Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Christie, J. M.,
N. E. Schoppa, and
G. L. Westbrook.
Tufted Cell Dendrodendritic Inhibition in the Olfactory Bulb Is
Dependent on NMDA Receptor Activity.
J. Neurophysiol. 85: 169-173, 2001.
Mitral and tufted cells
constitute the primary output cells of the olfactory bulb. While tufted
cells are often considered as "displaced" mitral cells, their
actual role in olfactory bulb processing has been little explored. We
examined dendrodendritic inhibition between tufted cells and
interneurons using whole cell voltage-clamp recording. Dendrodendritic
inhibitory postsynaptic currents (IPSCs) generated by depolarizing
voltage steps in tufted cells were completely blocked by the
N-methyl-D-aspartate (NMDA) receptor antagonist
D,L-2amino-5-phosphonopentanoic acid
(D,L-AP5), whereas the
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor
antagonist 2-3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f] quinoxaline-7-sulfonamide (NBQX) had no effect. Tufted cells in the external plexiform layer (EPL) and in the periglomerular region (PGR) showed similar behavior. These results indicate that NMDA receptor-mediated excitation of interneurons drives inhibition of
tufted cells at dendrodendritic synapses as it does in mitral cells. However, the spatial extent of lateral inhibition in
tufted cells was much more limited than in mitral cells. We suggest
that the sphere of influence of tufted cells, while qualitatively
similar to mitral cells, is centered on only one or a few glomeruli.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The two classes of
primary output neurons in the main olfactory bulb, mitral cells and
tufted cells, share several common features. Each receives sensory
input from olfactory nerve axons in the neuropil of the glomerular
layer and in turn project their axons to piriform cortex. Within the
bulb, dendrodendritic synapses form between interneurons and the
primary and secondary dendrites of both mitral and tufted cells. Mitral
cells are located in a compact layer referred to as the mitral cell
layer (MCL), whereas tufted cells are dispersed throughout the external
plexiform layer (EPL) and the periglomerular region (PGR) (Cajal
1911). Tufted cells are also considerably more diverse in their
morphology (Scott and Harrison 1991
).
Our understanding of olfactory bulb function is largely based on
studies of mitral cells and the dendrodendritic synapses between mitral
and granule cells. At this unique synapse, release of glutamate from
mitral cell dendrites drives GABA release from granule cells that then
leads to recurrent and lateral inhibition of mitral cells.
Dendrodendritic inhibition of mitral cells can be quite prolonged and
follows the slow kinetics of
N-methyl-D-aspartate (NMDA) receptors on granule
cells (Mori and Takagi 1978; Schoppa et al.
1998
). Dendrodendritic inhibition provides the first step in
the network processing of odorant responses that are mapped onto
glomeruli in a highly ordered manner (Vassar et al.
1994
). As a result, the duration and spatial extent of
GABAergic inhibition is likely to have a major impact on sensory integration.
The role of tufted cells in olfactory processing is not well
characterized, but there is reason to think their function is distinct
from mitral cells. For example, the distribution of dendrites in the
bulb suggests that the dendrites of principal cells may preferentially
interact with different sets of interneurons. Granule cell dendrites
are present either in the deep zones of the EPL where secondary mitral
cell dendrites are located, or in more superficial zones of the EPL
where tufted cell secondary dendrites are located (Mori et al.
1983; Orona et al. 1983
). Furthermore, dendrodendritic synapses of tufted cells in the PGR may be restricted to periglomerular cell interneurons (Pinching and Powell
1971a
; Price and Powell 1970a
,b
). Distinct
interneuronal circuits may also underlie the differences in inhibitory
postsynaptic potential (IPSP) size in these two types of principal
cells (Ezeh et al. 1993
). We examined dendrodendritic
inhibition in tufted cells in both the EPL and PGR using whole cell
recording from slices of young rats. Tufted cells were identified
visually by their location; morphological subtypes were classified
using intracellular dye injections.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Slices of the main olfactory bulb (400 µM) were prepared from
Sprague-Dawley rat pups [postnatal day 10 to 14 (P10-P14)] as described (Schoppa et al.
1998). Tufted cells, visualized by infrared DIC optics, were
identified by location and morphology (Scott and Harrison
1991
). All experiments were performed at room temperature (21-24°C). Whole cell voltage-clamp recordings were made in an oxygenated, magnesium-free solution containing (in mM) 125 or 140 NaCl,
25 NaHCO3, 25 glucose, 2.5 KCl, 1.25 NaH2PO4, and 2 CaCl2, pH 7.3. Patch pipettes (4-8 M
) were
filled with an intracellular solution containing (in mM) 140 KCl, 10 EGTA, 10 HEPES, 2 MgCl2, 2 CaCl2, 2 NaATP, and 0.5 NaGTP, pH 7.3. In some
cases, tufted cells were filled with the dye Alexa 568 hydrazide
(Molecular Probes, Eugene, OR; 0.1 mg/ml) and visualized after fixation
using confocal microscopy (Odyssey XL, Noran Instruments, Middleton, WI). To verify the location of tufted cell dendrites, slices were counterstained with the nucleic acid stain SYTO-13 (1:4,000, Molecular Probes, Eugene, OR).
To evoke dendrodendritic inhibitory postsynaptic currents (IPSCs), a
depolarizing voltage step (0 mV, 5 ms) was applied to the soma of a
voltage-clamped tufted cell. To examine lateral inhibition, IPSCs were
evoked by stimulating the glomerular layer with a tungsten electrode
(0.5 M, WPI, Sarasota, FL) centered on a single glomerulus.
Stimulation pulses (100 V, 100 µs) were generated by a stimulation
isolation unit (Winston Electronics, Millbrae, CA). IPSCs were recorded
with an Axopatch 1B (Axon Instruments, Foster City, CA), filtered with
the built-in 4-pole Bessel filter and digitized at 2 kHz. Access
resistance (Rs) was constantly monitored; recording was terminated when
Rs was >15 M
. All data were
analyzed using AXOGRAPH (Axon Instruments). IPSC charge was estimated
by integrating the baseline-subtracted current. Statistical significance was determined using standard Student's
t-tests or repeated measures ANOVA as appropriate (Microsoft
Excel, Redmond, WA).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Dendrodendritic inhibition in tufted cells is driven by NMDA receptors
We first examined tufted cells with cell bodies located in the
intermediate zone of the EPL (Fig.
1A). In whole cell voltage clamp, a depolarizing voltage step (0 mV, 5 ms) elicited a slowly decaying IPSC due to activation of dendrodendritic synapses. Bath application of bicuculline methiodide (40 µM) completely blocked the
IPSC, consistent with activation of GABAA
receptors (5 ± 2.9% of control, mean ± SE,
n = 3). The selective NMDA receptor antagonist D,L-2-amino-5-phosphonopentanoic acid (D,L-AP5;
50 µM) completely and reversibly abolished the IPSC, whereas the
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor
antagonist 2-3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f] quinoxaline-7-sulfonamide (NBQX) (10 µM) had no effect
(Fig. 1, B-D). The dependence of dendrodendritic
inhibition on NMDA receptor activation is also observed in mitral cells
under the same conditions (see Schoppa et al. 1998
). The
amplitude and time course of the IPSCs were similar to those recorded
in mitral cells (not shown) (see Schoppa et al. 1998
).
|
As for mitral cells, dendrodendritic inhibition of tufted cells may
occur either on primary dendrites in the glomerular layer or on
secondary dendrites in the EPL (Macrides and Schneider
1982; Mori et al. 1983
; Orona et al.
1984
). However, tufted cells can be subtyped into three groups
based on their somatic location and dendritic arbors (Scott and
Harrison 1991
): Ts, soma and
dendrites in the superficial EPL; Tm,
soma and dendrites in the intermediate EPL (Fig.
2A); and
Ti, soma in internal portion of the
EPL with dendrites in intermediate EPL. In addition, there is at least one morphologically distinct subtype in the PGR (Fig. 2B)
that is characterized by a single large primary dendrite that projects to a single glomerulus and branches repeatedly within it. Secondary dendrites are rare on PGR tufted cells (Pinching and Powell
1971a
), thus inhibitory input must be largely from
periglomerular cells, rather than granule cells.
|
Dendrodendritic IPSCs generated in tufted cells with somata in the EPL (Fig. 3A1, top) shared similar characteristics to those generated in tufted cells located within the PGR (Fig. 3A1, bottom). The IPSC decay time constant, time-to-peak, peak amplitude, and IPSC charge were similar for both groups as shown in Fig. 3A2. D,L-AP5 (50 µM) completely and reversibly abolished the dendrodendritic IPSCs in tufted cells in the periglomerular region and in all zones of the EPL. In contrast, NBQX (10 µM) had no effect (Fig. 3B). Thus despite the differences in morphology, all types of tufted cells generate dendrodendritic inhibition with similar receptor pharmacology and kinetics.
|
Spatial extent of lateral inhibition in tufted cells is less than mitral cells
Our results demonstrate that the general features of
dendrodendritic inhibition are similar in mitral and in tufted cells. However, morphological differences, such as the longer secondary dendrites of mitral cells, could affect the extent of lateral inhibition. Hence, we compared the spatial extent of lateral inhibition in mitral cells with that of tufted cells in the intermediate EPL. A
bipolar electrode was used to stimulate individual glomeruli at varying
lateral distances (L) from the test cell (Fig.
4A1). Under our conditions,
the bipolar electrode was expected to directly activate the primary
dendrites of principal cells within an underlying glomerulus. Because
the primary dendrites of principal cells project only to a single
glomerulus (Scott and Harrison 1991
), lateral movement
of the stimulating electrode can be used to quantify the spatial extent
of lateral inhibition. As shown for an intermediate tufted cell
(Tm) in Fig. 4A2, an IPSC
generated by stimulation at a lateral separation (
L) of
250 µm was markedly smaller than at the control location
(
L = 0 µm). The relative degree of lateral inhibition in intermediate tufted cells
(Tm) was less than mitral cells for
all distances examined (Fig. 4, B and C).
Inhibition in tufted cells was completely eliminated at
L
400 µm, while inhibition in mitral cells was
approximately half-maximal at the same distance (Fig. 4B).
For all intervals (100-400 µm, binwidth 100 µm), the extent of
lateral inhibition in tufted cells was significantly less than that of
mitral cells (Fig. 4C).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
NMDA receptor dependence is a general property of dendrodendritic inhibition in the olfactory bulb
Previous reports have demonstrated an unusual dependence of mitral
cell dendrodendritic inhibition on NMDA receptor activity in granule
cells (Isaacson and Strowbridge 1998; Schoppa et
al. 1998
). Our results indicate that inhibition in tufted cells
shares this property regardless of their morphology or location. Mitral and tufted cells receive inhibitory input from granule and
periglomerular cells. Direct recordings from granule cells have
demonstrated that excitatory postsynaptic currents (EPSCs) from single
mitral cells are conventional in that they have both AMPA and NMDA
components, but that the AMPA receptors act primarily to facilitate the
depolarization necessary to relieve magnesium block of NMDA receptors.
Both the long duration of the NMDA receptor-mediated EPSC as well as
its calcium permeability appear to contribute to the release of GABA at
dendrodendritic synapses in the bulb (Chen et al. 2000
;
Halabisky et al. 2000
; Schoppa and Westbrook
1999
).
Although we did not examine EPSCs in periglomerular (PG) cells
evoked by stimulation of single tufted cells, PG cells also express
NMDA receptor subunits (Giustetto et al. 1997). Thus we expect that tufted cells also drive the activation of NMDA receptors on
these interneurons, leading to GABA release. As for mitral cells,
stimulation of only a single tufted cell was sufficient in the absence
of extracellular magnesium to evoke a dendrodendritic IPSC. In
physiological magnesium, it is expected that stronger stimulation such
as activation of many mitral or tufted cells by olfactory nerve
activity will be required to depolarize the principal cell sufficiently
to activate recurrent and lateral inhibition. Our recordings were
performed in slices from rat pups (P10-P14). However, a
similar pattern of NMDA receptor dependence has been observed in young
adult rats using c-fos mRNA expression as a measure of cell
activity (Schoppa et al. 1998
). In those experiments,
pretreatment with MK-801 increased c-fos mRNA expression in
mitral cells consistent with their disinhibition. Close examination also revealed increased cellular activity in scattered large cells throughout the EPL and in PGR, suggesting that there was simultaneous disinhibition of tufted cells.
NMDA receptors are also present on mitral cell dendrites and contribute
an autoexcitatory response that may influence the excitability of the
mitral cell dendrite as well as resulting interneuronal activity
(Isaacson 1999; Nicoll and Jahr 1982
). We
have also recorded autoexcitatory responses in tufted cells in the EPL
(Christie, unpublished observation), providing further evidence that
the organization of synaptic glutamate receptors is common to both
mitral and tufted cells.
Morphological differences between classes of principal cells have functional consequences on signal processing
Although morphological differences between subtypes of
interneurons (Kosaka et al. 1998; Mori et al.
1983
; Orona et al. 1983
) as well as principal
cells in the olfactory bulb have been long recognized, the functional
consequences of this cellular diversity remains poorly understood. In
our experiments, tufted cells located in the PGR, whose dendrodendritic
synapses are limited to PG cells (Pinching and Powell
1971b
), were not functionally distinct from tufted cells in the
EPL that contact PG cells and granule cells (Mori et al.
1983
; Orona et al. 1983
). Tufted cells in the
EPL, while similar in overall morphology to mitral cells, have shorter secondary dendrites (Mori et al. 1983
; Orona et
al. 1984
), which might predict differences in lateral
inhibition. Consistent with this idea, we found that the spatial extent
of lateral inhibition in tufted cells located in the intermediate zone
of the EPL was less than mitral cells. Secondary dendrites of tufted
cells in superficial zones of the EPL are even shorter than those in
deeper zones (Mori et al. 1983
; Orona et al.
1984
). Thus the spatial extent of lateral inhibition may be
even less in superficial tufted cells than in the tufted cells in the
intermediate EPL. We were not able to explore this issue using direct
glomerular stimulation because of the likelihood of electrical
artifacts in superficial tufted cells adjacent to the stimulator. Our
results suggest that the extent of lateral inhibition of intermediate
tufted cells is local, extending for several glomeruli (
400 µM),
whereas the extent of lateral inhibition of mitral cells extends more
broadly (
750 µM). Our experiments were performed in magnesium-free
solutions that enhance NMDA receptor responses. However, lateral
inhibition is also present in mitral cells in physiological
extracellular magnesium, although the spatial extent of lateral
inhibition is somewhat reduced (Schoppa, unpublished observation).
The precise spatial map of odorant receptors onto glomeruli in the bulb
is thought to provide the basis for the odorant code. However, lateral
inhibition is likely to be important in tuning of the glomerular map.
Several lines of evidence suggest that local interglomerular
interactions mediated by tufted cells could occur. For example, odorant
receptors that respond to similar odorants are highly homologous
(Malnic et al. 1999). Likewise, olfactory receptor
neurons that express highly related odorant receptors appear to project
their axons to glomeruli that are in close proximity (Tsuboi et
al. 1999
). Consistent with active local inhibitory
interactions, neighboring principal cells that respond to n-aliphatic
aldehydes are often inhibited by aldehydes whose aliphatic chain is one
or more carbon shorter (Yokoi et al. 1995
). Such issues
are presumably important in odor detection and discrimination as
odorants activate multiple glomeruli (Mori et al. 1999
;
Rubin and Katz 1999
) to generate the combinatorial code
that constitutes a perceived odor (Malnic et al. 1999
).
![]() |
ACKNOWLEDGMENTS |
---|
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-26494.
![]() |
FOOTNOTES |
---|
Address for reprint requests: J. M. Christie, Vollum Institute, Oregon Health Sciences University, 3181 SW Sam Jackson Park Rd., Portland, OR 97201 (E-mail: christij{at}ohsu.edu).
Received 13 June 2000; accepted in final form 29 September 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|