produced a robust long-duration IPSP (control trials, left). D: after saclofen application, the IPSP
showed a small reduction in peak amplitude and duration.
Approximate resting potential was 070 mV ( – – – ).
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FIG . 10. Effect of GABAA antagonist bicuculline on
NBP cell IPSPs. A: control responses (left) of a NBP
cell to tetanic stimulation of DFL (200 Hz) were obtained during current injection (0.45-, 0.15-, 0-, 0.15-,
0.3-, 0.45-, and 0.6-nA step pulses). – – – , resting
membrane potential ( –69 mV). r, strong compound
IPSP. After bicuculline was applied to the slice surface
and the responses retested (right), the compound IPSP
was abolished except for a small component (0.3 nA
trial, r ). Effect of bicuculline on the capacity of the
compound IPSPs to inhibit cell firing is measured from
the traces obtained during depolarizing current injection
(0.45 and 0.6 nA). Instantaneous firing rates during control and bicuculline conditions are plotted in B. Under
), the cell showed some adaptacontrol conditions (
tion to current injection (slow decline in firing rate over
time). At the onset of the tetanic stimulus ( t Å 0), the
evoked IPSP reduces the firing rate, strongly during 0.45nA injection and weakly during 0.6-nA injection. After
bicuculline application ( – – – ), the tetanic stimulus
caused a much smaller reduction in firing rate (0.45 nA;
compared with current injection alone, not shown). C:
response of NBP to tetanic stimulation before and after
bicuculline application; in this cell there was a substantial
( Ç25%) component of the slow IPSP that was insensitive to bicuculline. Bicuculline caused the reversal potential of the slow IPSP to shift from 078 to less than
085 mV.
PRIMARY AFFERENT-EVOKED INHIBITION
3185
FIG . 12. Effects of excitatory-amino-acid (EAA)
transmission blockade on NBP cell IPSPs. Tetanic
stimulation ( ø ) produced strong long-duration IPSPs
in the recorded cell. Under control conditions (left),
current injection ( –0.75–1.0 nA, 0.25-nA increments) reversed the polarity of the IPSP during the
initial portion of the stimulus; reversal potential just
below rest ( –72 mV, – – – ). After 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) / APV application,
a small inhibitory effect was left during the tetanic
stimulus. This inhibitory effect was only apparent during current injection (1 and 1.25 nA) as a small reduction in spike firing frequency during the tetanic stimulus. CNQX / APV also hyperpolarized the cell by 2
mV and reduced its input resistance; note the smaller
voltage deflections evoked by the current steps.
Electrophysiological and pharmacological characteristics
of synaptically evoked inhibition in BP cells
BP CELL INHIBITION. Electrophysiology. Cells were classified as BP cells if their response to DFL stimulation included
a depolarizing and excitatory potential (EPSP, single stimulus) or a compound EPSP in their response to tetanic stimuli.
As in NBP cells, tetanic stimulation was more effective than
single stimuli at producing the strongest response. A representative range of responses to DFL stimulation is shown in
Fig. 13.
The cell in Fig. 13A (1–3) responded mostly with excitation to both single (with a bimodal EPSP) and tetanic stimulation. The tetanic stimulation was most effective, evoking
several spikes (Fig. 13A3). However, in most cells, IPSPs
also were evoked by single and tetanic stimulation (e.g.,
Fig. 13, B and C). The IPSPs appeared to curtail the summating EPSPs (e.g., Fig. 13B), resulting in a net hyperpolarization. The interaction of EPSPs and IPSPs (clearly separable
in the response to single pulses, Fig. 13C1) could produce
quite complex responses. For instance, in the cell in Fig. 13
C, a short tetanus of five pulses had a net inhibitory effect
(Fig. 13C, 2 and 3) that prevented cell spiking, whereas
tetani with more than five pulses (Fig. 13C4) were strongly
excitatory.
In cells in which a single EPSP could be discerned after
a single DFL stimulus, the first EPSP peaked at 1.2 { 0.8
ms (n Å 50). This was followed by a decaying depolariza-
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tion (27 cells), which in some cells had a clear later EPSP
peak at 18.8 { 22.4 ms (n Å 14) or by a clear early IPSP
after the EPSP (23 cells), similar to the cell in Fig. 13C1.
These early IPSPs peaked at 8.7 { 7.6 ms (n Å 6) and
reversed at 71.7 { 8.4 mV (n Å 8). Slow IPSPs, such as
those in the cells in Fig. 13, B and C, peaked at 197 { 102
ms (n Å 17) and reversed at 088.5 { 0.5.2, with a mean
duration of 813 { 396 ms (n Å 15). Compared with NBP
cells (see Electrophysiological and pharmacological characteristics of synaptically evoked inhibition in NBP cells ),
BP-cell IPSP characteristics (early and late IPSPs) were not
significantly different except for the slow IPSP duration,
which was significantly shorter in NBP cells (NBP cell slow
IPSP duration was 501 { 371 ms, n Å 17, P õ 0.05).
To determine whether the BP-cell IPSPs evoked by DFL
stimulation showed pharmacological sensitivities similar to
NBP cell IPSPs, the effects of GABA antagonists and EAA
antagonists were tested on the BP-cell responses.
Pharmacology. 1) GABAA antagonism. The effects of
GABAA antagonists were tested on the response of 10 BP
cells to DFL stimulation. All of these cells showed varying
degrees of mixed excitatory and inhibitory responses to DFL
stimulation (see above). The example in Fig. 14 shows a
typical effect of bicuculline. This BP cell responded to single-pulse stimulation of DFL with a large EPSP, followed
by a fast IPSP (Fig. 14A), whereas tetanic stimulation (Fig.
14B) evoked a mixed excitatory and inhibitory response.
Bicuculline completely blocked the fast IPSP (Fig. 14A)
and the inhibitory component of the mixed response (Fig.
14B). It is noteworthy that the peak of the EPSP is unaffected by bicuculline, suggesting that the IPSP is delayed
so as to overlap only the slower phase of the EPSP (see
Berman and Maler 1998b for discussion of EPSP/IPSP timing). The small slow depolarizing response that followed
the tetanus at rest (Fig. 14B, left) increased in magnitude
after the bicuculline treatment.
In all 10 cells GABAA antagonists increased the cells’ net
excitatory response to DFL stimulation. The nature of the
effect depended on whether there was clear evidence of fast
and slow IPSPs. Where fast IPSPs followed a DFL-evoked
EPSP (n Å 4), they were blocked completely by GABAA
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IPSPs. Non-NMDA (CNQX) and NMDA receptor antagonists (CPP or APV) were applied to the slice surface. The
NBP cell in Fig. 12 responded to tetanic stimulation of the
DFL with a strong compound IPSP. After CNQX and APV
(1 mM) application, the IPSP mostly was abolished; during
the stimulus period (Fig. 12, right, ø ), there remained a
small inhibitory effect on current-evoked spiking. CNQX,
with or without CPP/APV, reduced (n Å 3) or eliminated
(n Å 2) the IPSPs produced by tetanic stimulation. Hence
the inhibition evoked via primary afferent stimulation is
mostly due to EAA-mediated synaptic activation of granule
cell interneurons.
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antagonists (e.g., Fig. 14A). Long-lasting IPSPs evoked by
single or tetanic stimulation were blocked partially by
GABAA antagonists (4 of 4 cells). Slow EPSPs (e.g., Fig.
14B), present in three BP cells, were augmented by GABAA
antagonism. Two of these cells responded with a fast EPSP
without evidence of a fast IPSP; bicuculline increased the
amplitude of the fast EPSP, suggesting the presence of a
masked IPSP. Hence in BP cells, primary afferent-evoked
EPSPs always will be followed immediately by a fast
GABAA IPSP.
2) GABAB antagonism. Unlike NBP cells, BP cells did
have a saclofen/phaclofen-sensitive component of the slow
IPSP. Figure 15 shows one such cell in which single-pulse
stimulation evoked a EPSP followed by IPSPs (Fig. 15A).
Tetanic stimulation evoked a mixed EPSP/IPSP where the
EPSP had a short-lived effect on cell excitability; the summating IPSP quickly gained control over the cell after only
two pulses of the stimulus (Fig. 15B, note the spike on the
1st pulse in the tetanus). Application of saclofen partially
antagonized the slow component of the IPSP but had no
effect on the early component. Of five BP cells tested, saclo-
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fen or phaclofen completely blocked (n Å 3) or reduced the
amplitude (n Å 2) of the slow IPSP. Two additional BP
cells were exposed to both bicuculline and saclofen; the
effects of the two drugs were additive but failed to fully
block the slow IPSP.
Effect of EAA antagonists on BP-cell IPSPs
Non-NMDA (CNQX) and NMDA (APV or CPP) receptor antagonists were tested on the responses of 16 BP cells
to DFL stimulation. In all 16 cases, the antagonists reduced
or blocked the EPSPs. Where present under control conditions, evoked IPSPs were augmented by the drugs (n Å 6)
or appeared in the response where previously masked or
absent (n Å 4). The two cells featured in Fig. 16 (A and
B) show such an effect. One cell (Fig. 16A) responded with
vigorous excitation during the tetanus that gave way to a
slow IPSP. After CNQX / APV treatment, the excitatory
component was completely abolished, whereas the IPSP was
augmented. There may be two components to the IPSP as
the membrane potential was clamped at around rest during
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FIG . 13. Responses to single and tetanic
stimulation of the DFL in 3 BP cells. Responses ranged from excitatory to mixed excitatory and inhibitory. A: BP cell with mostly
excitatory response. A1: DFL stimulation (single stimulus) evokes EPSP with 2 peaks; at
1 and 4.8 ms. A2: current-clamp experiment
(0.25-nA increments and as indicated) shows
the voltage sensitivity of the EPSPs ( 00.5-nA
trace has the largest amplitude EPSP). A small
slow IPSP appears to be present on the depolarized trace (sag toward end of response to
current step), but it cannot prevent spiking in
the more depolarized trials (0.5 and 0.75 nA).
A3: response of same cell to a 200-Hz tetanic
stimulus ( m ). Several spikes are evoked during the stimulus, whereas a single stimulus
could not generate a spike ( A1). There is no
evidence of IPSPs. B: BP cell responded with
similar EPSPs (see inset, 5-trial average) as
the cell in A to a single stimulus; however, this
cell also showed a slow (400-ms duration)
IPSP after the EPSPs (single, 5-trial average).
Tetanic stimulation (3-trial average) produced
a mixed response with the EPSPs summating
at first but then were overwhelmed by a strong
IPSP(s) that also persisted for 400 ms. C1:
BP cell responding to single stimulus mostly
with an EPSP followed by a fast (peak at 2.5
ms) and slow IPSP (peak at Ç260 ms). In
some trials a spike was evoked by the stimulus
(1 trial shown). C2: tetanic stimulation at 200
Hz but with only 5 stimulus pulses produced
a mixed EPSP/IPSP response, with the inhibitory effect dominating; current clamp (rest,
00.25 and 00.5 nA) showed the reversal of
the IPSPs just below resting potential ( 071
mV), which prevents the EPSPs from reaching
threshold. C3: net effect of the EPSP/IPSP
mix is inhibitory as the tetanic stimulus ( m,
200 Hz, 5 pulses) interrupts a current-evoked
spike train (0.5 nA). C4: tetanic stimulation
of 30 pulses at 200 Hz produces a summating
EPSP that overcomes the inhibition to generate
4 spikes followed by a slow IPSP.
PRIMARY AFFERENT-EVOKED INHIBITION
3187
the tetanic stimulus, then hyperpolarized after the stimulus.
A more depolarized trace revealed the effect of this early
part of the IPSP (Fig. 16A, inset). In the second cell (Fig.
16B), a large component of the mixed response to tetanic
stimulation was inhibitory (r ). The CNQX / APV application blocked the EPSP (see inset) evoked by single stimulus
pulses. After drug treatment, tetanic stimulation evoked a
large IPSP.
In two of the preceding experiments, the GABAB antagonist saclofen reduced the amplitude of the slow IPSP that
had been augmented/unmasked by CNQX / CPP. An example is shown in Fig. 16C where the peak amplitude of the
slow compound IPSP (during exposure to CNQX / CPP)
was reduced by ú30% after application of saclofen.
In summary, EAA antagonism blocks primary afferent
EPSPs but enhances the compound IPSP in BP cells. Given
that EAA antagonism blocked inhibition in NBP cells, it is
likely that the EAA antagonism removed GC1 and GC2
inhibition of BP cells and the enhanced IPSP originates from
ovoid cells, because they will be activated directly by the
DFL stimulating electrode. As the enhanced IPSP is sensitive
to GABAB antagonism, this is further evidence that it originates from a source other than GC1 and GC2 (their inhibition
was insensitive to GABAB antagonism).
GC2 firing patterns may determine slow IPSP time course
in NBP cells
FIG . 15. Effect of saclofen on BP cell IPSPs. A: cell shown responded to a
single stimulus (inset) with a short latency (peak at 1.8 ms) EPSP, followed
by a small slow IPSP. B: tetanic stimulation (200 Hz, 10 pulses) evoked only
1 spike followed by a strong, slow ( ú800 ms long) summating IPSP. Application
of saclofen to the slice surface resulted in a partial antagonism of the slow IPSP
but left the early component of the IPSP unaffected.
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The duration of the GABAA IPSPs evoked by tetanic stimulation was unexpectedly long. Classical GABAA IPSPs are
usually 20–50 ms in duration, whereas the bicuculline-sensitive IPSP after tetanic stimulation could last for hundreds
of milliseconds in NBP (e.g., Fig. 10) and BP cells (not
shown). To determine whether the duration of the IPSP is
due to the receptor-transmitter kinetics, passive postsynaptic
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FIG . 14. Effect of bicuculline on a BP cell
response to DFL stimulation. A: response to a
single DFL stimulus before (control) and after
(bicuculline) bicuculline application to the
slice surface. EPSP is unaffected, whereas the
IPSP is abolished completely by the drug, revealing a slow depolarizing potential. Note that
in the control trace, the IPSP was followed by
a slow depolarizing potential. Stimulus artifact
blanked. B: responses to tetanic (200 Hz, 10
pulses) stimulation under current-clamp conditions (amplitude indicated, depolarized traces
offset for clarity) before (control) and after
(bicuculline) drug application. In the control
case, the stimulus produced a mixed EPSP/
IPSP response that was mostly inhibitory; only
2 spikes occur during the stimulus ( ø ) despite
the strong spiking evoked by the 0.75-nA current. At rest ( 075 mV, – – – ), a slow depolarizing potential is evoked by the tetanic stimulus. After bicuculline, the response to tetanic
stimulation was purely excitatory. Spikes were
evoked during the stimulus in all depolarized
traces. A pronounced depolarizing potential
followed the stimulus.
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N. J. BERMAN AND L. MALER
membrane properties (see appendix), or sustained firing of
the presynaptic cell (most likely the GABAergic GC2), we
recorded extracellular unit activity (10 units) in the GCL
where GC2s are in the majority (Maler and Mugnaini 1994).
The results from three experiments are presented in Fig. 17.
First, the response of a typical NBP cell is shown in Fig.
17A. The IPSP summated during the tetanus and then slowly
returned to baseline after 400 ms. The duration of the IPSP
was not just due to the time constant of the cell because
measurements of cell input resistance (using the voltage
deflections caused by 30 ms, 00.2-nA current pulses, Fig.
17B) showed that the input resistance of the cell dropped
to 75% of normal at the peak of the IPSP and then returned
to baseline with a time course similar to the IPSP in Fig.
17A. Recordings of granule cell unit activity are shown in
Fig. 17, C–E. The units responded with bursts of sustained
activity lasting from 50 to 800 ms. The responses were
highly variable (e.g., Fig. 17, D and E) between units and
from trial to trial (Fig. 17E, cell 8, 3 trials shown). Some
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units (e.g., Fig. 17, E and D, cell 3) had a high rate of
spontaneous firing. Thus the kinetics of unit response in the
GCL may account for the long and variable time course of
the IPSP evoked by tetanic stimulation of NBP cells.
DISCUSSION
The aim of this study was to characterize the inhibition
evoked in ELL pyramidal cells by stimulation of primary
afferents. The source of the various IPSPs then could be
assigned to the inhibitory circuitry previously identified by
our laboratory (Maler and Mugnaini 1994). An identification strategy was developed by correlating the responses of
recorded cells to DFL stimulation with their morphological
type (NBP or BP) as determined by intracellular staining.
NBP cells responded with IPSPs only, whereas BP cells
responded with EPSPs and IPSPs. This agrees with the presence or absence (NBP cells) of direct primary afferent glutamatergic excitatory input (Maler 1979; Wang and Maler
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FIG . 16. Effect of EAA antagonists on BP cells’ responses to DFL stimulation. A: under control conditions,
this BP cell responded to 200-Hz (20 pulse) stimulation
with a summating EPSP that crossed threshold for spiking
only after 17 pulses (4-trial average), this was followed
by a slow IPSP. After CNQX / APV treatment, the depolarizing response was abolished completely, and the amplitude of the slow IPSP was increased (6-trial average,
spikes truncated by averaging). Response to tetanic stimulation actually contained a ‘‘silent’’ IPSP that was obvious in some trials in which the cell was somewhat depolarized (1 trial shown in inset); stimulus brings the potential down to rest ( – – – ). B: under control conditions,
this BP cell responded to a single stimulus pulse with an
EPSP followed by a IPSP (inset, 8-trial average). Tetanic
stimulation ( ø ) produced a predominantly inhibitory response that also consisted of a slow IPSP after the tetanus
(6-trial average). Treatment with CNQX / APV antagonized the early EPSP (see inset) and augmented the inhibitory response to the stimulus (6-trial average). C: BP
cell responded to single pulse DFL stimulus with and
EPSP and IPSP (inset). EPSP was blocked completely
by CNQX / CPP. With EAA transmission blocked
(CNQX / CPP, main plot) tetanic stimulation evoked a
typical slow IPSP. This slow IPSP was partially blocked
by the GABAB antagonist saclofen.
PRIMARY AFFERENT-EVOKED INHIBITION
3189
1994) via the basal dendritic bush (BP cells) and the in vivo
classification of BP and NBP cells as E and I cells (excited
vs. inhibited by increased electrosensory input) (Bastian
1981b; Saunders and Bastian 1984).
The discussion focuses on 1) the similarity in ELL pyramidal cell responses to GABA with mammalian pyramidal
cells, 2) the strong voltage dependence of GABA responses
and its implications, 3) evidence for GABAA and GABAB
receptors on BP and NBP cells and the interneurons associated with these receptor subtypes, and 4) functional implications of the parsed inhibitory circuitry for first stage electrosensory processing in the ELL.
Effect of GABA on pyramidal cells
Consistent with mammalian pyramidal cells, the response
of ELL pyramidal cells to exogenous GABA consisted of
more than one phase. The early component was usually depolarizing and reversed at about –60 mV. The later component, which outlasted the GABA iontophoresis pulse, was
hyperpolarizing and reversed at very negative potentials
( ú100 mV). The early component was blocked by GABAA
antagonists, and the late hyperpolarizing component was
sensitive to GABAB antagonists. The latter effect varied from
cell to cell and between applications. This is perhaps not
surprising given the low potency and variable efficacy of
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GABAB antagonists in the mammalian CNS (Baumfalk and
Albus 1987) and the possibility of pharmacologically distinct subsets of GABAB receptors (Matthews et al. 1994;
Scherer et al. 1988). Thus the reversal potential, time course,
and pharmacology of the response of pyramidal cells to
GABA strongly suggest that both BP and NBP cells express
classic fast GABAA-receptor-gated (Cl 0 ) channels and slow
GABAB-receptor-gated (K / ) channels.
The hyper- versus depolarizing nature of the early GABA
response and the presence of two phases with slightly different reversal potentials may be due to action of GABA on
somatic versus dendritic GABAA receptors. In our experiments, the iontophoretic pipette was placed in the PCL,
hence GABA applications would reach the soma, the somatic
dendrites, and the proximal apical and basal (in BP cells)
dendritic trunks. There is much evidence that dendritic applications of GABA result in depolarizing potentials while applications that recruit somatic GABAA receptors result in
mixed or purely hyperpolarizing potentials due to conductance increases to Cl – (Alger and Nicholl 1982; Andersen
et al. 1980; Connors et al. 1988). There is no consensus on
the ionic mechanisms mediating the dendritic response (see
Alger and Nicholl 1982; Djørup et al. 1981; Smith et al.
1995; Thompson et al. 1988; Wong and Watkins 1982) although there are suggestions it involves some ion in addition
to Cl – (Thompson et al. 1988) and perhaps extrasynaptic
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FIG . 17. Time course of NBP IPSP and unit activity in the granule cell layer evoked by tetanic stimulation (100 Hz, 100
ms, ø ) of DFL. A: response of NBP cell to stimulation of the DFL produces a compound, long-lasting (400 ms) IPSP. B:
input resistance (Rin ) of the same cell monitored (30 ms, 00.2 nA current pulses) under the same conditions in A (4 trials).
C: single trial showing a granule layer unit response to stimulation of DFL. Positive-going stimulus artifacts removed in all
traces. D: histograms showing the average firing rate for 3 such units. Stimulation artifacts prevented reliable monitoring of
spikes during stimulation. E: single trial records from 2 of the cells in D showing the trial-by-trial variability in the responses.
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N. J. BERMAN AND L. MALER
There are, however, striking differences between the slow
IPSP in these two cell types: slow IPSPs in BP cells were
significantly longer than those in NBP cells (mean of 813
vs. 501 ms), only the BP slow IPSP was sensitive to GABAB
antagonists, and the slow IPSP of BPs was enhanced by
blockade of excitatory transmission, whereas NBP IPSPs
were blocked under the same conditions. As discussed later,
we attribute the slow IPSP of NBPs to activation of GC1
interneurons; the transmitter/receptor involved is unknown,
but the channel is likely to be K / selective (due to its relatively hyperpolarized reversal potential). We propose that
the slow GABAB IPSP in BP cells is mainly due to the
GABAergic (Maler and Mugnaini 1994) ovoid cell fibers
that densely invest the basal dendrite of this cell type and
can be stimulated directly from the DFL.
Voltage dependence of the GABAA response
NBP CELLS. Nonbasilar pyramids receive input from GC1
and GC2 cells, but not from ovoid cells (Bastian et al. 1993;
Maler 1979; Maler and Mugnaini 1994). As the type 2 granular cell is GABAergic, primary afferent input therefore can
cause GABAergic inhibition only via the type 2 interneuron.
Although the transmitter used by GC1 cells is unknown,
morphological studies suggest that it is an inhibitory interneuron (Maler et al. 1981). Single-pulse electrical stimulation of primary afferents always evoked fast GABAA IPSPs
in NBPs and often evoked slow long-lasting IPSPs as well,
consistent with this interpretation. Tetanic stimulation with
frequencies similar to those of probability coders in vivo
(Bastian 1981a; Hopkins 1976) generated prominent longlasting IPSPs that were very effective in reducing currentevoked spiking. The EAA antagonist block of DFL-evoked
IPSPs confirmed that primary afferents can only affect NBPs
via GC1 and GC2 cells and that the gap junction input to
these interneurons (from primary afferents) (Maler et al.
1981) does not strongly activate them.
Based on the GABAA antagonist block of the fast IPSPs
and its reversal potential of 071 mV, we conclude that it
originates from GC2 cells activating GABAA receptors on
NBP somata and somatic dendrites. The slow IPSP, which
was reduced by GABAA but not GABAB antagonists and
had a more negative reversal potential ( 084 mV), is likely
to be due to prolonged activation (see Slow IPSP kinetics)
of the same GC2 input and input from non-GABAergic GC1
cells. The intermediate reversal potential of the slow IPSP
is simply due to combined activation of GABAA channels
( 062 to 070 mV: exogenous GABA and fast IPSP) and the
K / channels putatively activated by GC1 cell input; the true
reversal potential of the GC1 cell contribution to the IPSP
is thus presumably even more negative than 084 mV. GC1
cells are less numerous than GC2 cells (Maler and Mugnaini
1994) and have sparse long-range projections to pyramidal
cells and may not always be activated by our focal stimulation in DFL. This explains why the non-GABAergic component of NBP cell IPSPs was small and variable.
The question then remains: what drives the NBP GABAB
receptors responsible for the GABAB-like responses evoked
by exogenous GABA? Our companion study (Berman and
Maler 1998a) suggests that it is the descending bipolar cell
inhibitory pathway (Maler and Mugnaini 1994) as it produces GABAB-receptor-like inhibition in NBP and BP cells.
Slow IPSP kinetics. The duration of the GABAA compo-
The conductance changes due to GABA applications were
larger when the neuron was depolarized and smaller when
the neuron was hyperpolarized. This is consistent with the
voltage sensitivity of ligand-gated chloride channels
(GABA: Segal and Barker 1984; glycine: Faber and Korn
1987; Legendre and Korn 1995). GABA-activated chloride
channels increase their conductance (Segal and Barker
1984) and decrease their rate of desensitization (Yoon 1994)
with depolarization. This net increase in GABAA-receptormediated inhibition with depolarization may have important
consequences for sensory processing in the ELL.
GABAA-mediated IPSPs usually are described as
‘‘shunting’’ inhibition (Andersen et al. 1980) because the
opening of these Cl 0-permeable channels results in large
decreases in input resistance with relatively small voltage
changes (due to the close proximity of Cl 0 equilibrium potential and resting membrane potential). However, the voltage sensitivity of these channels implies that the shunting
effect is nonlinear. For example, the voltage sensitivity of
GABAA channels might provide for a simple form of gain
control or normalization of primary afferent input: stronger
input will produce greater depolarization of BP cells and
thus automatically make GABAA (GC2 cell) inhibition more
effective by augmenting the associated conductance increase. This simple view does not take into account the
possible interactions of GABAA input with that of the more
hyperpolarizing K / -mediated IPSPs (GC1 and ovoid cells)
that may attenuate the conductance changes due to GC2
IPSPs, the autoinhibition of GABA synapses via GABAB
receptors and the complex kinetics of granule cell firing.
Although detailed modeling studies are needed, it is clear
that GABAA-receptor-mediated inhibition by GC2 cells
might mediate more complex computations than previously
anticipated.
GABAA and GABAB inhibition of BP and NBP cells
Electrical stimulation of the primary afferents in the DFL
produced fast GABAA-type IPSPs (bicuculline/SR-sensitive
and reversed close to rest) in both BP and NBP cells; as
discussed later, we attribute these IPSPs to activation of the
GC2 interneuron.
Both BP and NBP cells also responded to DFL stimulation
with a slow IPSP with a very negative reversal potential.
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receptors that are not usually activated by physiological levels of synaptic GABA release (Wong and Watkins 1982).
The late component of the GABA response and the hyperpolarization induced by baclofen likely both were mediated
by K / -linked GABAB receptors (see Bormann 1988); they
were both hyperpolarizing at rest and were associated with
more modest conductance changes than the early GABA
response (but see Ogata et al. 1987). It should be emphasized that exogenous GABA will activate a mix of somatic
and dendritic GABAA and GABAB receptors and that the
final response will be a complex mix determined by the
receptors’ activation and desensitization time constants (Otis
and Mody 1992; Tia et al. 1996; Wong and Watkins 1982)
and affinity for GABA (Sodickson and Bean 1996).
PRIMARY AFFERENT-EVOKED INHIBITION
3191
nent of the NBP slow IPSP was too long ( ú1 s) to be
accounted for by typical GABAA receptor kinetics (Berman
et al. 1992; Mody et al. 1994; Pearce 1993) or by the electrotonic properties of the thin somatic dendrites of NBPs: conductance changes tracked the IPSP time course, suggesting
that the GABAA receptors remained active throughout the
IPSP, and modeling the effect of IPSPs on pyramidal cells
demonstrated that IPSPs generated on distal thin somatic
dendrites were only slightly prolonged compared with those
on the soma (see APPENDI X ). Unit recording of granule cells
suggested that sustained firing of throughout the IPSP period
accounts for the long time course of this slow IPSP in NBP
cells. The response of granular interneurons was highly variable; we do not know whether this is due to slice conditions
or to intrinsic properties of these interneurons (see Phasic
properties).
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FIG . 18. Current view of GABAergic circuitry activated by electroreceptor input (this study; Maler 1979; Maler and Mugnaini 1994; Maler et
al. 1981). BP and NBP cells receive inhibitory input from the GABAergic
GC2 via GABAA receptors. Strength of this input (indicated by line thickness) is substantially greater than the diffuse, sparse, possibly K / -mediated
inhibitory input from non-GABAergic GC1. GABAergic ovoid cells provide strong GABAB ergic to the basilar dendrite of BP cells. This input also
may involve a smaller GABAA component. Electrotonic, inhibitory, and
excitatory synaptic connections are indicated in the key. GC1 and GC2
cells both provide sparse, diffuse electrotonic dendro-dendritic input to BP
somatic dendrites via an ascending dendrite (omitted for clarity) (see Maler
1979; Maler et al. 1981).
strong GABAB-mediated input, and this is due to fibers from
GABAergic ovoid cells, which climb up the basal dendrite
of these cells. This input also may involve a GABAA component.
Functional interpretations
Our current understanding of the role of inhibition in generating ELL pyramidal-cell receptive fields and temporal
response characteristics (Maler and Mugnaini 1994; Shumway and Maler 1989) has been based on ELL circuitry
(Maler 1979; Maler and Mugnaini 1994; Shumway and
Maler 1989), GABA immunohistochemistry (Maler and
Mugnaini 1994), in vivo physiology (Bastian 1981b; Bastian and Courtright 1991), and pharmacology (Bastian 1993;
Shumway and Maler 1989).
GC2 CELLS CONTROL GAIN AND TEMPORAL RESPONSES TO
ELECTROSENSORY INPUT. Blockade of feedback input to the
ELL (Bastian 1986b,c) or application of bicuculline to the
PCL (Bastian 1993; Shumway and Maler 1989), increased
the responsiveness of pyramidal cells and made them more
tonic in their response to electrosensory input. Because the
GC2 is the only GABAergic interneuron type that receives
both primary afferent and feedback input, it was hypothesized that this cell was primarily responsible for gain and
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Unlike NBP cells, BP cells, in addition to the
input from GC1 and GC2, also receive an input from GABAergic ovoid cells that invests their basal dendrite (Maler
and Mugnaini 1994). BP cells responded to DFL stimulation
with a complex mixture of EPSPs and IPSPs that complicates
the interpretation of the response during tetanic stimulation.
The response to single stimuli and tetani demonstrates that
primary afferent stimulation evokes both fast and slow
IPSPs. The fast IPSP has the same reversal potential and
time to peak as the fast IPSP seen in NBP cells and was
blocked completely by GABAA antagonists. We therefore
propose that the fast IPSP of BP cells is also due to GC2
cell activation of postsynaptic GABAA receptors. The late
phase of the IPSP has a reversal potential of about 088 mV
and, unlike the case for NBP cells, was antagonized partially
by saclofen. Further, applications of EAA antagonists enhanced the slow IPSP, which then could be reduced by saclofen. In the presence of EAA antagonists, granular interneurons are not activated (see preceding section for NBP cells)
but ovoid cell axons, which run in the DFL (Maler and
Mugnaini 1994), still can be stimulated. Therefore this
GABAB component is likely to be due to activation of ovoid
cells.
Thus we propose that the IPSP that can be evoked in the
absence of EAA transmission probably is entirely due to
ovoid cell input to BP cells; the increase in the evoked IPSP
(during EAA antagonism) can be attributed to the fact that,
in the absence of GABAA (Cl 0-mediated) inhibition and
EAA excitation, the reversal potential of the IPSP moves
in the negative direction toward that of the GABAB (K / mediated) channel. Tetanic stimulation of commissural fibers (ovoid cell axons) in vivo evokes a similar slow hyperpolarizing IPSP in BP cells (Bastian et al. 1993). It is not
clear whether the IPSP due to ovoid cell input to BP cells
has a GABAA as well as a GABAB component. The fact that
saclofen could never completely antagonize the DFL-evoked
response in the presence of EAA antagonists suggests this
to be the case. Our current view of the GABAergic circuitry
associated with electroreceptor input is summarized in Fig.
18 the major granule cell inhibitory input to BP and NBP
cells is from the GABAergic GC2 and is mediated by
GABAA receptors, there is a lesser input to pyramidal cells
from the non-GABAergic GC1 that probably involves a
K / –mediated hyperpolarization, and BP cells receive a
BP CELLS.
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and more precise recent studies have supported this idea
(Nelson et al. 1997).
The same manipulations (blockade
of feedback input or bicuculline in the pyramidal cell layer)
that caused pyramidal cells to become more tonic also caused
their inhibitory surrounds to increase in strength and size
(Bastian 1986b, 1993; Shumway and Maler 1989). This
implies that surround inhibition in the ELL is mediated by
either GABAB receptors or by a non-GABAergic mechanism. Further, because bicuculline actually increased surround inhibition, it was likely that the interneuron responsible received GABAergic input via GABAA receptors. The
GC1 cell is not GABAergic (Maler and Mugnaini 1994),
although the morphology of its contacts onto pyramidal cells
have the characteristics of inhibitory synapses (Maler et al.
1981). Because GC1 itself receives GABAergic input (from
polymorphic cells) we hypothesized (Maler et al. 1981;
Shumway and Maler 1989) that it was primarily responsible
for surround inhibition in the ELL: bicuculline would block
inhibition of GC1 cells, thus increasing their activity, while
blocking feedback input to polymorphic cells (these interneurons have extensive apical dendrites in receipt of feedback input) (Maler 1979; Maler et al. 1981) also would
reduce inhibition of GC1 interneurons; in either case, increased activity of GC1 would cause increased surround
inhibition.
Our results are also consistent with this hypothesis. NBP
cells receive input from primary afferents only indirectly
via GC1 and GC2 cells (Maler 1979). After application of
GABAA antagonists, stimulation of primary afferents still
evoked a saclofen-insensitive hyperpolarizing potential in
five of nine NBP cells. We hypothesize that this potential
was an IPSP generated by activation of GC1 cells. This
response is not always seen, perhaps because these cells are
relatively rare (1 GC1:3.7 GC2 cells) (Maler and Mugnaini
1994) and therefore may be difficult to stimulate.
SURROUND INHIBITION.
BP
cells can be inhibited disynaptically by primary afferent input via ovoid cells as well as by GC1 and GC2 interneurons.
For these cells, GABAA antagonists block part of the evoked
IPSP; we hypothesize this is, as in NBP cells, due to GC2.
GABAB antagonists block a slow component; we attribute
this to ovoid cell activation. The remaining rather variable
component of the IPSP is presumably due to activation of
GC1. The ovoid cell is believed to mediate common mode
rejection in the electrosensory system (Bastian et al. 1993;
Maler and Mugnaini 1994). Both the rapid synaptic transmission and fast conduction velocity of the ovoid cell (Bastian et al. 1993) were proposed to adapt it to this role. It
was therefore surprising to find that ovoid cell synaptic input
to basilar pyramidal cells is likely to be mediated by both
GABAA and GABAB receptors because the GABAB component has a slow time course. Consistent with our in vitro
data, commissural stimulation of ovoid cell fibers in vivo
produces a slow IPSP in pyramidal cells (Bastian 1993).
Further, this slow IPSP is effective in quenching weak electrosensory stimuli, but strong stimuli are unaffected (Bastian
1993), suggesting that the input may act via conditional
inhibition (see Berman and Maler 1998b). Bastian et al.
(1993) also reported that ovoid cells had a high level of
OVOID CELLS: COMMON MODE AND NOISE REJECTION?
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temporal properties of pyramidal cell responses: bicuculline
would block directly GC2 synaptic input to pyramidal cells,
while blocking feedback input would eliminate a major excitatory input to these interneurons and thus reduce their
activity. The results of this study are entirely consistent with
this interpretation.
Gain control. We have demonstrated that the synapses
made by GC2 use only GABAA receptors; thus the in vivo
application of bicuculline to the PCL will block the effects of
GC2 cell activity. The fast IPSPs generated by GC2 overlap
primary afferent input to basilar pyramidal cells and determine whether it will drive them to spike threshold; this presumably underlies the putative ability of the GC2 to control
the gain of the pyramidal cell response.
Temporal control: phasic properties. The inhibition due
to the GC2 cell is also very potent in terminating pyramidal
cell depolarization; this is consistent with its presumed role
in regulating pyramidal cell temporal response properties;
GC2 GABAA-mediated inhibition is likely to be a major
contributor to the phasicness of the pyramidal cell response
to sustained electrosensory input.
In the ELL slice, the GABAA-summed IPSP evoked by
primary afferent tetanic stimulation often outlasts the stimulus tetanus for ú500 ms due to the burst firing of granular
interneurons. Although it is possible that this burst firing is
an artifact of our in vitro preparation, it is noteworthy that
Tharani et al. (1996) have suggested that ELL granular neurons express N-type Ca 2/ channels and that these might
regulate their oscillatory and burst activity (Turner et al.
1991, 1996; R. W. Turner, personal communication). It is
therefore possible that feedback (Bastian 1986b,c) and intrinsic ELL circuitry (Maler and Mugnaini 1994) regulate
temporal properties of pyramidal cells in part via the voltagedependent Ca 2/ channels of GC2 cells. This may be especially important with regard to the different temporal properties of pyramidal cells in the three ELL tuberous segments
because Shumway (1989a) has shown that, although cells
in the centromedial segment are low-pass filters (consistent
with the long summed GABAA IPSPs we see in this segment), those in the lateral segment respond optimally to
high-frequency input (see also Metzner and Heiligenberg
1992). It may be instructive to investigate the time course
of DFL-evoked pyramidal cell inhibition in the lateral segment and the effect of local (GCL) application of N-channel
antagonists on this inhibition.
Temporal control: coincidence detection. The fast time
course of the primary-afferent-evoked IPSP may be important for coincidence detection. The primary afferent EPSP
is rapid and appears to be shortened considerably by the
GABAA IPSP (e.g., Fig. 15). As the EOD discharge frequency is extremely high in these fish ( °1,000 Hz), the
rapid repolarization of the EPSP by GABAA IPSP may be
essential to allow the cell to sample the environment at EOD
rates. The fast kinetics of the primary afferent EPSP/IPSP
favor the decoding of input spike patterns via coincidence
detection (Gabbiani et al. 1994; Softky and Koch 1993;
Traub et al. 1993) rather than via long-term temporal integration. Bastian (1981b) has already suggested, based on behavioral and electrophysiological data, that the detection of
weak electrosensory input requires coincidence detection,
PRIMARY AFFERENT-EVOKED INHIBITION
APPENDIX
Computer simulations of the effect of synaptic input
location on IPSP duration
In the experiments above, activation of primary afferents produced long ( ú300 ms) IPSPs in pyramidal cells; these IPSPs
were blocked by bicuculline/SR and therefore were attributed to
GABAA receptors. Based on the firing patterns of GABAergic
interneurons, we hypothesized that the duration of the IPSP most
likely was caused by sustained presynaptic firing and hence summation of many successive IPSPs rather than by some postsynaptic
process that produced a long IPSP from a single volley of presynaptic firing. One alternative hypothesis was that GABAergic inputs
on the thin ( õ1-mm diameter) dendritic processes around the soma
(Maler and Mugnaini 1994) would produce IPSPs that would be
smeared temporally by the cable properties of these dendrites (Jack
and Redman 1971; Jack et al. 1975) and therefore produce a prolonged IPSP when measured at the soma. To test the feasibility of
this explanation, we used a passive compartmental model of a
basilar pyramidal cell to simulate the effect these fine dendritic
diameters would have on IPSP time course.
Compartmental model
A simple passive 35 compartmental model neuron (Fig. A1)
was constructed based on measurements of a biocytin-filled ELL
basilar pyramidal cell (R. Turner, personal communication) and
representative measurements of the fine somatic dendrite processes
of the basilar pyramidal cell shown in Fig. A1. Membrane capacitance was 0.75 mF.cm – 2 and resistance 150 kV.cm – 2 throughout,
cytoplasmic resistance 250 V.cm. A passive leak current conductance (reversal potential –70 mV) was set to 0.05 mS.cm – 2 so as
to produce an input resistance of Ç20 MV when measured from
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the soma with hyperpolarizing current. Inhibitory synaptic conductances (7.6 mS.cm – 2 , reversal potential –70 mV) were modeled
as an alpha function (Jack et al. 1975) and were placed on the
soma, somatic dendrites, and proximal apical and basal dendrites.
These regions receive substantial GABAergic input (Maler and
Mugnaini 1994). GABAB inhibitory conductances were not specifically included in the model. The estimate of the conductance
load was derived from counts of GABAergic boutons on pyramidal
cells that were cut en face (Maler and Mugnaini 1994) and estimates of GABA receptor synapse conductance (Douglas and Martin 1990). The accuracy of the conductance parameter is not critical
because the aim of the simulations was to assess the factors affecting the duration of the IPSP. The somatic dendrite trunks were
difficult to measure accurately (LY-filled cells, see Fig. 1) because
of their thin diameters and varicosities, therefore two diameter
measures (1 and 2.5 mm) were used to span the average range of
thicknesses observed. A terminating compartment 15 mm long and
1 mm thick (referred to as somatic dendrite tip) was used to simulate inputs terminating on the ends of the somatic dendrites.
The model was implemented on Nodus (version 3.2.2), a Macintosh-based compartmental model simulator (De Schutter 1989; url
http://bbf-www.uia.ac.be/).
Simulations were performed to explore the effects that
the somatic dendrites’ fine diameters would have on IPSP duration.
To test this, two parameters were varied, the location of the input
and the thickness of the somatic dendrites. The performance of the
compartmental model (Fig. A1A) to a simple alpha function IPSP
is shown in Fig. A1B. The model cell was depolarized (0.5 nA to
the soma) to move the membrane potential away from IPSP reversal ( –70 mV). With synaptic inputs on the somatic dendrites activated, the IPSP amplitude scaled with dendrite diameter in part
because the GABA conductance was based on membrane area.
The duration of the IPSP was little changed. The different dendrite
diameters had little effect on IPSP amplitude when the soma and
proximal regions of the apical and basal dendrites were activated.
Combined activation of all these compartments, which may occur
in our slice experiments, caused the longest IPSP (up to Ç30 ms).
Varying the site of inhibition had little effect on IPSP time
course measured at the soma (Fig. A2A). Comparisons of the IPSP
profile at the soma with the conductance changes applied to the
dendritic compartments (Fig. A2A, compare top with bottom row)
showed that the soma voltage change closely tracked the synaptic
conductance change. There was little evidence that the IPSP voltage change outlasted the conductance change. Tetanic activation
of synapses (Fig. A2B) produced a summating IPSP that lasted
up to Ç60 ms after the onset of the last pulse in the stimulus;
however, this was due to the summating conductance increases
that also took that long to decay (not shown). IPSP time to peaks
of 2–5 ms were used as these approximate the range measured
electrophysiologically (single-pulse fast IPSPs, this study).
RESULTS.
DISCUSSION. The thin diameter and profusion of pyramidal cell
somatic dendrites seen in the filled cells raises the question as to
their function. The inputs to the soma and the somatic dendrites
arise from the same interneurons (Maler 1979; Maler et al. 1981);
morphological (Maler et al. 1981) and electrophysiological evidence suggests that this input is strictly inhibitory. The model
simulations show that inhibitory inputs on the fine somatic dendrites are effective in generating IPSPs at the soma, the thinness
of the dendrites do not greatly alter the time course of IPSPs
measured at the soma, and the IPSPs generated at the soma reach
the Cl 0 reversal potential and the addition of somatic dendritic
IPSPs produces little additional hyperpolarization or conductance
increase at the soma.
The degree of time course slowing due to membrane properties
( Jack et al. 1975 ) is insufficient to account for the electrophysiological data on GABAA ergic IPSP durations. Thus we hypothe-
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resting activity (73 spikes/s); hence there is tonic GABAB
inhibition of the BP basal dendrite. This points to a role for
ovoid cell input in reducing noise in addition to its role in
common mode rejection.
The complex mode of transmission at ovoid-basal dendrite
synapses (see discussion on voltage dependence of GABAA
transmission) (voltage dependence of GABAB transmission:
Berman and Maler 1998a; conditional inhibition: Berman
and Maler 1998b) suggests that our understanding of ovoid
cell function is incomplete. The presence of a large saclofensensitive component in ovoid-basilar pyramidal cell IPSPs
may provide a pharmacological tool for delineating the function of the ovoid cell in vivo.
In summary, our in vitro results are, on the whole, remarkably consistent with the interpretation of ELL pyramidalcell receptive fields based on in vivo physiology and circuit
analysis; fast GC2 inhibition is consistent with its role in
gain control and shaping of temporal responses. However,
two of our results were unexpected: granular interneurons
respond to electroreceptor input with very variable spike
bursts and ovoid cell-BP-cell IPSPs probably mediated by
both GABAA and GABAB receptors. These findings suggest
that GABA inhibition at this level of sensory processing
may be regulated more dynamically than previously thought.
Furthermore, the strong voltage dependency of GABAA-receptor-mediated inhibition, which is a common feature of
mammalian GABAA Cl 0 conductances as well, must be included in models if we are to fully understand the complex
nature of GABAergic inhibition.
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FIG . A2. Temporal characteristics of somatic dendrite inhibition. A: single IPSP activations (t Å 0 ms, Alpha function time to
peak Å 2 ms). Active compartments indicated
(see Fig. 1.). Top: soma voltage plots; bottom:
soma and somatic dendrite (trunk only) synaptic conductances. Note that the IPSP voltage
change tracks closely the conductance change.
There is little evidence of the IPSP voltage
change outlasting the conductance change (somatic dendrite trunks Å 2.5 mm diam). Steady
depolarizing current, 0.5 nA. B: tetanic IPSP
activation (10 pulses, 200 Hz). Tetanus activation produced a summating compound IPSP.
Smoothness depended on the time to peak of
the IPSP alpha function (2 and 5 ms tested).
Various traces are from simulation runs in
which the somatic dendrite tips alone or with
somatic dendrite trunks or all synaptic conductances were activated (see Fig. A1, A).
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FIG . A1. Compartmental model of ELL
basilar pyramidal cell. A: diagram of model
compartmental dimensions. Leak and GABA
conductances were applied as mS.cm02 of
membrane surface area. Calibration bars: vertical, compartment lengths; horizontal, compartment diameters. B: model response to
simulated inhibitory synaptic inputs. Single
IPSPs (Alpha function time to peak: 2 ms)
at t Å 0. Simulated current injection (begins
at t Å –29 ms) of 0.5 nA to depolarize cell
to 060 mV to enable the IPSP (reversal potential Å resting potential Å –70 mV) to produce a voltage change. Simulations were run
with 2 models having different somatic dendrite trunk diameters (1 and 2.5 mm). Left:
graphics indicate compartments with synaptic
inputs that were activated (blank: not activated; shaded: activated). Varying somatic
dendrite trunk diameter affected mainly the
amplitude of the IPSP but not the duration
(top). Alone, somatic dendrite inhibition produces a sizable IPSP. When added to somatic
and proximal dendrite inhibition, the somatic
dendrite inhibition causes a small increment
in the IPSP amplitude.
PRIMARY AFFERENT-EVOKED INHIBITION
We thank W. Ellis for excellent technical assistance and A. Vaillant for
operating the scanning confocal microscope.
L. Maler and N. J. Berman were supported by the Medical Research
Council of Canada.
Address for reprint requests: N. J. Berman, Dept. of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, 451 Smyth Rd.,
Ottawa, Ontario K1H 8M5, Canada.
Received 4 February 1998; accepted in final form 27 August 1998.
REFERENCES
ADAMS, J. C. Heavy metal intensification of DAB-based HRP product reaction product. J. Histochem. Cytochem. 29: 775, 1981.
ALGER, B. E. AND NICHOLL, R. A. GABA-mediated biphasic inhibitory response in the hippocampus. Nature 281: 315–317, 1979.
ALGER, B. E. AND NICHOLL, R. A. Pharmacological evidence for two kinds
of GABA receptor on rat hippocampal pyramidal cells studied in vitro.
J. Physiol. (Lond.) 328: 125–141, 1982.
ANDERSEN, P., DINGLEDINE, R., GJERSTAD, L., LANGMOEN, I. A., AND
LAURSEN, A. M. Two different responses of hippocampal pyramidal cells
to application of gamma-amino butyric acid. J. Physiol. (Lond.) 305:
279–296, 1980.
BASS, A. H. Electric organs revisited: evolution of a vertebrate communication and orientation organ. In: Electroreception, edited by T. H. Bullock,
and W. Heiligenberg. New York: Wiley, 1986, p. 13–70.
BASTIAN, J. Electrolocation. I. How the electroreceptors of Apteronotus
albifrons code for moving objects and other electrical stimuli. J. Comp.
Physiol. [A] 144: 465–479, 1981a.
BASTIAN, J. Electrolocation. II. The effects of moving objects and other
electrical stimuli on the activities of two categories of posterior lateral
line cells in Apteronotus albifrons. J. Comp. Physiol. [A] 144: 481–494,
1981b.
BASTIAN, J. Electrolocation: Behaviour, anatomy and physiology. In: Electroreception, edited by T. H. Bullock, and W. Heiligenberg. New York:
Wiley, 1986a, p. 577–612.
BASTIAN, J. Gain control in the electrosensory system mediated by descending inputs to the electrosensory lateral line lobe. J. Neurosci. 6: 553–
562, 1986b.
BASTIAN, J. Gain control in the electrosensory system. A role for descending
projections to the lateral electrosensory lateral line lobe. J. Comp. Physiol. [A] 158: 505–515, 1986c.
BASTIAN, J. The role of amino acid neurotransmitters in the descending
control of electroreception. J. Comp. Physiol. [A] 172: 409–423, 1993.
/ 9k2f$$de46
J080-8
BASTIAN, J. Pyramidal-cell plasticity in weakly electric fish: a mechanism
for attenuating responses to reafferent electrosensory inputs. J. Comp.
Physiol. [A] 176: 63–73, 1995.
BASTIAN, J. Plasticity in an electrosensory system. I. General features of a
dynamic sensory filter. J. Neurophysiol. 76: 2483–2496, 1996.
BASTIAN, J. AND COURTRIGHT, J. Morphological correlates of pyramidal
cell adaptation rate in the electrosensory lateral line lobe of weakly electric fish. J. Comp. Physiol. [A] 168: 393–407, 1991.
BASTIAN, J., COURTWRIGHT, J., AND CRAWFORD, J. Commissural neurons
of the electrosensory lateral line lobe of Apteronotus Leptorhynchus.
Morphological and physiological characteristics. J. Comp. Physiol. [A]
173: 257–274, 1993.
BAUMFALK, U. AND ALBUS, K. Phaclofen antagonizes baclofen-induced
suppression of visually evoked responses in the cat’s striate cortex. Brain
Res. 463: 398–402, 1987.
BERMAN, N. J., DOUGLAS, R. J., AND MARTIN, K. A. GABA-mediated inhibition in the neural networks of visual cortex. In: GABA in the Retina
and Central Visual System, edited by R. R. Mize, R. E. Marc, and A. M.
Sillito. Amsterdam: Elsevier, 1992, p. 443–476.
BERMAN, N. J. AND MALER, L. Interaction of GABAA-mediated inhibition
with voltage-gated currents of pyramidal cells: computational mechanism
of a sensory searchlight? J. Neurophysiol. 80: 3197–3213, 1998a.
BERMAN, N. J. AND MALER, L. Distal versus proximal inhibitory shaping
of feedback excitation in the electrosensory lateral line lobe: implications
for sensory filtering. J. Neurophysiol. 80: 3214–3232, 1998b.
BERMAN, N. J., PLANT, J., TURNER, R., AND MALER, L. Excitatory amino
acid transmission at a feedback pathway in the electrosensory system. J.
Neurophysiol. 78: 1869–1881, 1997.
BORMANN, J. Electrophysiology of GABAA and GABAB receptor subtypes.
Trends Neurosci. 11: 112–116, 1988.
BULLOCK, T. H. Species differences in effect of electroreceptor input on
electric organ pacemakers and other aspects of behaviour in electric fish.
Brain Behav. Evol. 2: 85–118, 1969.
CARR, C. E. AND MALER, L. Electroreception in gymnotiform fish. Central
anatomy and physiology. In: Electroreception, edited by T. H. Bullock,
and W. Heiligenberg. New York: Wiley, 1986, p. 319–373.
CARR, C. E., MALER, L., AND SAS, E. Peripheral organization and central
projections of the electrosensory organs in gymnotiform fish. J. Comp.
Neurol. 211: 139–153, 1982.
CONNORS, B. W., MALENK A, R. C., AND DA SILVA, L. R. Two inhibitory
postsynaptic potentials, and GABAA and GABAB receptor-mediated responses in neocortex of rat and cat. J. Physiol. (Lond.) 406: 443–468,
1988.
DE SCHUTTER, E. Computer software for development and simulation of
compartmental models of neurons. Comp. Biol. Med. 19: 71–81, 1989.
DJO/ RUP, A., JAHNSEN, H., AND L AURSEN, A. M. The dendritic response
to GABA in CA1 of the hippocampal slice. Brain Res. 219: 196 –
201, 1981.
DOUGLAS, R. J. AND M ARTIN, K.A.C. Control of neuronal output by
inhibition at the axon initial segment. Neural Comp. 2: 283 – 292,
1990.
FABER, D. S. AND K ORN, H. Voltage-dependence of glycine activated
Cl- channels: a potentiometer for inhibition? J. Neurosci. 7: 807 –
811, 1987.
GABBIANI, F., MIDTGAARD, J., AND K NÖPFEL, T. Synaptic integration
in a model of cerebellar granule cells. J. Neurophysiol. 72: 999 –
1009, 1994.
GRACE, A. A. AND LLINAS, R. Morphological artifacts induced in intracellularly stained neurons by dehydration. Circumvention using rapid dimethyl
sulfoxide clearing. Neuroscience 16: 461–475, 1985.
HEILIGENBERG, W. Neural Nets in Electric Fish, Cambridge, MA: MIT
Press, 1991.
HEILIGENBERG, W. AND DYE, J. Labelling of electrosensory afferents in a
gymnotid fish by intracellular injection of HRP: the mystery of multiple
maps. J. Comp. Physiol. [A] 148: 287–296, 1982.
HOPKINS, C. D. Stimulus filtering and electroreception: tuberous electroreceptors in three species of gymnotoid fish. J. Comp. Physiol. [A] 111:
171–207, 1976.
HORIK AWA, K. AND ARMSTRONG, W. E. A versatile means of intracellular
labeling injection of biocytin and its detection with avidin conjugates. J.
Neurosci. Methods 25: 1–11, 1988.
JACK, J.J.B., NOBLE, D., AND TSIEN, R. W. Electric Current Flow in Excitable Cells. London: Oxford Univ. Press, 1975.
JACK, J.J.B. AND REDMAN, S. J. An electrical description of the motoneu-
12-01-98 21:11:17
neupa
LP-Neurophys
Downloaded from http://jn.physiology.org/ by 10.220.33.4 on June 16, 2017
size that the long GABAA IPSP durations recorded in pyramidal
cells are caused by sustained presynaptic GABA release due to
burst firing of GC2 interneurons.
As to the function of the somatic dendrites, there are three options
where placing inhibitory inputs on the somatic dendrites may confer
a computational advantage: prolong the inhibitory input, provide
increased membrane real estate for inhibition, and perform local
AND-NOT computations (see Koch 1997) on gap junction input.
Our simulations suggested that the first option was not likely; somatic dendrite IPSPs were not significantly prolonged due to the
electrotonic characteristics of the fine dendrites. As for the second
option, the extra inhibitory synaptic drive onto somatic dendrites
significantly affects the overall inhibition only to the extent that the
somatic dendrite inhibitory inputs form the dominant fraction of
active inhibitory inputs. When there is a large fraction of somatic
inhibition, the soma saturates near the reversal potential of the inhibitory conductance and further somatic dendrite inhibition does not
significantly affect soma Vm or Rin . The available evidence indicates
that GC2 innervate both soma and somatic dendrites (Maler et al.
1981) and hence selective activation of somatic dendrite GABAergic
inputs is unlikely. However, the somatic dendrites would permit
more GC1 and GC2 cells to contact pyramidal cells (especially the
NBP). With regard to the third option, the colocalization of inhibitory and dendro-dendritic gap-junction inputs on NBP somatic dendrites (Maler et al. 1981) would make local computations feasible
on this extensive somatic dendritic arbor.
3195
3196
N. J. BERMAN AND L. MALER
/ 9k2f$$de46
J080-8
SHUMWAY, C. Multiple electrosensory maps in the medulla of weakly electric Gymnotiform fish. I. Physiological differences. J. Neurosci. 9: 4388–
4399, 1989a.
SHUMWAY, C. Multiple electrosensory maps in the medulla of weakly electric Gymnotiform fish. II. Anatomical differences. J. Neurosci. 9: 4400–
4415, 1989b.
SHUMWAY, C. A. AND MALER, L. GABAergic inhibition shapes temporal
and spatial response properties of pyramidal cells in the electrosensory
lateral line lobe of gymnotiform fish. J. Comp. Physiol. [A] 164: 391–
407, 1989.
SILLITO, A. M. Functional considerations of the operation of GABAergic
inhibitory processes in the visual cortex. In: Functional Properties of
Cortical Cells, edited by E. G. Jones and A. Peters. New York: Plenum
Press, 1984, p. 91–117.
SMITH, R. L., CLAYTON, G. H., WILCOX, C. L., ESCUDERO, K. W., AND STALEY, K. J. Differential expression of an inwardly rectifying chloride conductance in rat brain neurons: a potential mechanism for cell-specific
modulation of postsynaptic inhibition. J. Neurosci. 15: 4057–4067, 1995.
SOFTKY, W. R. AND KOCH, C. The highly irregular firing of cortical cells
is inconsistent with temporal integration of random EPSPs. J. Neurosci.
13: 334–350, 1993.
SODICKSON, S. L. AND BEAN, B. P. GABAB receptor-activated inwardly
rectifying potassium current in dissociated hippocampal CA3 neurons.
J. Neurosci. 16: 6374–6385, 1996.
SOMERS, D. C., NELSON, S. B., AND SUR, M. An emergent model of orientation selectivity in cat visual cortical simple cells. J. Neurosci. 15: 5448–
5465, 1995.
THARANI, Y., THURLOW, G. A., AND TURNER, R. W. Distribution of omegaconotoxin GVIA binding sites in teleost cerebellar and electrosensory
neurons. J. Comp. Neurol. 364: 456–472, 1996.
THOMPSON, S. M., DEISZ, R. A., AND PRINCE, D. A. Relative contributions
of passive equilibrium and active transport to the distribution of chloride
in mammalian cortical neurons. J. Neurophysiol. 60: 105–124, 1988.
THOMPSON, S. M. AND GÄHWILER, B. H. Comparison of the action of baclofen at pre- and postsynaptic receptors in the rat hippocampus in vitro. J.
Physiol. (Lond.) 451: 329–345, 1992.
TIA, S., WANG, J. F., KOTCHABHAKDI, N., AND VICINI, S. Distinct deactivation and desensitization kinetics of recombinant GABAA receptors. Neuropharmacology 35: 1375–1382, 1998.
TRAUB, R. D., MILES, R., AND JEFFERYS, J.G.R. Synaptic and intrinsic conductances shape picrotoxin-induced synchronized after discharges in the
guinea-pig hippocampal slice. J. Physiol. (Lond.) 461: 525–547, 1993.
TURNER, R. W., MALER, L., DEERINCK, T., LEVINSON, S. R., AND ELLISMAN,
M. H. TTX-sensitive dendritic sodium channels underlie oscillatory discharge in a vertebrate sensory neuron. J. Neurosci. 14: 6453–6471, 1994.
TURNER, R. W., PLANT, J. R., AND MALER, L. Conditional oscillatory discharge in topographic maps of the electrosensory lateral line lobe (ELL).
Soc. Neurosci. Abstr. 17: 1405, 1991.
TURNER, R. W., PLANT, J. R., AND MALER, L. Oscillatory and burst discharge across electrosensory topographic maps. J. Neurophysiol. 76:
2364–2382, 1996.
WANG, D. AND MALER, L. The immunocytochemical localization of glutamate in the electrosensory system of the gymnotiform fish. Apteronotus
leptorhynchus. Brain Res. 653: 215–222, 1994.
WONG, R.K.S. AND WATKINS, D. J. Cellular factors influencing GABA
response in hippocampal pyramidal cells. J. Neurophysiol. 48: 938–951,
1982.
XU, Z., PAYNE, J. R., AND NELSON, M. E. Logarithmic time course of sensory adaptation in electrosensory afferent nerve fibers in a weakly electric
fish. J. Neurophysiol. 76: 2020–2032, 1996.
YAMAMOTO, T., MALER, L., HERTZBERG, E. L., AND NAGY, J. I. Gap junction protein in weakly electric fish (Gymnotide): immunohistochemical
localization with emphasis on structures of the electrosensory system. J.
Comp. Neurol. 289: 509–536, 1989.
YOON, K.-W. Voltage-dependent modulation of GABAA receptor channel
desensitization in rat hippocampal neurons. J. Neurophysiol. 71: 2151–
2160, 1994.
ZAKON, H. H. The electroreceptive periphery. In: Electroreception, edited
by T. H. Bullock, and W. Heiligenberg. New York: Wiley, 1986, p. 103–
156.
ZUPANC, G. AND MALER, L. Neuronal control of behavioral plasticity: the
prepacemaker nucleus of weakly electric fish. J. Comp. Physiol. [A] 180:
99–111, 1997.
12-01-98 21:11:17
neupa
LP-Neurophys
Downloaded from http://jn.physiology.org/ by 10.220.33.4 on June 16, 2017
rone, and its application to the analysis of synaptic potentials. J. Physiol.
(Lond.) 215: 321–352, 1971.
KOCH, C. Computation and the single neuron. Nature 385: 207–210, 1997.
LEGENDRE, P. AND KORN, H. Voltage dependence of conductance changes
evoked by glycine release in the zebrafish brain. J. Neurophysiol. 73:
2404–2412, 1995.
MALER, L. The posterior lateral line lobe of certain gymnotiform fish.
Quantitative light microscopy. J. Comp. Neurol. 183: 323 – 363,
1979.
MALER, L. AND M UGNAINI, E. Organization and function of feedback
to the electrosensory lateral line lobe of gymnotiform fish, with emphasis on a searchlight mechanism. J. Comp. Physiol. [ A ] 173: 667 –
670, 1993.
MALER, L. AND MUGNAINI, E. Correlating gamma-aminobutyric acidergic
circuits and sensory function in the electrosensory lateral line lobe of a
gymnotiform fish. J. Comp. Neurol. 345: 224–252, 1994.
MALER, L., SAS, E., JOHNSTON, S., AND ELLIS, W. An atlas of the brain of
the weakly electric fish. Apteronotus Leptorhynchus. J. Chem. Neuranat.
4: 1–38, 1991.
MALER, L., SAS, E. K., AND ROGERS, J. The cytology of the posterior
lateral line lobe of high frequency weakly electric fish ( Gymnotoidei):
differentiation and synaptic specificity in a simple cortex. J. Comp. Neurol. 195: 87–139, 1981.
MARTIN, K.A.C. From single cells to simple circuits in the cerebral cortex.
Q. J. Exp. Physiol. 73: 637–702, 1988.
MASON, A. AND LARKMAN, A. Correlations between morphology and electrophysiology of pyramidal neurons in slices of rat visual cortex. II.
Electrophysiology. J. Neurosci. 10: 1415–1428, 1990.
MATHIESON, W. B., HEILIGENBERG, W., AND MALER, L. Ultrastructural studies of physiologically identified electrosensory afferent synapses in the
gymnotiform fish. Eigenmannia. J. Comp. Neurol. 255: 526–537, 1987.
MATHIESON, W. B. AND MALER, L. Morphological and electrophysiological
properties of a novel in vitro preparation: the electrosensory lateral line
lobe brain slice. J. Comp. Physiol. [A] 163: 489–506, 1988.
MATTHEWS, G., AYOUB, G. S., AND HEIDELBERGER, R. Presynaptic inhibition by GABA is mediated via two distinct GABA receptors with novel
pharmacology. J. Neurosci. 14: 1079–1090, 1994.
METZNER, W. AND HEILIGENBERG, W. The coding of signals in the gymnotiform fish Eigenmannia: from electroreceptors to neurons in the torus
semicircularis of the midbrain. J. Comp. Physiol. [A] 169: 135–150,
1992.
METZNER, W. AND JURANEK, J. A sensory brain map for each behavior?
Proc. Natl. Acad. Sci. USA 94: 14798–14803, 1997.
MODY, I., DE KONINCK, Y., OTIS, T. S., AND SOLTESZ, I. Bridging the cleft
at GABA synapses in the brain. Trends Neurosci. 17: 517–525, 1994.
NELSON, M. E., XU, Z., AND PAYNE, J. R. Characterization and modeling
of P-type electrosensory afferent responses to amplitude modulations in
a wave-type electric fish. J. Comp. Physiol. [A] 181: 532–544, 1997.
OGATA, N., INOUE, M., AND MATSUO, T. Contrasting properties of K /
conductances induced by baclofen and g-aminubutyric acid in slices of
the guinea pig hipoocampus. Synapse 1: 62–69, 1987.
OTIS, T. S. AND MODY, I. Modulation of decay kinetics and frequency of
GABAA receptor-mediated spontaneous inhibitory postsynaptic currents
in hippocampal neurons. J. Neurosci. 49: 13–32, 1992.
PEARCE, R. A. Physiological evidence for two distinct GABAA responses
in rat hippocampus. Neuron 10: 189–200, 1993.
SAUNDERS, J. AND BASTIAN, J. The physiology and morphology of two
classes of electrosensory neurons in the weakly electric fish. Apteronotus
Leptorhynchus. J. Comp. Physiol. [A] 154: 199–209, 1984.
SCHARFMAN, H. E. AND SARVEY, J. M. Physiological correllates of responses to gamma-aminobutyric acid (GABA) recorded from rat visual
cortical neurons in vitro. Synapse 2: 619–626, 1988a.
SCHARFMAN, H. E. AND SARVEY, J. M. Responses to GABA recorded from
identified rat visual cortical neurons. Neuroscience 23: 407–422, 1988b.
SCHERER, R. W., FERK ANY, J. W., AND ENNA, S. J. Evidence for pharmacologically distinct subsets of GABAB receptors. Brain Res. Bull. 21: 439–
443, 1988.
SCHWARTZKROIN, P. A. Characteristics of CA1 neurons recorded intracellularly in the hippocampal in vitro slice preparation. Brain Res. 85: 423–
436, 1975.
SEGAL, M. AND BARKER, J. L. Rat hippocampal neurons in culture: properties of GABA-activated Cl – ion conductance. J. Neurophysiol. 51: 500–
515, 1984.