1Neurological Sciences Institute, Oregon Health Sciences University, Portland, Oregon 97209; and 2Institute Alfred Fessard, Centre National de la Recherche Scientifique, 91190 Gif sur Yvette, France
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ABSTRACT |
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Han, Victor Z., Kirsty Grant, and Curtis C. Bell. Rapid Activation of GABAergic Interneurons and Possible Calcium Independent GABA Release in the Mormyrid Electrosensory Lobe. J. Neurophysiol. 83: 1592-1604, 2000. The primary afferent fibers from the electroreceptors of mormyrid electric fish terminate centrally in the granular layer of the electrosensory lobe (ELL). This study examines the excitatory and inhibitory processes that take place in this layer using an in vitro slice preparation and field potentials evoked by stimulation of primary afferent fibers in the deep fiber layer of ELL. The postsynaptic response to stimulation of the afferent fibers was still present after blocking chemical transmission in three different ways: by adding glutamate receptor antagonists to the medium, by substituting a nominally calcium-free medium for normal medium, and by blocking calcium channels with cadmium. Blockade of chemical transmission was demonstrated by disappearance of control responses to parallel fiber stimulation. The continued presence of a postsynaptic response in the absence of chemical excitation is consistent with previous anatomic and physiological evidence for electrical synapses between afferent fibers and granular cells in ELL. Granular cell activation by primary afferent fibers was followed by a powerful, short-latency inhibition mediated by GABA and GABAA receptors, as indicated by a large increase in the postsynaptic response to afferent fiber stimulation following application of the GABAA receptor antagonist, bicuculline. Bicuculline caused a marked increase of the postsynaptic response even after chemical synaptic excitation had been blocked by glutamate receptor antagonists, by a calcium-free medium, or by cadmium. Thus activation of the inhibitory interneurons responsible for GABA release did not require chemical excitation. Nonchemical excitation of the inhibitory interneurons could be mediated either by electrical synapses between afferent fibers and inhibitory interneurons, or by nonsynaptic activation of the large GABAergic terminals that are known to be present on granular cells. The marked increase of the postsynaptic response caused by bicuculline in a calcium-free medium or in the presence of cadmium suggests that the release of GABA by inhibitory terminals was not entirely dependent on calcium influx. This effect of bicuculline on the postsynaptic response in a calcium-free medium or in the presence of cadmium was markedly reduced by prior addition of the GABA transporter antagonist, nipecotic acid. Thus calcium-independent release of GABA may occur in ELL and may be partly dependent on reversal of a GABA transporter. Rapid and powerful inhibition at the first stage in the processing of electrosensory information could serve to enhance the small differences in latency among afferent fibers that appear to encode small differences in stimulus intensity.
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INTRODUCTION |
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The mormyrid electrosensory lobe (ELL) is a cerebellum-like
structure and receives the primary afferent fibers from
electroreceptors in the skin. The ELL is thus the first central stage
in the processing of sensory input from cutaneous electroreceptors. The
ELL, like many other sensory processing regions of the vertebrate
brain, receives extensive input not only from the periphery but also from other central structures (Bell and Szabo 1986).
This central input modulates the processing of sensory information in a
dynamic and plastic manner (Bell et al. 1997b
), and the
mormyrid ELL is a good site for investigating the roles of such central
inputs in sensory processing. Understanding sensory processing in ELL requires knowledge of the effects of both peripheral and central inputs
to the structure.
Primary afferent fibers from electroreceptors terminate on small
granular cells in the granular layers of ELL (Bell et al. 1989). These small granular cells relay the electrosensory
information on to larger cells of ELL that include the Purkinje-like
medium ganglion cells and the two types of efferent cells, large
ganglion and large fusiform cells. The physiology of the larger cells
of ELL has been examined both in vivo (Bell et al.
1997a
) and in vitro (Grant et al. 1998
), but
very little is known about the physiology of granular cells and about
interactions in the granular layer that condition the electrosensory
information received by the larger cells of ELL. This study uses field
potentials and pharmacology to investigate responses and cellular
interactions in the granular layer that are evoked by primary afferent
stimulation. The focus is on a powerful and short-latency inhibition
that takes place in this layer. All experiments were done in an in
vitro slice preparation.
The ELL has six layers: molecular, ganglion, plexiform, granular,
intermediate, and deep fiber (Fig.
1A) (Bell and Szabo
1986; Meek et al. 1999
). Primary afferent fibers
from electroreceptors terminate on granular neurons with mixed chemical
and electrical synapses (Bell et al. 1989
; Meek
et al. 1999
) (Fig. 1B, right). The same granular
cells are also contacted by large
-aminobutyric acid
(GABA)-containing terminals that cover one-third to one-half of the
small somas of granular neurons (Fig. 1, A and
B). These terminals arise from large multipolar intermediate
layer neurons (LMI cells) that have their cell bodies in the
intermediate layer (Meek et al. 1999
). The axons of LMI
cells have local branches that terminate in the ELL granular layer near
the cell body as well as branches that travel some distance to
terminate in the ELL granular layer on both the ipsilateral and
contralateral sides. The LMI cells are unusual in that their dendrites,
which extend up into the overlying granular layer, become myelinated as
they exit from the cell body. It has been suggested further that the dendrites retain their myelin as they branch and terminate on granular
cells as presynaptic structures (Meek et al. 1999
). Thus the large GABA-containing terminals on granular cells may arise from
the dendrites as well as the axons of LMI cells.
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Intra-axonal recordings from primary afferent fibers near their
terminals in ELL show synaptic potentials as well as orthodromic spikes
from electroreceptors (Bell 1990). The synaptic
potentials are probably due to synaptic input to postsynaptic granular
cells that is observed inside the primary afferent because of the
electrical synapses that the afferent makes with granular cells. The
synaptic potentials include the following: excitatory postsynaptic
potentials (EPSPs) evoked by a centrally originating corollary
discharge signal associated with the motor command that drives the
electric organ to discharge; EPSPs evoked by stimulation of
electroreceptors near the one from which the recorded afferent arises
that are due to convergence of different afferents onto the same
granular cells; and an inhibitory postsynaptic potential (IPSP) that is evoked by stimulation of more distant electroreceptors. The IPSP is
prominent and is probably mediated by the large GABAergic terminals of
LMI cells on granular cells.
This study demonstrates that stimulation of afferent fibers from electroreceptors evokes a powerful, GABA-mediated inhibition in the granular layer of the mormyrid ELL. Primary afferent activation of the inhibitory interneurons that release the GABA is very rapid and does not depend on excitatory chemical synaptic transmission. In addition, the evoked release of GABA may be partly independent of external calcium.
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METHODS |
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Slice preparation
Experiments were carried out using 26 fish of the mormyrid
species Gnathonemus petersii. The fish were first deeply
anesthetized with MS 222 (Sigma; concentration 1:10,000 in aquarium
water). The brain was removed and cooled rapidly by immersion in an
ice-cold balanced salt solution containing (in mM) 0 NaCl, 2.0 KCl,
1.25 KH2PO4, 24 NaHCO3, 2.6 CaCl2, 1.6 MgSO4.7H2O, 20 glucose, and 213 sucrose [low sodium artificial cerebrospinal fluid (ACSF)], saturated with a gas mixture containing 95%
O2-5% CO2. Three to four
hundred micrometer-thick slices were cut in the frontal (transverse) plane using a custom-made horizontal rotating circular blade microtome, under the same ice-cold salt solution. After cutting, the slices were
transferred to a bath containing equal parts of low sodium ACSF and
normal ACSF (in mM: 124 NaCl, 2.0 KCl, 1.25 KH2PO4, 24 NaHCO3, 2.6 CaCl2, 1.6 MgSO40.7H2O, and 20 glucose) at room temperature. The slices remained in this solution for
30 min and were then moved to a standard interface recording chamber
where they were continuously superfused with normal ACSF at a flow rate
of ~2 ml/min, at room temperature. The normal ACSF was also bubbled with 95% O2-5% CO2 (pH
7.2-7.4, osmolarity 290 mosM). Complete or partial replacement of
sodium with sucrose during cutting, and for a short time afterward,
appears to reduce cell death during slice preparation
(Aghajanian and Rasmussen 1989
).
Recording and stimulation
Field potentials were recorded in the granular layer of the
medial zone of the electrosensory lobe (Fig.
2A) using glass
microelectrodes with tip diameters of 3-5 µm and filled with 3 M
NaCl (resistance 5-10 M). Signals were recorded directly to a
computer using the Axon Instruments interface and Axoscope software.
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Electrical stimulation of the tissue was delivered through tungsten microelectrodes (A-M systems) insulated except at the tip and plated with gold to minimize electrode polarization. One stimulus electrode was placed in the middle of the molecular layer to activate parallel fibers (SM, Fig. 2A). The two other stimulus electrodes were placed in the intermediate (SI) or deep fiber (SD) layers to activate lateral GABAergic terminals and primary afferent fibers, respectively. The three stimulus electrodes were monopolar. Each stimulus electrode was paired with a second tungsten microelectrode in the recording chamber outside the brain slice that served as the indifferent electrode. Stimuli were 0.1 ms in duration and delivered at 0.5-1 Hz through a stimulus isolation unit. Stimulus intensities ranged from a few microamperes to 50 µA.
The field potential evoked by parallel fiber stimuli served as a
monitor of excitatory chemical transmission. Parallel fiber stimuli
evoke an EPSP in the apical dendrites of ELL medium ganglion and
efferent cells (Grant et al. 1998). This response is
mediated by the release of glutamate and by postsynaptic receptors of
both the N-methyl-D- aspartate (NMDA) and
-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) types.
The field potential evoked by parallel fiber stimulation is negative in
the molecular layer where the EPSP is generated but is positive in the
ganglion and granular layers where the cell bodies and basilar
dendrites of medium ganglion and efferent cells act as a current source
for the current sink in the molecular layer.
Pharmacology
All drugs were bath applied by adding them to the perfusate. The following blocking agents of excitatory or inhibitory chemical synaptic transmission were tested: the non-NMDA glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 30 µM; RBI), the NMDA glutamate receptor antagonist D-2-amino-5-phosphonopentanoate (AP5; 35 µM; RBI), the GABAA receptor antagonist bicuculline methiodide (30 µM; Sigma) the GABAB receptor antagonist 2-hydroxysaclofen (500 µM; RBI), and the glycine receptor antagonist strychnine hydrochloride (15 µM; Sigma). In some experiments, the slices were bathed with ACSF in which the calcium ions were replaced by an equivalent increase in magnesium ions ("calcium-free" ACSF). Cadmium (100 µM) was used to block voltage-sensitive calcium channels, and tetrodotoxin (TTX; 0.5 µM) was used to block voltage-sensitive sodium channels.
Data analysis
Data were analyzed off-line using Clampfit (Axon Instruments) and Origin (Microcal) software. All of the responses illustrated in the figures are averages of 15 individual responses. Responses in the granular layer to primary afferent stimulation consist of an initial positive-negative wave due to activation of the presynaptic fibers and a subsequent negative wave due to activation of postsynaptic granular cells (Fig. 2B; see RESULTS). The amplitude of the postsynaptic component was measured by calculating the area between the baseline and the response trace in the averaged record, beginning at the transition between pre- and postsynaptic components of the response at a latency of ~2 ms and ending 10-25 ms later (see shaded area in Fig. 2B, trace 5). (The recorded response varied in different slices, and the delay of the endpoint used in measuring the area varied accordingly.) Means ± SD of these areas were calculated across different replicates of the same experiment in different slices and were compared using Student's t-test. Differences with P values smaller than 0.01 were judged to be significant.
When a conditioned-test paradigm was used, as in measuring paired-pulse depression (Figs. 7 and 8) or lateral inhibition (Figs. 9 and 10), the average response to the conditioning stimulus alone (control) was subtracted from the average response to conditioning plus test stimuli to obtain the effect of the test stimulus in isolation. The response to the test stimulus was expressed as a percent of the response to the same stimulus in the absence of a conditioning stimulus. Results are plotted as means ± SD.
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RESULTS |
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Field potentials in the granular layer evoked by primary afferent fiber stimulation
The field potential recorded in the granular layer in response to
weak stimulation (3-10 µA) of the deep fiber layer has two negative
components (Fig. 2B). The initial component has been identified as the incoming presynaptic volley ("n pre" in Fig. 2B) and the later component as the postsynaptic response of
granular cells ("n post" in Fig. 2B), based on their
latencies, spatial distribution, and abilities to follow high-frequency
stimulation (Grant et al. 1998).
The responses to weak stimulation of the deep fiber layer may be
attributed to activation of primary afferent fibers (Grant et
al. 1998). Although there are other types of fibers in the deep
fiber layer besides primary afferents (Meek et al.
1999
), these other fiber types are either unmyelinated or much
smaller in diameter than the primary afferents, and thus are likely to have a higher threshold. In addition, the laminar distribution of field
potentials (Fig. 2B) is consistent with the laminar
termination pattern of primary afferents but not with the termination
pattern of the other fiber types. Primary afferent fibers terminate on cells in the granular layer and do not extend beyond the boundary between the granular and plexiform layers (Bell and Szabo
1986
; Meek et al. 1999
). The region in which the
postsynaptic response (n post) is observed thus corresponds
anatomically to the region in which primary afferent fibers terminate.
The postsynaptic response shows marked paired-pulse depression (see
Paired-pulse depression of responses to deep fiber layer stimulation and Fig. 7) and does not follow frequencies
>100 Hz (Grant et al. 1998). The latter is illustrated
in Fig. 2C by the almost complete lack of a postsynaptic
response to all but the first stimulus in a train of stimuli at 200 Hz.
Primary afferent fibers make electrical synapses on granular cells
(Bell et al. 1989
; Meek et al. 1999
), and
an electrotonic EPSP would be expected to follow much higher rates of
stimulation than 100 Hz. Perhaps an electrotonic EPSP contributes to
the slowly rising negativity that follows the presynaptic volley in
later responses (double arrows in Fig. 2C). The larger
portion of the postsynaptic response, that does not follow high
frequencies, is possibly due to postsynaptic sodium spikes. It is not
due to chemical EPSPs or calcium spikes because it is still present
when calcium inflow is prevented in calcium-free medium (Fig.
5Ab) or by blocking calcium channels with cadmium (Fig.
5Bb). The poor following capacity of the larger portion
of the postsynaptic response could be due to refractoriness of the
postsynaptic spike.
Pharmacology of field potentials in granular layer
A pharmacological approach was used to understand the functional organization of primary afferent projections and synaptic transmission in the granular layer.
The presence of large GABAergic terminals on the granular cells that
receive primary afferent input (Fig. 1B) suggests that GABAergic inhibition plays an important role in shaping the spatial and
temporal properties of granular cell responsiveness. Bath application
of the GABAA receptor blocker bicuculline
markedly increased both the amplitude and duration of the negative
field potential corresponding to the postsynaptic response (Figs.
3B and 4B),
confirming that the initial excitation of granular cells is normally
followed by a powerful, primary afferent-evoked inhibition, mediated
via GABAA receptors. The difference between
responses in the absence and in the presence of bicuculline was highly
significant (n = 28 slices, P 0.001),
with the average response under bicuculline being 193% of the control
response.
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Subtraction of averaged traces obtained in the presence of bicuculline from those obtained in its absence showed that the GABAergic inhibition began at a short latency of only 1 ms after the peak of the presynaptic volley (Fig. 3C). Adding the glycine receptor antagonist strychnine to the bath did not cause a significant change in the postsynaptic response (11 slices, P = 0.27; not illustrated). Thus glycine does not seem to be involved as a transmitter at this initial stage of processing in ELL.
Primary afferent terminals are excitatory (Bell 1990)
and form mixed chemical-electrical synapses on granular neurons (Fig. 1B) (Bell et al. 1989
; Meek et al.
1999
). Glutamate is the most common excitatory transmitter, and
antagonists of glutamate receptors were therefore applied to examine
the role of chemical transmission in the excitatory response evoked in
the granular layer by primary afferent fiber stimulation (Fig.
4, D-F). Application of
CNQX, an antagonist of the AMPA type of glutamate receptors, caused a
reduction in the postsynaptic response to deep fiber layer stimulation (Fig. 4D). Subsequent addition of the NMDA receptor
antagonist, AP5, caused a slightly additional reduction that was
particularly evident in the late components of the response (Fig.
4E). On average, the two glutamate receptor antagonists
together reduced the control response by 30% relative to the control
response recorded in normal ACSF (Fig. 4, A and
C) and the difference between responses in the absence and
in the presence of the antagonists was statistically significant
(n = 8 slices, P
0.001). Responses to
molecular layer stimulation (SM in Fig. 4), recorded simultaneously,
were completely blocked by addition of the two antagonists (Fig.
4E), indicating that the drugs penetrated the slice and were
effective in blocking glutamatergic synaptic transmission. The
reduction of the postsynaptic response to deep fiber layer stimulation
in the presence of the glutamate receptor antagonists indicates that the effect of primary afferent activation on granular cells is due in
part at least to glutamate-mediated chemical transmission.
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When bicuculline was added to the bath solution in the presence of CNQX and AP5, despite the existing blockade of glutamate receptors, there was nevertheless a large increase in the postsynaptic response (Fig. 4F). The increase caused by bicuculline in the eight slices tested was significant (P = 0.003) and averaged 207%. This increase was less, however, than that observed in the absence of the glutamate antagonists when only bicuculline was added to the bath (Fig. 4B). The prominent postsynaptic response in the presence of the glutamate receptor antagonists and bicuculline indicates that synaptic transmission from primary afferent fibers to granular cells also takes place by some mechanism in addition to glutamate-mediated chemical transmission, with the most probable such mechanism being electrical transmission via the morphologically demonstrated gap junctions.
The marked increase in the postsynaptic response caused by bicuculline in the presence of the glutamate receptor antagonists also suggests that primary afferent activation continued to evoke the release of GABA despite the blockade of chemical excitatory transmission. This in turn suggests that excitation of the inhibitory interneurons responsible for GABA release can also take place by some other means than glutamate-mediated chemical synaptic transmission.
Role of calcium in generating the postsynaptic response
Transmitter release is generally believed to depend on influx of calcium through voltage-gated calcium channels. The role of chemically mediated transmission was therefore investigated further by replacing calcium in the medium with an equimolar concentration of magnesium, and by blocking voltage-gated calcium channels with cadmium (100 µM). The primary afferent volley evoked by stimulation in the deep fiber layer continued to evoke a clear postsynaptic response, both in nominally calcium-free medium (Fig. 5Ab) and in the presence of cadmium (Fig. 5Bb). The continued presence of a postsynaptic response in the absence of calcium, or when calcium channels are blocked by cadmium, provides further evidence for a role of electronic EPSPs in the excitation of granular cells by primary afferent fibers. The simultaneous disappearance of responses to parallel fiber stimulation (SM; Fig. 5, Ab and Bb) confirmed the effective blockade of excitatory chemical transmission under these conditions.
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Addition of bicuculline to the bath still caused a marked increase in
the amplitude and duration of the postsynaptic responses to stimulation
of the deep fiber layer in a calcium-free medium (Fig. 5Ac)
or in the presence of cadmium (Fig. 5Bc). Bicuculline caused
an average increase of 220% (n = 5 slices,
P 0.001) in the postsynaptic response recorded in a
calcium-free medium and an average increase of 272% (n = 9 slices, P
0.001) in the postsynaptic response
recorded in the presence of cadmium. The effects of a calcium-free
medium and of bicuculline on responses to molecular layer and deep
fiber layer stimuli were reversed after returning to normal ACSF (Fig.
5Ad). The effects of cadmium on responses to parallel fiber
stimuli did not reverse, however, at least within a period of 1 h
after removing the cadmium (Fig. 5Bd). As expected, addition
of the sodium channel blocker, TTX, abolished both the pre- and
postsynaptic components of the response to primary afferent stimulation
(Fig. 5Ae).
The large increases in postsynaptic responses caused by bicuculline, in calcium-free medium and in the presence of cadmium, suggest that GABA release continues to be evoked even though chemical excitatory chemical transmission is blocked. This result is consistent with the results obtained in the presence of glutamate receptor antagonists, in suggesting that excitation of the inhibitory interneurons responsible for GABA release does not require chemical synaptic transmission. In addition, and more surprisingly perhaps, the results also suggest that some of the evoked GABA release at these synapses is calcium independent because the GABA appears to have been released in a calcium-free medium and when voltage-gated channels were blocked with cadmium.
Calcium-independent release of transmitter has been observed at other
synapses, and evidence has been obtained that transporter molecules
have a role in such release (Attwell et al. 1993;
Schwartz 1987
). The proposed mechanism is one in
which the normal direction for the transport of transmitter, from
outside to inside, is reversed when the inside of the cell becomes
depolarized or experiences a large increase in sodium ion
concentration. We therefore tested the effects of the GABA transporter
blocker, nipecotic acid (NA) in nine slices (Fig.
6). Addition of NA in a calcium-free
medium caused a small but not statistically significant increase in the postsynaptic response to deep fiber layer stimulation
(n = 9 slices). The effect of subsequent addition
of bicuculline to such slices was variable. Addition of bicuculline
under these conditions yielded a reduction in the postsynaptic response
in three slices, no change in one slice, some enhancement but clearly
less than that observed in the absence of NA in three slices, and
enhancement similar to that observed without NA in two slices. The
average increase caused by the addition of bicuculline to a
calcium-free medium in the presence of NA caused an average increase of
9% in the postsynaptic response, a change that was not significant
(n = 9 slices, P = 0.7). This
small increase is in contrast to the highly significant increase of
220% caused by bicuculline in a calcium-free medium that did not
contain NA, as described above. The results suggest a possible role of
GABA transporters in the calcium-independent release of GABA in ELL.
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Paired-pulse depression of responses to deep fiber layer stimulation
As reported previously (Grant et al. 1998 and
above), most of the postsynaptic response to deep fiber layer
stimulation does not follow repetitive stimulation at frequencies of
>100 Hz. The interaction between successive stimuli was examined in
more detail with a paired-pulse protocol. The amplitude of the
presynaptic volley was reduced for stimulus intervals of <10 ms (Fig.
7B, bottom graph),
due presumably to primary afferent fiber refractoriness. However, the
postsynaptic component of the response showed a more prolonged
depression (Fig. 7, A and B), which lasted
between 25 and 40 ms in the 15 different slices that were tested.
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This depression of the second response in the paired-pulse protocol could be due to release of GABA by the first stimulus, and this possibility was first tested by applying bicuculline to the bath. Surprisingly, however, the addition of bicuculline greatly increased the degree and duration of the depression (Fig. 7, C and D), rather than reducing it. Under bicuculline, paired-pulse depression of the postsynaptic response lasted for >200 ms (Figs. 7D and 8) in the 12 different slices that were tested, in marked contrast to the 25-40 ms of depression observed in the absence of bicuculline. The brief, paired-pulse depression of the presynaptic component was not affected by bicuculline (Fig. 7D, bottom), however, indicating that the increased duration of paired-pulse depression caused by bicuculline is a postsynaptic phenomenon.
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Bicuculline blocks GABAA receptors but does not affect GABAB receptors. Inhibition due to activation of GABAB has a long duration of action, suggesting that they might be involved in the long-lasting depression after a single stimulus. Accordingly, the role of GABAB receptors was tested with the GABAB antagonist 2-hydroxy-saclofen. The increase in paired-pulse depression observed with bicuculline in the bath was partially blocked by the further addition of 2-hydroxy-saclofen (Fig. 8). Postsynaptic responses to stimuli delivered 50 ms after a preceding stimulus in the presence of both bicuculline and hydroxy-saclofen were significantly larger than such responses in the presence of bicuculline alone (5 slices, P = 0.009). Thus the prolonged paired-pulse depression in the presence of bicuculline appears to be caused in part by a large increase in the GABA released by the first stimulus followed by activation of GABAB receptors.
Lateral inhibition of responses to deep fiber layer stimulation
As described in the INTRODUCTION, granular cells are
inhibited by electrosensory stimuli delivered to the skin surface at some distance from the electroreceptors that excite those same granular
cells (Bell 1990). Similar lateral inhibitory phenomena could be observed in the slice preparation by evoking a postsynaptic granular layer response with a stimulus in the deep fiber layer (SD in
Fig. 2A) and preceding such a stimulus with a stimulus to
the granular or intermediate layer placed 200-300 µm lateral to the
recording site (SI in Fig. 2A). SI alone evoked only a very
small response at the recording site in the granular layer (top
trace, Fig. 9A). However,
delivery of such a stimulus just before a stimulus to the deep fiber
layer (SD in Fig. 2A) caused a clear reduction in the
postsynaptic response to the deep fiber layer stimulus, with only
minimal effects on the presynaptic response (Fig. 9A,
left, superimposed bottom traces). The reduction was observed in all nine slices that were tested. The conditioned postsynaptic response was examined in isolation by subtracting the
response to the lateral stimulus alone [(SI + SD)
SI]. Such measurements showed that the inhibitory effect of the lateral stimulus
on the postsynaptic response to deep fiber layer stimulation lasted
25-40 ms (Fig. 9A, right).
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Bicuculline abolished the inhibitory effect of a lateral stimulus (Fig. 9, B and C), in marked contrast to the enhancement that the same drug caused in paired-pulse depression (Fig. 7, C and D; see DISCUSSION). Blockade of the inhibitory effect of a lateral stimulus by bicuculline was observed in all eight slices in which the effects of the drug were tested.
However, the inhibitory effect of a lateral stimulus was still present in a calcium-free medium (Fig. 10B) in six of nine slices tested. Addition of bicuculline to the calcium-free medium blocked lateral inhibition in all six of these slices (Fig. 10C), just as it did in normal medium. These results indicate that lateral inhibition at this first stage of the system is mediated by GABA and suggest that the GABA may be released, in part at least, by a calcium-independent mechanism.
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DISCUSSION |
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This study used pharmacological tools and field potentials to analyze synaptic transmission in the granular layer of the mormyrid ELL, where the primary afferent fibers from electroreceptors terminate. The following aspects are discussed: 1) synaptic transmission at the mixed chemical-electrical synapse between primary afferent fibers and granular cells, 2) activation of GABAergic interneurons, 3) paired-pulse depression, 4) calcium-independent release of GABA in the ELL granular layer, 5) comparison with gymnotid electric fish, and 6) functional implications of the large and rapid release of GABA for processing of electrosensory information.
Synaptic transmission at the mixed chemical-electrical synapse between primary afferent fibers and granular cells
The present findings are consistent with previous anatomic
(Bell et al. 1989; Meek et al. 1999
) and
physiological (Bell 1990
) evidence for electrical
transmission between primary afferent fibers and granular cells of ELL.
Thus a postsynaptic response was still present in the granular layer
after manipulations that blocked chemical excitatory transmission.
These manipulations included the following: the addition of the
glutamate receptor blockers, CNQX and AP5, to the medium; the addition
of cadmium to the medium; and substitution of calcium-free (with
magnesium) medium for normal medium. In each case, the effective
blockade of chemical synaptic transmission was indicated by
disappearance of the postsynaptic response to parallel fiber stimulation.
The postsynaptic response to primary afferent stimulation in the
granular layer was much reduced by addition of the glutamate receptor
antagonists to the medium (Fig. 4E), and in fact became obvious only after the further addition of bicuculline to the bath
(Fig. 4F). The reduction caused by glutamate receptor
antagonists indicates that glutamate-mediated excitatory chemical
transmission contributes significantly to transmission at the primary
afferent to granular cell synapse. Such a contribution is consistent
with morphological evidence for chemical as well as electrical
transmission at this synapse (Bell et al. 1989;
Meek et al. 1999
).
Somewhat surprisingly, the postsynaptic response to primary afferent
stimulation was not much affected by substitution of a calcium-free
medium for normal medium (Fig. 5Ab) or by addition of
cadmium to the medium (Fig. 5Bb). These manipulations
blocked chemical excitatory transmission, as indicated by disappearance of the parallel fiber responses, and would therefore be expected to
reduce the postsynaptic responses to afferent stimulation just like the
glutamate receptor antagonists. Perhaps the substitution of
calcium-free medium and the addition of cadmium both increased the
excitability of granular cells, compensating for the reduction in
synaptic current. The well-known enhancement of neuronal excitability that is present in low calcium (Hille 1992) may not have
been fully opposed by substitution with equimolar magnesium. In
addition, manipulations that interfere with calcium influx could also
interfere with calcium-activated potassium channels or with release of
inhibitory transmitter. These latter possibilities have been used to
explain the finding that hippocampal cells in slices are depolarized
and more excitable in a medium with low calcium and equimolar
substitution with magnesium or manganese (Jefferys and Haas
1982
; Taylor and Dudek 1982
) and might also
explain an increased excitability of ELL granular cells under similar conditions.
Activation of GABAergic interneurons
The postsynaptic response to primary afferent stimulation in the ELL granular layer was dramatically increased after addition of the GABA receptor antagonist, bicuculline, but was not much affected by addition of the glycine receptor antagonist, strychnine. Thus GABA release and GABA receptors appear to have important roles at this first stage of electrosensory processing in ELL, but glycine release and glycine receptors do not.
The marked increase in the postsynaptic response caused by bicuculline
suggests that GABA release is normally evoked by primary afferent
stimulation and that such release causes a large and rapid inhibition
of granular cells. The latency of the GABA-mediated inhibition,
determined by subtracting the prebicuculline control response from the
response under bicuculline, was 1 ms. This is very short for the
presumed disynaptic pathway from primary afferents through GABAergic
inhibitory interneurons to granular cells, considering that delays at
chemical synapses in cold-blooded vertebrates at room temperature are
~0.5 ms (Katz and Miledi 1965). However, our results
suggest that chemically mediated excitation of the GABAergic
interneuron may not be necessary for GABA release, because bicuculline
still causes a marked increase in the postsynaptic response when
chemical synaptic transmission has been blocked by glutamate receptor
antagonists, by a calcium-free medium, or by the presence of cadmium.
How might GABA release be evoked under such circumstances?
The granular cells that receive primary afferent input from the
periphery also receive very large GABA-containing synaptic terminals
that arise from the axons, and possibly the presynaptic myelinated
dendrites also, of large multipolar intermediate layer neurons (LMI
cells) (Bell et al. 1989; Meek et al.
1999
). These multipolar interneurons are therefore a prime
candidate for the source of the GABA that is released in the granular
layer by stimulation of afferent fibers. Two mechanisms for activating
LMI cells without chemical excitatory transmission may be suggested:
nonsynaptic activation of LMI cell terminals following granular cells
excitation and electrical synapses between primary afferent fibers and
LMI cell dendrites.
Morphological work indicates that the somas of LMI cells are contacted
by only a few excitatory terminals and that none of these terminals
appear to originate from primary afferent fibers (Meek et al.
1999). Moreover, the dendrites of LMI cells become myelinated
as they exit from the soma, and it is possible that the dendritic
branches retain their myelin until they terminate as large presynaptic
endings on granular cells. If the entire dendritic arbor of these cells
is indeed myelinated, there would be little if any opportunity for
excitatory synaptic input to the dendrites. The possible absence of
excitatory synaptic input on the soma and dendrites of LMI cells has
lead to the suggestion that the large LMI terminals on granular cells
might be excited directly and nonsynaptically following granular cell
excitation by input from primary afferent fibers (Meek et al.
1999
). Two mechanisms may be suggested for nonsynaptic
excitation of the terminals: ephaptic excitation in which current
generated by the granular cells passes through the terminal,
hyperpolarizing it at entry and depolarizing it at exit; and
depolarization of the terminal due to potassium accumulation in the
synaptic cleft following granular cell excitation. These two mechanisms
have also been suggested for nonsynaptic activation of the calyx type
afferent terminals on type I vestibular hair cells (Goldberg
1996
). Alternatively, parts of the dendritic arbor of LMI cells
may be free of myelin and contacted by electrical or mixed
chemical-electrical synapses from primary afferent fibers or granular
cells. More detailed knowledge of LMI and granular cells is needed to
distinguish these different means of exciting LMI cells without
chemical excitatory transmission.
Paired-pulse depression
The postsynaptic granular layer response to the second of two
identical fiber layer stimuli showed was depressed for ~30 ms after
the first stimulus. The duration of the paired-pulse depression was
greatly enhanced by addition of the GABAA
antagonist, bicuculline. This long duration of paired-pulse depression
after addition of bicuculline appeared to be due in part to activation
of GABAB receptors, because the depression was
reduced by the further addition of the GABAB
receptor antagonist, 2-hydroxy-saclofen. The prolongation of
paired-pulse depression following addition of bicuculline is somewhat
surprising because one would suppose that GABA release and activation
of GABAA receptors after the first stimulus would contribute to depression of the response to the second stimulus. Results similar to ours have also been obtained in other systems, however. In the frontal cortex (Kang 1995) and
hippocampus (Higgins and Stone 1993
), bicuculline
increased paired-pulse depression, and this depression was partially
reduced by GABAB antagonists. GABAB antagonists were also found to reduce
paired-pulse depression in the olfactory bulb (Keller et al.
1998
).
The blockade of GABAA receptors with bicuculline results in a large increase in the response of granular cells to afferent stimuli, as indicated by the large increase in the postsynaptic response. The increased granular cell response under bicuculline would result in a longer refractory period for these cells and thus lead to a longer duration of paired-pulse depression. In addition, the increased granular cell response could result in a large increase in the amount of GABA released by LMI cells, supposing that granular cell excitation activates LMI cell terminals by synaptic or nonsynaptic mechanisms, as described in the preceding section. Increased GABA release would result in strong activation of GABAB receptors.
A single stimulus to the intermediate layer, at a site lateral to the
recording point in the granular layer where a postsynaptic response to
afferent stimulation was recorded, caused a reduction in the
postsynaptic response that lasted ~30 ms (Fig. 9A).
Bicuculline blocked this lateral inhibitory effect (Fig.
9B). This blockade is in contrast to the enhancement of
paired-pulse depression caused by the same drug. The lateral inhibitory
stimulus did not excite the granular cells at the recording site and
may have caused the release of only a small amount of GABA that was
sufficient to activate GABAA receptors but not
sufficient to activate GABAB receptors.
GABAB receptors are known to require release of
greater amount of GABA for activation than GABAA
receptors (Nicoll et al. 1990), due perhaps to location
of GABAB receptors outside the synaptic cleft.
Calcium independent release of GABA in the ELL granular layer
Bicuculline caused a marked increase in the postsynaptic response
to afferent stimulation in calcium-free medium and also after addition
of cadmium to the medium. These results imply that GABA release was
evoked when entry of calcium into the presynaptic terminal was either
prevented or greatly reduced. Such calcium-independent release of GABA
is unusual but has been demonstrated in the distal retina
(Schwartz 1987), in the striatum (Bernath and
Zigmond 1988
), in the hippocampus (During et al.
1995
), and in the cerebellum (Rossi and Hamann
1998
). The suggested mechanism in each case is a reversal in
the normal direction of the GABA transporter; instead of carrying GABA
into the cell, as normally occurs, the transporter carries GABA out of
the cell. Reversal of the transport direction occurs when the terminal
is depolarized or contains an elevated concentration of sodium (for
review see Attwell et al. 1993
). Nipecotic acid is an
antagonist of the GABA transporter and has been shown to block
calcium-independent release of GABA in several of the above systems.
Similarly, in the ELL, nipecotic acid prevented or reduced the marked
increase in the granular layer response to afferent stimulation caused
by bicuculline, under conditions in which calcium entry was blocked or
reduced (Fig. 6). Some might argue that the rapid onset of GABA release following primary afferent activation precludes a mechanism that involves reversal of the GABA transporter. However, experiments with
the glutamate transporter have shown that the initial phase of
transporter action can be activated within less than a millisecond when
transmitter concentrations are high (Wadiche and Kavanaugh 1998
), and the same might be true of the GABA transporter. Thus calcium-independent release of GABA in the ELL granular layer may be
present and may be dependent at least in part on the GABA transporter.
The evoked release of GABA is not measured directly in our experiments but is inferred on the basis of the large increase in postsynaptic responses that occurs when the GABA receptor antagonist, bicuculline, is added to the medium. It might be argued, however, that the effect of bicuculline is not due to blocking the effect of the evoked GABA release, but is instead due to blocking the effect of GABA that is present in the slice in the absence of afferent activation, due to tonic rather than evoked release. Blocking the tonic inhibition caused by GABA with bicuculline would make the granular layer cells more excitable, causing them to respond more vigorously to excitatory afferent input.
Two of our results suggest that GABA release in the ELL granular layer is evoked, and that the bicuculline effect cannot be explained by a blockade of tonic GABAergic inhibition. First, the fact that a GABAB antagonist reduced the depression of the postsynaptic response caused by a preceding stimulus implies that GABA release was evoked by the first stimulus. Second, the fact that bicuculline blocked the inhibition caused by a stimulus to the intermediate layer, just lateral to the recording site, means that GABA release was evoked by the lateral stimulus. Moreover, the lateral inhibitory effect was still present in calcium-free medium and was still blocked by bicuculline (Fig. 10).
The marked effects of bicuculline on the postsynaptic response in a calcium-free medium or in the presence of cadmium are evidence that the evoked release of GABA may occur in a calcium-independent manner in the granular layer of the mormyrid ELL. Field potentials are subject to a variety of interpretations, however, and the clear demonstration of calcium-independent transmitter release and analysis of its mechanisms will require intracellular recording from the cellular elements involved in the process.
Comparison with gymnotid electric fish
The electrosensory systems of gymnotid electric fish from South
America and mormyrid fish from Africa have a surprisingly large number
of similar features, given that the two electrosensory systems almost
certainly evolved independently (Finger et al. 1986).
Such similarities are present at the initial stages of central
processing of electrosensory information in ELL. Thus the synapses made
by primary afferent fibers in gymnotids have the morphology of mixed
chemical-electrical synapses, like those in mormyrids (Maler et
al. 1981
). In addition, the primary afferent fibers in
gymnotids activate large inhibitory neurons in the deeper layers, which
are known as ovoid cells. These ovoid cells have large widely branching
axons that terminate both ipsilaterally and contralaterally on granular
neurons and other cell types of ELL (Bastian et al.
1993
; Maler and Mugnaini 1994
) and are therefore similar to the inhibitory large multipolar interneurons (LMI cells) of
mormyrids. Thus afferent activation in both systems appears to result
in a powerful and widespread inhibition at this first stage of
electrosensory processing.
Differences between the two types of fish are also present at this
first stage of processing. First, in vitro studies of the gymnotid ELL
show that the excitatory effect of primary afferents on postsynaptic
cells is largely blocked by glutamate receptor antagonists, indicating
that transmission is mainly chemical, in spite of the morphological
evidence of mixed synapses (Berman and Maler 1998).
However, our results in mormyrids showed that synaptic excitation still
occurs when chemical transmission is blocked by glutamate receptor
antagonists blockers, by calcium-free medium, or by cadmium. The
recording of large synaptic potentials inside primary afferents in
mormyrids (Bell 1990
) also argues for the importance of
electrical transmission in these fish. Second, the inhibition caused by
ovoid cells in gymnotids has a very slow onset (Bastian
1993
) and appears to be mediated by receptors of the
GABAB type (Berman and Maler 1998
). This
again is in contrast to the mormyrid ELL, where the primary
afferent-induced inhibition was shown to have a very rapid onset and
to be mediated by GABAA receptors.
Functional implications of the large and rapid release of GABA for processing of electrosensory information
Electric fish generate an electric organ discharge (EOD) and sense
nearby objects by the distortions that such objects cause in the
pattern of self-generated electrical current that flows through their
skin, a process known as active electrolocation. In mormyrid electric
fish, the magnitude of current flowing through different regions of the
skin is sensed by mormyromast electroreceptors. The response latency of
afferent fibers from these electroreceptors is exquisitely sensitive to
the magnitude of the current (Szabo and Hagiwara 1967).
Behavioral (Hall et al. 1995
) results suggest that this
response latency, as measured from the time of a centrally originating
corollary discharge signal associated with the motor command that
drives the EOD, is the code for stimulus intensity. The behavioral
results indicate that fish are sensitive to changes in response latency
as small as 0.1 ms.
Strong and rapid lateral inhibition of the granular cells, mediated by the laterally spreading processes of large multipolar interneurons, as described in this paper, would enhance small differences in latency between the responses of electroreceptor afferents from adjacent skin regions and would minimize repetitive firing in response to the EOD. Responses of granular cells would therefore convey a sharpened electrical image to second-order cells in ELL.
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ACKNOWLEDGMENTS |
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This research was supported by grants from the National Science Foundation and the National Institute of Mental Health to C. C. Bell, and by funds from the Center National de Recherche Scientifique and a National Atlantic Treaty Organization traveling fellowship to K. Grant.
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FOOTNOTES |
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Address for reprint requests: V. Z. Han, Neurological Sciences Institute, Oregon Health Sciences University, 1120 N.W. 20th Ave., Portland, OR 97209.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 24 September 1999; accepted in final form 17 November 1999.
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REFERENCES |
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