Department of Zoology, University of Oklahoma, Norman, Oklahoma 73019
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ABSTRACT |
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Wilson, Donald A.. Comparison of Odor Receptive Field Plasticity in the Rat Olfactory Bulb and Anterior Piriform Cortex. J. Neurophysiol. 84: 3036-3042, 2000. Recent work in the anterior piriform cortex (aPCX) has demonstrated that cortical odor receptive fields are highly dynamic, showing rapid changes of both firing rate and temporal patterning within relatively few inhalations of an odor, despite relatively maintained, patterned input from olfactory bulb mitral/tufted cells. The present experiment examined the precision (odor-specificity) of this receptive field plasticity and compared it with the primary cortical afferent, olfactory bulb mitral/tufted cells. Adult Long-Evans hooded rats, urethan anesthetized and freely breathing, were used for single-unit recording from mitral/tufted and aPCX layer II/III neurons. Partial mapping of receptive fields to alkane odors (pentane, heptane, and nonane) was performed before and immediately after habituation (50-s exposure) to one of the alkanes. The results demonstrated that odor habituation of aPCX responses was odor specific, with minimal cross-habituation between alkanes differing by as few as two carbons. Mitral/tufted cells, however, showed strong cross-habituation within the odor set with the most profound cross effects to carbon chains shorter than the habituating stimulus. The results suggest that although mitral/tufted cells and aPCX neurons have roughly similar odor receptive fields, aPCX neurons have significantly better odor discrimination within their receptive field. The results have important implications for understanding the underlying bases of receptive fields in olfactory system neurons and the mechanisms of odor discrimination and memory.
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INTRODUCTION |
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Current theories of
odor discrimination by mammalian olfactory systems begin with molecular
feature recognition by a large set of odor receptors (Buck
1996; Mombaerts 1999
). A single receptor neuron,
expressing perhaps a single receptor protein, may respond to many odors
(Duchamp-Viret et al. 1999
; Kaluza and Breer
2000
; Malnic et al. 1999
) if each of those odors
includes the appropriate feature. This feature recognition is refined
through precise receptor axon project patterns to glomeruli in the main
olfactory bulb (MOB) and through lateral interactions between those
glomeruli and between their output neurons (mitral/tufted cells;
Bozza and Kauer 1998
; Vassar et al. 1994
;
Yokoi et al. 1995
). The individual features of a
particular odorant may then be combined through precise temporal
synchrony in mitral/tufted cell spike trains (Kashiwadani et al.
1999
) and/or through convergence within the primary olfactory
(piriform) cortex (Haberly 1985
; Lynch
1986
; Mori et al. 1999
).
Similar to olfactory receptor neurons, MOB mitral/tufted cells
(Katoh 1993; Meredith 1986
; Wellis
et al. 1989
) and anterior piriform cortex (aPCX) neurons
(Haberly 1969
; McCollum et al. 1991
;
Tanabe et al. 1975
; Wilson 1998a
) have
relatively broad receptive fields, responding to multiple odorants.
Within a particular class of odorants (e.g., aliphatic acids or
aldehydes), receptive fields can be mapped along a dimension of carbon
chain length (Katoh et al. 1993
). Using this paradigm,
molecular or odor receptive fields can be described wherein a single
receptor or mitral/tufted cell responds to a sequential series of chain
lengths (e.g., 3-7 carbons) but not to chain lengths longer (
8) or
shorter (
2). Mitral/tufted cell receptive fields are further
frequently characterized by inhibitory surrounds (Katoh et al.
1993
; Sato et al. 1994
), with suppressive
responses evoked by the outlier chain lengths (8 and 2 carbon chain
lengths in this example). Receptive fields of aPCX neurons have been
less thoroughly described but appear to be similar to those of
mitral/tufted cells with responsiveness occurring within a sequential
series of carbon chain lengths (Wilson 2000
).
The breadth of receptive fields in the olfactory receptors and MOB
mitral/tufted cells has been interpreted as due to coding of specific
molecular features or odotopes that may be a component of many odors
(Malnic et al. 1999; Mori et al. 1999
;
Shepherd 1994
). In contrast, cross-habituation studies
suggest that aPCX receptive fields may be due to relatively independent
odor inputs, which can be selectively modified by experience, leaving
responses to other odors stable (Wilson 1998a
, 2000
).
The present study used a cross-habituation paradigm with alkane odors
of varying carbon chain length to specifically compare receptive field
dynamics and odor discrimination between MOB mitral/tufted cells and
aPCX neurons. The results suggest that while mitral/tufted cells show cross-habituation between odors within their receptive fields, aPCX
neurons show very little cross-habituation, significantly enhancing
odor discrimination at the cortical level.
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METHODS |
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Male Long-Evans hooded rats (150-450 g) from Charles River Labs were used as subjects. Animals were housed in polypropylene cages with food and water available ad libitum. Lights were maintained on a 12:12 light: dark cycle and testing occurred during the light portion of the cycle.
Single-unit recordings from mitral/tufted cells and aPCX layer II/III
neurons in urethan-anesthetized, freely breathing rats were obtained as
previously described (Wilson 1998a, 2000
). Briefly, electrical stimulation of the lateral olfactory tract (LOT) was used to
identify mitral/tufted cells by antidromic activation and layer II/III
aPCX neurons from extracellular evoked field potentials and synaptic
activation (Fig. 1) (Haberly and
Shepherd 1973
). Respiratory activity was monitored via a
piezoelectric device strapped to the animal's chest. Single units were
isolated directly, or were extracted through template matching (10-kHz sampling rate) using Spike2 software for the Macintosh (CED). The
single-unit nature of the recordings were confirmed with
autocorrelograms showing at least a 2-ms refractory period.
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Odor stimulation
A continuous stream (600 ml/min) of air, passed through an
activated charcoal filter and humidified, was blown across the nares of
the freely breathing animal. A computer controlled Picospritzer forced
air through odorant saturated syringe filters (2.7 µ glass microfiber, Whatman), which mixed with the clean airstream to create
odor concentrations of
~101 of saturated
vapor. Odorants used were pentane, heptane, and n-nonane
(Sigma-Aldrich). Stimulus onset was triggered on the respiratory cycle
(at the inhalation/exhalation transition).
Mitral/tufted cell or aPCX neuron partial receptive fields to these
three alkanes were mapped with 2-s odor pulses, separated by 60 s
between each stimulus. Order of stimuli were randomly varied within and
between cells. Stability of receptive fields were determined by a
second presentation of each stimulus. In cells responding to at least
two of the stimuli, one of the stimuli was pseudo-randomly chosen to
serve as the habituating stimulus (50-s duration). The receptive field
was then remapped 15-50 s after the termination of the habituation
exposure and recovery was tested at least 2 min later. In most cases,
only one habituating series was used for a single odor in individual animals.
Spike counts during the 2-s stimuli were compared with counts during the immediate 2 s preceding the stimulus. Response magnitude to posthabituation stimuli were expressed relative to the mean response magnitude prehabituation. Response magnitude during the 50-s odor stimuli were determined in 10-s bins, by comparing odor-evoked spike counts (difference from preodor activity) in each bin with those evoked during the first 10 s of odor stimulation. Data are plotted as means ± SE.
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RESULTS |
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A total of 24 mitral/tufted cells and 21 aPCX neurons responded to
at least two of the test odors and were included in the data set. Both
mitral/tufted cells and aPCX layer II/III neurons displayed receptive
field properties to alkanes similar to those described by
Imamura et al. (1992) for rabbit olfactory bulb
mitral/tufted cells (Fig. 1A). Individual cells responded to
sequential series of chain lengths; that is, there was no cell observed
that responded to both pentane and nonane without also responding to
heptane. These responses to 2-s odor pulses were relatively stable as
long as interstimulus intervals exceeded
60 s (Fig. 1B).
Following partial mapping of mitral/tufted and aPCX neuron receptive
fields to alkanes, one of the three odors was pseudo-randomly chosen to
be the 50-s habituating stimulus. Responses to the 50-s alkane stimuli
revealed a marked difference between mitral/tufted cells and aPCX
neurons. As previously reported for other odors (Wilson
1998a), aPCX neurons rapidly habituated to alkanes compared with mitral/tufted cells (Fig. 2). Thus
although both cell types displayed some response decrement during the
50-s stimulation, there was a significant difference in odor-evoked
response magnitude during the final 10 s of stimulation between
cell types [t(43) = 2.99, P < 0.01]. This
difference in habituation rate was apparent for all three alkane
stimuli (Fig. 2B).
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Interestingly, after the termination of the 50-s habituating stimulus,
while aPCX responses began to recover almost immediately (Wilson
1998a), mitral/tufted cell responses to 2-s test stimuli continued to decline for ~30 s before recovery onset. This difference in posthabituating stimulus activity fortuitously allowed comparison of
cross-habituation at a time when self-habituation levels in mitral/tufted cells and aPCX neurons were roughly similar (e.g., Fig.
4, 0 carbon chain length difference).
In addition to habituation rate/magnitude, the extent of cross-habituation between alkanes also differed significantly between MOB mitral/tufted cells and aPCX neurons. Figure 3 shows examples of odor receptive fields in a mitral/tufted cell and aPCX neuron before and after habituation. The mitral/tufted cell in Fig. 3A showed strong responses (number of odor-evoked spikes) to all three odors prior to habituation to heptane. Following heptane habituation, responses to all three odors were reduced, until recovery ~2 min later. In contrast, the aPCX neuron, which also responded to all three alkanes, showed a selective decrease in odor-evoked spiking to nonane following habituation to nonane (Fig. 3, B and D); responses to pentane and heptane were relatively unchanged.
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A plot of cross-habituation as a function of carbon chain length difference (Fig. 4) shows significantly greater cross-habituation between alkanes differing by two or four carbons in mitral/tufted cells compared with aPCX neurons [ANOVA, main effect of cell type, F(1,101) = 7.79, P < 0.01. Post hoc Fisher tests revealed a significant difference between mitral/tufted cells and aPCX neurons in cross-habituation between stimuli differing by two or four carbons, P < 0.05]. Self-habituation levels were similar between mitral/tufted cells and aPCX neurons at this time point (15-50 s posttermination of the habituating stimulus).
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The specific effects of habituation on MOB mitral/tufted and aPCX neuron odor receptive fields is shown in Fig. 5. Habituation to a single alkane had much more selective effects on aPCX receptive fields than on mitral/tufted cell receptive fields. Thus while habituation to one chain length produced marked self-habituation in aPCX neurons, responses to other chain lengths were relatively unaffected. Mitral/tufted cells, however, showed suppression of responses across the receptive field following habituation to a single odor.
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Furthermore the cross-habituation expressed by MOB mitral/tufted cells was asymmetrical. For example, habituation to heptane produced more cross habituation to pentane than to nonane (Fig. 5). In fact, significantly greater cross-habituation occurred to carbon chains shorter than the habituating stimulus compared with cross-habituation to carbon chains longer than the habituating stimulus in the MOB [Fig. 6; ANOVA, main effect of relative chain length; F(1,61) = 7.68, P < 0.01]. Post hoc Fisher tests revealed significantly (P < 0.05) greater cross-habituation to shorter chains than longer chain in mitral/tufted cell responses but no significant asymmetry in aPCX cross-habituation.
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DISCUSSION |
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The present results demonstrate that while MOB mitral/tufted cells
and aPCX neurons have superficially similar static odor receptive
fields, examination of receptive field dynamics reveals important
differences between these two cell types. Neurons in the aPCX showed
more rapid and odor-specific habituation than MOB mitral/tufted cells.
The rapid habituation in the aPCX relative to its primary afferent, MOB
mitral/tufted cells, may in part be due to synaptic depression of LOT
afferents (Wilson 1998b) and could allow cortical
filtering of prolonged stimuli. The enhanced habituation in cortex
compared with more peripheral structures is similar to that reported
for both vision (Miller et al. 1991
; Sanchez-Vives et al. 2000
) and audition (Condon
and Weinberger 1991
). The reduced cross-habituation apparent in
the aPCX relative to MOB mitral/tufted cells suggests that computations
occurring within the aPCX enhance odor discrimination, similar to the
MOB enhancement of odor discrimination compared with the olfactory receptor sheet (Duchamp and Sicard 1984
; Yokoi et
al. 1995
). Just as the rat aPCX is capable of discriminating
between 2 carbons chain length differences, rats are capable of making
behavioral odor discriminations of 1-2 carbon chain length differences
(Linster and Hasselmo 1999
). Given the differences in
odor single-unit discrimination shown here, this behavioral
discrimination may be more dependent on the aPCX than on the MOB.
In the rodent MOB, single mitral/tufted cells receive olfactory nerve
input within a single glomerulus and, thus given the precise projection
patterns of receptor cell axons, potentially receive direct input from
receptor neurons all expressing the same receptor protein
(Ressler et al. 1994; Vassar et al.
1994
). Despite expressing a single receptor protein type,
individual vertebrate olfactory receptor neurons respond to multiple
odors (Duchamp-Viret et al. 1999
; Firestein et
al. 1993
; Malnic et al. 1999
; Sato et al.
1994
; Sicard and Holley 1984
; Touhara et
al. 1999
) and show cross-adaptation between odors
(Caprio and Byrd 1984
; Lipschitz and Michel
1999
; although see Kashiwayanagi and Kurihara
1994
) as reported here for mitral/tufted cells. Thus it appears
likely that mitral/tufted cell odor receptive field dynamics stem, in
part, from a loose coding of molecular features at the receptor level,
such that exposure to one stimulus affects responses widely through the
receptive field. Similar results have been reported in awake rats where
repeated, 20-min stimulation with a single odor produced long-term
decreased responding of mitral/tufted cells to that odor, as well as
molecularly dissimilar odors (Buonviso et al. 1998
).
However, the specificity of habituation effects on aPCX neuron
receptive fields is in sharp contrast to cross-habituation expressed at
the receptor and MOB levels. The question arises then if more
peripheral structures have broadly depressed receptive fields following
exposure to a single odor, how can the aPCX display such selective
habituation? While precise mechanisms of enhanced odor discrimination
in the cortex remain to be clarified, feedback/association systems
within the cortex and between the cortex and MOB are most likely
involved (Bower 1991; Haberly 1985
;
Hasselmo and Barkai 1995
).
At another level of analysis, perhaps the difference in cross-habituation is evidence that mitral/tufted cells and aPCX neurons respond to the same odors but for different reasons. For example, in the visual system, neurons in the lateral geniculate nucleus of the thalamus respond maximally to points of light that fall on their center-surround receptive fields. Visual cortex neurons with simple receptive fields respond maximally to bars of light or contrast edges (an ensemble of many points of light) in particular orientations. Thus both of these cell types would respond to bars of light if the bar overlaps with their specific receptive field but for different reasons.
A similar difference in the level of analysis performed by MOB and aPCX
neurons may occur in the olfactory system with mitral/tufted cells
responsive to specific odorant features expressed by many odorants and
aPCX neurons responsive to feature ensembles (Bower 1991; Haberly 1985
; Lynch 1986
;
Mori et al. 1999
). This difference in coding between the
two cell types could account for the observed difference in
cross-habituation. The schematic shown in Fig.
7 shows three odors each composed of
three odorant/molecular features. Individual odorant receptor neurons
and MOB mitral/tufted cells respond to particular features. Thus
mitral/tufted cell A responds to all three odors because they each
contain the A feature. The aPCX neuron, however, responds to all three
odors because it is driven by the feature ensembles ABC, ADE, and AFG
due to patterns of mitral/tufted cell afferent convergence and/or
association fiber convergence.
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With this difference in the underlying basis of receptive fields,
habituation to odor 2, for example, would produce effects in
the mitral cells and in the aPCX neuron similar to that shown in the
data reported here. Mitral cell A would show cross-habituation to both
odors 1 and 3 because they both contain the A
feature to which this cell is primarily responsive and to which it is now habituated. The aPCX neuron, on the other hand, while habituated to
the ADE ensemble should continue to respond well to both the ABC and
AFG ensembles given that many of the features in those odors are still
effective. The reduction in input from the A component of these
ensembles could be compensated for by cortical processing. Recent
models of piriform cortex suggest that association circuitry and
neuromodulatory inputs enhance the cortical ability to appropriately interpret partially degraded signals (Hasselmo and Barkai
1995). The minimal cross-habituation between binary odor
mixtures and their components previously reported (Wilson
2000
) further supports this cortical ensemble coding hypothesis.
Asymmetry in cross-habituation
The cross-habituation expressed by MOB mitral/tufted cells was
asymmetrical with chain lengths shorter than the habituating stimulus
expressing greater cross-habituation than chain lengths longer than the
habituating stimulus. Although in the present study the aPCX did not
demonstrate asymmetry, a similar asymmetry in cross-habituation between
carbon chains of different length has been demonstrated in human
psychophysics. Habituation to the alcohol pentanol (C5) produces
greater cross-habituation to the shorter alcohol propanol (C3) than
visa versa (Cain 1970).
Olfactory receptor neurons discriminate differences in carbon chain
length (Kaluza and Breer 2000; Sato et al.
1994
), and as chain length increases, the probability of
receptor cell activation increases (Sato et al. 1994
).
Similarly as carbon chain length increases, there is an increase in the
complexity of odor-evoked spatial patterns of MOB glomerular layer
activation (Johnson et al. 1999
). In fact, examination
of the published spatial maps of glomerular activation to aliphatic
acids (Johnson et al. 1999
) suggests that odors with
shorter chain lengths may activate a subset of the same glomeruli
activated by longer chains (e.g., the response to a 7 carbon chain
stimulus may include within it the response elements for a 5 or 6 carbon chain). If this is the case, then habituation to the longer
chain stimulus might be expected to produce strong cross-habituation to
shorter chain stimuli, as observed here in mitral/tufted cells.
What does the aPCX contribute to odor processing?
The present results, combined with previous work (Wilson
1998a, 2000
), suggest that the aPCX is a rapidly adapting
system with excellent odor-discrimination capabilities. Rapidly
adapting systems are maximally responsive to change, allowing filtering of background or currently nonrelevant stimuli. In addition, the aPCX
demonstrates significantly greater odor discrimination capabilities than its primary afferent, MOB mitral/tufted cells, as determined by
cross-habituation (Fig. 4). This combination of rapid habituation and
precise odor discrimination would allow the aPCX to filter background
stimuli yet maintain responsiveness to similar, novel odors or change.
In addition to allowing precise odor habituation, the highly specific
modulation of odor receptive fields in aPCX could enhance precision of
odor discrimination, identification, and memory. Experience can produce
either decreases or increases in cortical responsiveness to specific
stimuli. For example in the auditory system, habituation causes highly
frequency-specific decreases in cortical responses to the repeated tone
(Condon and Weinberger 1991), while associative
conditioning produces highly frequency-specific increases in both
thalamic and cortical responses to the conditioned tone (Edeline
and Weinberger 1992
; Weinberger 1993
). It is
hypothesized that aPCX receptive fields could similarly be modulated in
either direction with experience, and receptive field dynamics could play a major role in olfactory discrimination and memory (Bower 1991
; Haberly 1985
; Lynch 1986
).
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ACKNOWLEDGMENTS |
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The author thanks Dr. Regina Sullivan for comments on an earlier draft of the manuscript.
This work was supported by National Institute on Deafness and Other Communication Disorders Grant DC-03906.
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FOOTNOTES |
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Author E-mail: dwilson{at}ou.edu
Received 19 June 2000; accepted in final form 25 August 2000.
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REFERENCES |
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