Chem. Senses 27: 1-6,
2002
© Oxford University Press 2002
The Minimum Number of Neurons in the Central Olfactory Pathway in Relation to its Function
a Retrograde Fiber Tracing Study
Department of Anatomy, Shinshu University School of Medicine, Matsumoto 390-8621, Japan 1 Department of Neurosurgery, Shinshu University School of Medicine, Matsumoto 390-8621, Japan
Correspondence to be sent to: Tetsuji Moriizumi, Department of Anatomy, Shinshu University School of Medicine, Matsumoto 390-8621, Japan. e-mail: moriizum{at}sch.md.shinshu-u.ac.jp
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Abstract |
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The present study was aimed at determining the functionally essential size of the neuronal population in the central olfactory nervous system. Using conditioned rats who had learnt to avoid repellent (cycloheximide) solution by olfaction, varying degrees of injuries were made to the lateral olfactory tract, a major central olfactory pathway connecting the olfactory bulb to the olfactory cortex. After examining their olfactory ability to discriminate cycloheximide solution from water, intact bulbar projection neurons (mitral cells) with fiber connections to the olfactory cortex were quantified using a retrograde fiber tracing technique. The numbers of retrogradely labeled mitral cells from the rats with normal olfaction ranged between 20 and 92% of the control value, while those numbers from the anosmic rats ranged between 0 and 22%. We conclude that the functionally essential neuronal population is approximately one-fifth of the total in the central olfactory pathway, a presumed threshold value in terms of the ability to avoid cycloheximide solution by olfactory discrimination.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Sensation is perceived by neuronal networks from sensory organs to the brain. In the brain, many central sensory pathways transmit peripheral sensory information to specific cortical areas, depending on the different senses. However, the minimal number of sensory neurons and fibers (central sensory pathways) needed for a specific sensation has yet to be determined.
The olfactory nervous system is a hierarchically ordered neuronal pathway
that consists of three main stations: the olfactory epithelium, olfactory bulb
and olfactory cortex. Olfactory receptor neurons in the olfactory epithelium,
transforming chemical stimuli to electric signals, send their axons (olfactory
nerves) to the olfactory bulb and make synapses with bulbar projection neurons
(mitral cells) (Lancet, 1988
;
Buck and Axel, 1991). The
mitral cells, developmentally similar to cortical pyramidal neurons, are the
biggest neurons in the bulb, and project to the olfactory cortex via the
lateral olfactory tract (LOT) (Scott,
1986; Greer, 1991;
Kratskin, 1995;
Moriizumi et al.,
1995). Thus the LOT is a major central olfactory pathway
connecting the olfactory bulb to the olfactory cortex, and is made up of
myelinated fiber bundles of mitral cells.
We have already established a simple and accurate method to examine
olfactory function in rodents by odor aversion behavior
(Kimura et al., 1991;
Moriizumi et al.,
1994). This is based on the ability to discriminate repellent
(cycloheximide) solution from water by olfaction. Since its taste is so
disgusting, rodents remember the smell of the solution when they drink it, and
thereafter learn to avoid it by olfaction. Thus an olfactory conditioned
reaction is established that enables us subsequently to examine olfactory
function.
The present study was undertaken to determine neuroanatomically the minimum number of mitral cells required for the ability to avoid cycloheximide solution by olfaction. For this purpose, a retrograde neuronal tracer was employed to quantify the numbers of intact mitral cells with fiber connections to the olfactory cortex among LOT-lesioned rats that were functionally divided into two groups: one with normal olfaction and one with loss of olfaction in terms of cycloheximide solution.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The experiments were carried out on adult male Wistar rats (11-13 weeks old, 250-300 g). All surgical manipulations were done under general anesthesia with sodium pentobarbital (50 mg/kg, i.p.). First, unilateral olfactory bulbs were completely ablated by aspiration within a 21G needle. The unilaterally bulbectomized rats (n = 31) were trained to avoid cycloheximide solution. The detailed experimental procedures of the olfactory discrimination test were similar to those in our previous paper (Moriizumi et al., 1994
) but with the slight modification mentioned below. When the
rat sniffed the cycloheximide solution and drank it, the response was
interpreted as a wrong response. When the rat sniffed the cycloheximide
solution, avoided it and drank water, the response was regarded as a correct
response. The number of correct responses was divided by the number of total
responses, and the percentage of correct responses was calculated. All rats
were successfully conditioned to avoid cycloheximide solution, and gave 100%
correct responses. Transection of the LOT ipsilateral to the remaining olfactory bulb was performed in the conditioned rats (n = 25). The LOT transection was produced by aspiration with a 27G needle. Retrospective examination showed that the LOT lesions were localized between 2-4 mm from the posterior end of the bulb. In the control rats (n = 6), the LOT was exposed and no lesions were made on it. After deprivation of water for 2-3 days, the rats were submitted to odor aversion behavior to examine their olfactory ability to discriminate cycloheximide solution from water. Then correct responses were estimated for each rat from the control and LOT-lesioned groups.
After the discrimination test, the rats received injections of Fluoro-Gold (FG), a fluorescent retrograde neuronal tracer, into the olfactory cortex (piriform cortex) on the side of the remaining olfactory bulb. A relatively large volume (1.5 µl) of 2% FG (Fluorochrome, Denver, CO) dissolved in distilled water was injected to cover the whole olfactory cortex. The animals were allowed to survive for 4 days. They were then deeply anesthetized with sodium pentobarbital (80 mg/kg, i.p.) and perfused through the heart with 350-400 ml of 4% paraformaldehyde in 0.1 M phosphate buffer. The brains were removed, postfixed overnight in the same fixative and soaked in 30% sucrose for 2 days. The olfactory bulb and the olfactory cortex were cut serially at 30 µm thickness on a freezing microtome (olfactory bulb: horizontal plane; olfactory cortex: coronal plane). Parts of the sections from the control rats were counterstained with propidium iodide (PI) (20 µg/ml) (Sigma, St Louis, MO), because PI visualized the total mitral cell populations. The sections were mounted and examined under a fluorescence microscope. In the olfactory cortex where FG was injected, care was taken to examine whether the tracer involved the whole olfactory cortex, which was easily identified by its characteristic S-shaped configuration of the pyramidal cell layer. In cases where the tracer involved only part of the olfactory cortex and did not cover the whole olfactory cortex, these animals were excluded from the experiments.
In the olfactory bulb, the numbers of FG-positive mitral cells were counted in the three bulbar sections. These sections were selected from the center of the upper one-third, middle one-third and lower one-third portions of the bulb. After measuring the length of the mitral cell layer in each section, the average number of FG-positive mitral cells per unit length (mm) of the mitral cell layer was calculated in each animal. Thus the numbers of mitral cells with fiber connections to the olfactory cortex were determined in the control rats, the LOT-lesioned rats with normal olfaction and those rats with anosmia. The experiments were carried out in accordance with institutional guidelines for the care and use of animals established by the Animal Care and Use Committee at Shinshu University, and every effort was made to minimize animal suffering and pain.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Since unilateral bulbectomy does not affect odor aversion learning, as shown previously (Moriizumi et al., 1994
), rats (n = 31) were subjected to the
unilateral removal of an olfactory bulb to assess relationships between
lesions of the LOT, olfactory function and numbers of lesion-sparing mitral
cells by limiting the examination of the olfactory system to one side. The
unilaterally bulbectomized rats were conditioned to avoid cycloheximide
solution and gave 100% correct responses, as mentioned previously. The control
rats (n = 6) remained intact after the LOT was exposed
(Figure 1A). The other rats
(n = 25) were then subjected to transection of the LOT ipsilateral to
the remaining olfactory bulb, and varying degrees of lesions were made on the
LOT (Figure 1B).
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After deprivation of water, the rats were subjected to the discrimination
test. The control rats gave 90-100% (97 ± 5%) correct responses. The
LOT-lesioned rats were sharply divided into two groups. One group (n
= 7) showed 90-100% (94 ± 5%) correct responses, indicating normal
olfaction. The other group (n = 18) showed 0-20% (3 ± 7%)
correct responses, indicating loss of olfaction. To confirm soundness of the
discrimination test as a method of examining olfactory function, we removed
the remaining olfactory bulb in the unilaterally bulbectomized, conditioned
rats (n = 3). These rats with bilateral bulbectomy gave 0% correct
responses. Following the experimental procedures mentioned in detail in our
previous paper (Moriizumi et al.,
1994), we used 0.01% cycloheximide solution to examine the
olfactory function of the experimental animals throughout this study. The
anosmic rats with the LOT lesions (n = 8) were also examined with the
higher concentrations (0.05 and 0.1%) of cycloheximide solution and water, and
were unable to discriminate between these two solutions.
After the discrimination test, the rats received injections of FG into the olfactory cortex (piriform cortex) on the side of the remaining olfactory bulb to obtain quantitative data about mitral cell populations with fiber connections to the olfactory cortex (Figure 1C,D). FG injections into the olfactory cortex resulted in total labelings of mitral cells with FG that was retrogradely transported from the olfactory cortex (Figure 1E,F). PI staining also confirmed that the FG-positive cells completely matched the PI-positive mitral cells (Figure 2). The tufted cells in the external plexiform layer were not labeled with FG, except for a few deep tufted cells in the vicinity of the mitral cell layer (Figures 1F, 2 and 3). In the control rats, numerous mitral cells were labeled with FG, forming a well-defined mitral cell layer (Figure 3A,B). In contrast, FG-positive mitral cells were reduced in number in the LOT-lesioned rats. No preferential distribution of those FG-positive cells was found in the bulb. Compared with the LOT-lesioned rats with normal olfaction (Figure 3C,D), numbers of FG-positive mitral cells were much lower in the LOT-lesioned rats with anosmia (Figure 3E,F). The average numbers of FG-positive mitral cells per unit length of the mitral cell layer were 47-54 (50 ± 3) cells/mm in the control rats, 10-46 (21 ± 13) cells/mm in the LOT-lesioned rats with normal olfaction and 0-11 (3 ± 4) cells/mm in the LOT-lesioned rats with anosmia (Figure 4). The Mann—Whitney U test showed statistically significant differences between the control and normal groups (P = 0.0027), control and anosmic groups (P = 0.0003), and normal and anosmic groups (P = 0.0002).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Some methodological problems had to be overcome to pursue this study. One was how to approach the LOT. Since the LOT is located ventrolaterally to the retrobulbar olfactory stria, it is difficult to reach it from the vertical direction that is generally used in stereotaxic brain experiments. In this study, we could access the LOT from the lateral direction, which is often accepted in skull base surgery. Consequently the LOT was clearly identified as a white continuous band connecting the olfactory bulb to the olfactory cortex. Among the many central sensory pathways of the brain, the LOT has the advantage that we can access it and make a well-localized lesion on it without involving the surrounding tissues. Although the anosmic rats generally had larger LOT lesions than the rats with normal olfaction, it should be mentioned that even small LOT lesions of <1 mm diameter caused anosmia in cases where the lesions were made on the more posterior LOT, where the relatively broad band of the retrobulbar LOT gradually converges to a narrow band.
A second problem was whether we could totally label the mitral cell population by a retrograde neuronal tracer. Of many fluorescent tracers, FG turned out to be a good tracer for this purpose because it easily diffused far from an injection site to cover the whole olfactory cortex. Further, complete overlapping of FG-positive cells with PI-positive mitral cells in the control rats indicates that FG injections into the olfactory cortex successfully produced total labeling of mitral cells, which is a necessary prerequisite for measuring the quantity of olfactory projection neurons connected to the olfactory cortex. Since neuronal cell bodies of the axotomized mitral cells survived in the bulb, it is absolutely necessary to use a neuroanatomical tracer to estimate the mitral cells with fiber connections to the olfactory cortex. In the LOT-lesioned cases, neuronal fiber tracing gave objective scores for the degree of LOT lesioning by visualizing healthy mitral cells that transported the fluorescent tracer retrogradely from the olfactory cortex.
The present study showed that in anosmic animals there were <11 (0-11)
healthy mitral cells/mm, and that in animals with normal olfaction there were
>10 (10-46) cells/mm. Therefore, it is concluded that the essential mitral
population required for olfactory function is 
20% of the control value
(50 ± 3 cells/mm), a threshold value for olfaction. Recently, Lu and
Slotnick (Lu and Slotnick,
1998) examined olfactory function in animals with varying degrees
of bulbectomy. In this connection, it should be mentioned here that removal of
the anterior olfactory bulb often causes damage to the olfactory nerves
connecting to the posterior bulb, and that removal of the posterior olfactory
bulb often causes damage to the axon fibers derived from the mitral cells in
the anterior bulb. Thus the volume of intact bulb with partial bulbectomy is
not necessarily proportional to the number of intact mitral cells that relay
olfactory information from the olfactory epithelium to the olfactory cortex.
To know the neuronal number of intact mitral cells precisely, our experiments
have an advantage that lesions were made only on the central olfactory pathway
(LOT), leaving both the olfactory epithelium and the olfactory bulb totally
intact. However, it is interesting that our results turned out to match well
with those of the previous study (Lu and
Slotnick, 1998) reporting that bulbectomized rats with bulbar
savings scores of <21% had deficits in odor detection or discrimination
tasks. Considering that the total number of mitral cells in each olfactory
bulb has been reported to range between 45 000 and 75 000 in the rat
(Meisami and Safari, 1981;
Rosselli-Austin and Williams,
1990; Bonthius et al.,
1992; Royet et al.,
1998), the absolute number of olfactory projection neurons
required to discriminate between cycloheximide solution and water can be
simply calculated to be 9000-15 000 in the unilateral olfactory system. When
the bilateral olfactory system is intact, normal olfactory function can be
preserved with <20% of the olfactory projection neurons in terms of the
ability to discriminate cycloheximide solution from water. Furthermore, it
should be mentioned here that the olfactory function examined in the present
study is based upon the fact that the anterior part of the olfactory cortex
(piriform cortex) located between the bulbar posterior end and the lesioned
LOT is intact.
In the past decade, there have been important advances in olfactory
cognition. Each type of odorant receptor expressed in olfactory receptor
neurons has been reported to correspond to a single specific bulbar glomerulus
(Buck and Axel, 1991;
Mombaerts et al.,
1996). Rubin and Katz (Rubin
and Katz, 1999) have revealed that odorants are encoded by
distinct spatial patterns of bulbar glomerular activation. These studies
strongly suggest the existence of distinct topographies between olfactory
receptor neurons and bulbar glomeruli, depending on odorants. Although we
proposed that the essential neuronal population in the central olfactory
pathway is about one-fifth of the total population, the exact proportion
should be determined for different odors. An important question about the
existence of receptor-related topographic pathways in the higher olfactory
centers should be explored in the near future.
Many scientists are paying great attention to stem cells of the brain for
the realistic purpose of treating brain disorders
(Reynolds and Weiss, 1992;
McKay, 1997;
Roy et al., 2000).
However, there are no data available about the number of essential neurons for
the functional repair by transplanted brain stem cells. The present results
might provide fundamental data on a presumed quantity of replaceable neurons
required for the functional recovery of the injured brain.
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Acknowledgments |
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We thank Drs T. Katsuyama and J. Nakayama for their valuable comments on the manuscript, and Dr S. Kobayashi and Ms M. Kaido for their excellent technical assistance. This work was supported by a Grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.
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Accepted August 22, 2001
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