Chemical Senses 2005 30(Supplement 1):i101-i102; doi:10.1093/chemse/bjh134
Chemical Senses Vol. 30 No. suppl 1 © Oxford University
Press 2005; all rights reserved
Selective Imaging of the Receptor Neuron Population in the Olfactory Bulb of Zebrafish and Mice
Sigrun Korsching
Institute of Genetics, University at Cologne, Cologne, Germany
Correspondence should be addressed to: Sigrun Korsching, e-mail:
sigrun.korsching{at}uni-koeln.de
Key words: aliphatic aldehyde, amino acid, calcium
indicator dye, olfactory code, optical
imaging, pheromone, zebrafish
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Convergence of the olfactory projections allows analysis of receptor repertoires in the olfactory bulb
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The neuronal processing of odors takes place in several types
of neurons, including
the sensory neurons, projection neurons
and interneurons. To understand the neuronal
representation
of odors and eventually the encoding of those odors it is important
to
selectively measure the contributions of the different neuron
populations to odor-induced
neuronal activity. We are analyzing
the odor responses of the population of olfactory
receptor
neurons in two experimental systems, zebrafish and mouse. Odor
responses are
measured in the receptor neuron terminals within
the olfactory bulb. Thus, the response
properties of many different
odorant receptors can be visualized simultaneously by
optical
imaging of neuronal activity in the olfactory bulb, since olfactory
receptor
neurons expressing the same odorant receptor converge
onto common neuropil structures in
the olfactory bulb, the
glomeruli (cf.
Korsching, 2002
). This transition from
presumably
stochastic expression of odorant receptor genes in scattered
olfactory
receptor neurons within the sensory epithelium to
an ordered map in the olfactory bulb
(Figure
1) allows a
unique view on the size
and composition of the receptor repertoires
that are activated by particular
odorants.
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Figure 1 Monogenic expression and convergence of odorant
receptors. The top panel shows a schematic representation of three olfactory receptor
neurons (ORNs), each expressing a different odorant receptor (OR): the one
neuron—one receptor concept. The bottom panel depicts the convergence of same
receptor-expressing neurons on single glomeruli (glo) in the olfactory
bulb.
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Selective analysis of odor-induced presynaptic activity
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We have introduced activity indicator dyes selectively into
olfactory receptor neurons
by local application in the nose.
We use the voltage-dependent dye ANEPPQ (Friedrich and Korsching,
1998
) and the calcium
indicator dye CalciumGreen (Friedrich
and
Korsching, 1997
), both of which distribute in the whole
cell, up to and
including the axon terminals in the glomerular
layer of the olfactory bulb. By this means
we have analyzed
the tuning properties of major receptor populations selectively
in the
presynaptic compartment of glomeruli (Friedrich
and
Korsching, 1998
;
Fuss and Korsching, 2001
;
Fried
et al.,
2002
).
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Chemotopic representation of odorants in the olfactory bulb
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We report that responses to different chemical groups of odors
are segregated within
subregions of the zebrafish olfactory
bulb. Amino acids—important feeding
stimuli—are
represented in the anteriolateral olfactory bulb, whereas nucleotides
elicit responses in the posteriolateral olfactory bulb and
bile acids are represented
medially. Pheromone responses are
localized in central and medial regions. Amino acid
responses
are found in microglomerular structures reminescent of the termination
areas of
microvillous olfactory receptor neurons in the mammalian
accessory olfactory system. On
the other hand, pheromone responses
were detected in standard size glomeruli such as
those formed
by terminals of ciliated receptor neurons in the mammalian main
olfactory
system.
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Odorant features may be either required, tolerated or prohibitive
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We have performed a detailed analysis of the chemical tuning
of the
amino-acid-responsive microglomeruli. We report that
the amino acid head region is
required to elicit a response,
i.e. neither amines nor carboxylic acids of equivalent
chain
length function as odorants in the fish system (interestingly,
both groups of
chemicals are very strong odorants for air-breathing
vertebrates).
Using neutral amino acids of various side chain lengths as stimuli we find
that—barring few exceptions—most odorant receptors react stronger to longer
side chain amino acids, even extending beyond the size-range present in the 20
proteinaceous amino acids. Different microglomeruli are differently tuned to side chain
length, so that new odorant receptors are recruited to the response pattern even with
additions as small as a single methylen group. As predicted from these differences in
tuning we find that each concentration of each amino acid elicits a unique response
pattern not matched by any other combination of chain length and concentration.
When testing the recruitment of odorant receptors by polar and charged amino acids
(serine, threonine, and ornithine, lysine, respectively) we observe characteristic
differences in the response patterns. Some receptors are only activated by neutral amino
acids, some only by basic amino acids, and some are activated by both. There exist also
receptors that tolerate both neutral and polar amino acids, but none that specifically
require polar amino acids.
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Mammalian aldehyde receptors are oligo-specific
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In another project we have analyzed the tuning properties of
a major mammalian odorant
receptor population using the same
method of high resolution calcium imaging (Fried
et al., 2002
).
We show that eighty
different odorant receptors projecting
to the dorsal olfactory bulb of mouse respond to
high concentrations
of aliphatic unbranched aldehydes with limited specificity.
Different
ensembles of ~10–20 receptors encode any particular
aldehyde at low stimulus
concentrations with high specificity.
Pronounced differences in affinity were observed
within the
aldehyde receptor repertoire. Again, a unique response pattern
of activated
glomeruli is observed for each chain length and
(non-saturating) concentration.
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Concept of odorant/receptor interaction
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We conclude that odorant detection is combinatorial, i.e. requires
several receptors
even for relatively simple odorants such
as amino acids and aldehydes, but may be
mono-specific for
pheromones (Figure
2). Furthermore, we find that
individual
odorant receptors require the presence of some molecular features,
the absence
of others, and tolerate still other molecular features.
Thus, odorant receptors appear
not to be simple ‘feature
detectors’ but to detect particular combinations of
molecular
features—odotopes (Figure
3).
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Figure 2 Two coding strategies for odorants. Common odorants and
odors: combinatorial representation by activation of a characteristic subset of odorant
receptors—a unique ‘finger print’ for each odorant. Pheromones:
mono-specific activation of a single odorant receptor—labeled line
concept.
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Figure 3 The combinatorial model of odorant/receptor binding.
Even a simple odorant may consist of several odotopes. A particular receptor requires a
particular combination and positioning of odotopes for maximal activation. Odotopes may
be not tolerated (triangle), tolerated (twin triangle) or even required
(rectangle).
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Current work
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An interesting application of our imaging method will be the
study of odor responses
of genetically labeled glomeruli. Several
mouse lines expressing green fluorescent
protein (GFP) under
the control of a specific odorant receptor promoter are available.
We
are currently investigating the compatibility of the CalciumGreen
signal detection with
the GFP labeling in such a mouse line
(Feinstein
and Mombaerts, 2004
).
To address receptor neurons sharing an identified receptor and to address
interneurons and projection neurons separately we are characterizing suitable promoter
regions and using them to drive expression of genetically encoded calcium dyes as
indicators of odor-induced neuronal activity.
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References
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Feinstein, P. and
Mombaerts, P. (2004)
A contextual model for axonal sorting into glomeruli in the mouse olfactory system. Cell, 117, 817–831.
[CrossRef][Web of Science][Medline]
Fried, H.U., Fuss, S.H. and Korsching, S.I. (2002) Selective imaging of presynaptic activity in the mouse olfactory bulb shows concentration and structure dependence of odor responses in identified glomeruli. Proc. Natl Acad. Sci. USA, 99, 3222–3227.[Abstract/Free Full Text]
Friedrich, R.W. and Korsching, S.I. (1997) Combinatorial and chemotopic odorant coding in the zebrafish olfactory bulb visualized by optical imaging. Neuron, 18, 737–752.[CrossRef][Web of Science][Medline]
Friedrich, R.W. and Korsching, S.I. (1998) Chemotopic, combinatorial, and noncombinatorial odorant representations in the olfactory bulb revealed using a voltage-sensitive axon tracer. J. Neurosci., 18, 9977–9988.[Abstract/Free Full Text]
Fuss, S.H. and Korsching, S.I. (2001) Odorant feature detection: Activity mapping of structure response relationships in the zebrafish olfactory bulb. J. Neurosci., 21, 8396–8407.[Abstract/Free Full Text]
Korsching, S. (2002) Olfactory maps and odor images. Curr. Opin. Neurobiol., 12, 387–392.[CrossRef][Web of Science][Medline]
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