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Neurogenesis in the procerebrum of the snell Helix aspersa: a Quantitative analysis.

Introduction

The order Stylommatophora in terrestrial pulmonate molluscs has a region of the brain specialized for olfaction that is not found in marine or freshwater gastropods (Bullock and Horridge, 1965). This part of their nervous system, the procerebrum, is unique among gastropod nervous systems in that it has 20,000 to 50,000 small neurons of uniform size (Zs.-Nagy and Sakharov, 1970; Chase, 2000; Zaitseva, 2000) that process sensory information from the olfactory tentacles (Gelperin and Tank, 1990; Ratte and Chase, 2000). This olfactory center has similarities to olfactory centers in other phyla (Ache and Young, 2005): it is capable of odor recognition and one-trial learning of aversive stimuli (Gelperin, 1975; Sahley et al., 1981; Teyke, 1995), it has oscillations (Gelperin and Tank, 1990; Gelperin, 1999; Nikitin and Balaban, 2000), and it adds neurons during growth (Zakharov et al., 1998; Watanabe et al., 2008).

Neurons are added to the adult nervous system in several phyla (reviewed by Lindsey and Tropepe, 2006). Neurogenesis, which is seen in all vertebrate species examined, may affect the processing of sensory information. In mice, if mitosis is blocked in the lateral ventricle at 28 days to prevent neuron addition to the olfactory bulb, short-term olfactory memory is drastically reduced (Breton-Provencher et al., 2009). Unlike mammals, where neurogenesis is limited to the hippocampus and the olfactory bulb, adult songbirds have neuron recruitment throughout their forebrain (Vellema et al., 2010). In decapod crustaceans, neurogenesis occurs in adults in the olfactory pathway but not in mechanosensory or chemosensory pathways (Schmidt, 2007). In adult crickets, there is an increase in neurogenesis in mushroom bodies of crickets reared in an enriched sensory environment (Cayre et al., 2007). In the opisthobranch mollusc Aplysia, neurons of varying size in the central nervous system increase in number with age (Hickmott and Carew, 1991). In the terrestrial snail Helix luco rum, migration of neurons into the procerebrum has been shown during the first 30 days of post-hatch growth (Zakharov et al., 1998). A similar increase of neurons has been indicated in the procerebrum of the slug Limax maximus (Watanabe et al., 2008).

In the initial development of the nervous system in Helix aspersa Muller (1774), cerebral ganglia are formed by de-lamination of epithelial cells, but cerebral tubes, which arise from local invaginations of the ectoderm at the base of the tentacles, also appear as ganglia are being formed. These cerebral tubes form the accessory lobes of the cerebral ganglia (Raven, 1958). In H. aspersa, they contain dividing cells and are still present after hatching, but without a peripheral opening; however, in L. maximus, Henchman (1890) reported a cerebral tube with an epithelial opening 2 weeks after hatching.

In this paper I show three possible sources of neuron addition to the procerebrum. Cerebral tubes, described here in H. aspersa as a source of procerebral neurons, persist in adult Helix (Bang, 1917; Bullock and Horridge, 1965; Zs.-Nagy and Sakharov, 1970). Also shown here is that, several weeks post-hatch in H. aspersa, cells appear to migrate into the procerebrutn from both the peritentacular and olfactory nerves.

The small, densely packed neurons in the procerebrum tend not to be separated by glial processes (Zs.-Nagy and Sakharov, 1970). The cytoplasm of the perikarya, as described by Bullock and Horridge (1965), is so scanty it is difficult to visualize in ordinary preparations. Unlike larger neurons in the Helix ganglia, the DNA content of the nuclei in the procerebrum remains diploid during growth (Chase and Tolloczko, 1987). Measurements here of neuronal nuclear volume in H. aspersa indicate that the size of procerebral neurons remains constant throughout the life of the snail. It is possible to estimate the number of neurons in the procerebrum from measurements of the procerebral volume. It is shown here that the number of neurons in the procerebrum depends on the weight of the individual.

The time dependence of the weight of H. aspersa throughout its life is given by the logistic equation (CzarnolOki et al., 2008), but during the first few weeks after hatching, this equation can be approximated by an exponential equation (Garcia et al., 2006). Growth of the procerebrum can be divided into two periods: the initial exponential growth period and the subsequent period when the weight of snails follows the logistic equation. During exponential growth of Helix, addition of cells to the procerebrum comes primarily from the cerebral tube (Zakharov et al., 1998). Later, when snail weight is better approximated by the logistic equation, cells that are contiguous with neurons in the procerebral apex are seen in the peritentacular and olfactory nerves. These cells appear to be an additional source of neurons that are being added to the procerebrum. The result is a marked increase in the rate of neuron addition to the procerebrum during this second growth phase, but during both periods of growth, the number of neurons in the procerebrum can be described by mathematical equations that are dependent on snail weight.

Materials and Methods

Collection and culture of snails

Individuals of Helix aspersa were collected from local gardens in three ways. Eggs were collected in the field in November and brought to the laboratory where they were allowed to hatch. The following year adult snails were collected from the field and brought to the laboratory where they were maintained in aquaria on garden soil and fed lettuce. Eggs from these snails were collected before they hatched. Also, snails were collected in the field for immediate dissection in March (weight 0.35 g, 1.77 g, 2.74 g, 4.64 g, 10.6 g) and in June (2.55 g, 6.35 g, 9.82 g). Both field-collected eggs and eggs from aquaria were hatched in petri dishes. Limax maximus eggs were collected locally and cultured in the laboratory similarly to H. aspersa. Hatching in H. aspersa required about 16-20 days at room temperature, after which the snails were fed lettuce and maintained in petri dishes on moist filter paper. Filter paper and lettuce were changed daily. During the exponential growth phase, snails from three clutches hatched in the laboratory were weighed at 1, 9, 18, 35, and 49 days, n = 15, 16, 30, 23, and 14, respectively. In all cases, snail weight was wet weight with shell. In addition, laboratory-raised snails were maintained beyond the exponential growth phase until 71 days post-hatch for histological examination.

Histology

The cerebral ganglia and olfactory nerves of all snails were removed in Tris-buffered saline (80 mmol 1-1 NaCl, 4 mmol 1-1KC1, 8 mmol 1-1CaC12, 5 mmol 1-1MgCl, 5 mmol 1-1Tris, pH 7.8) and fixed overnight in 4% formalin in 0.1 mol 1-1 phosphate buffer (PB). After fixation, ganglia were stored for a day in PB containing 2% Triton X-100 and 0.1% sodium azide. Ganglia from all snails were processed to show fluorescence of the nuclear DNA. They were placed in 1% RNase (Sigma) in PBS at 30 [degrees]C for 1 h and then placed in Sytox Green (Molecular Probes/Invitrogen, 5 mmol 1-lsolution) diluted to 1:10,000 in 10 mmol 1-1 Tris, 1 mmol 1-1 EGTA, pH 7, for 30 min. Several of the SytoxGreen-stained ganglia were also processed using previously described techniques (Longley and Longley, 1986) with antibodies to anti-phospho-histone H3 (Upstate Biotechnology, cat. #06-570) and the peptide neurotransmitters SCP (University of Washington, Masinovsky et al., 1988), TPep (from Dennis Willows, Friday Harbor Laboratories), and FMRFamide (Immunostar) all diluted 1:200. The procerebrum of Limax maximus was dissected in HEPES buffered saline (70 mmol r' NaCl, 2 mmol 1-1 KC1, 4.9 mmol 1-1 CaCl2 4.7 mmol r' MgCl2, 5 mmol 1-1 HEPES, pH 7.8), fixed overnight in 4% formalin in 0.1 mmol l PB, and then processed with Sytox Green and TPep antibodies prior to anti-phospho-histone H3 antibodies. Secondary antibodies from Jackson ImmunoResearch were tetramethyl rhodamine isothiocyanate (TRITC) at 1:200 dilution. After treatment with Sytox Green and antibodies, ganglia were washed for one day in PBS containing 2% Triton X-100 and 0.1% sodium azide and then washed in reverse osmosis water to remove sodium azide. Ganglia were mounted in glycerol with 0.5% n-propyl gallate (Giloh and Sedat, 1982) for examination with a confocal microscope.

Microscopy

For consistency, all images are oriented as dorsal, right procerebrum views with anterior to the left or up. Microscopy of the green fluorescence of Sytox Green and the red fluorescence of TRITC, the secondary antibody for antiphospho-histone H3, SCP, TPep, and FMRFamide, was with a Biorad Radiance 2000 confocal microscope. Confocal microscope stacks were analyzed using ImageJ ver. 1.410 and Photoshop Extended C3.

Estimate of neuronal volume

The volume occupied by the neuronal nucleus in the procerebrum was measured indirectly by first estimating the maximum nuclear area. Measurements of nuclear area were quantified with Photoshop C3 Extended software from con-focal frames made with a 60X 1.4 na objective. To determine the maximum area of optically sectioned nuclei in a procerebrum, 80 randomly selected measurements of area were ranked in descending order and truncated to the largest 60 nuclei. These areas decreased linearly with rank and were fitted with a straight line to estimate the maximum area. Polar and equatorial axis lengths of a prolate spheroid approximating the shape of the nuclei were calculated from the mean of these maximum nuclear areas. From these lengths a nuclear volume was estimated. A maximum neuronal volume was estimated by multiplying the nuclear volume by the ratio of the frame area to the sum of in-focus areas of nuclei in the frame.

Measurement of procerebral volume

To calculate the neuronal volume of the procerebrum, confocal stacks in ganglia treated with Sytox Green were made with a 20X objective with z steps of 2 [micro]m for small ganglia and 4 [micro]m for larger ganglia. In large ganglia, laser intensity was varied during the scan to maintain a constant exposure. The procerebral area containing neurons could be recognized in each frame by the fluorescence of the densely packed neuronal nuclei (supplemental video 1 available at http://www.biobull.org/content/supplemental/). The neuronal volume of the procerebrum was measured using ImageJ by summing the neuronal area of individual frames of the confocal stack and then multiplying by the z step of the stack. The number of neurons in each procerebrum was estimated by dividing the procerebral volume by the volume of an individual neuron. To avoid bias, measurements of procerebral areas were not repeated.

Statistics

Measurements were graphed with Graphpad Prism, ver. 5.0c, which gave statistics of curves fitted to the data.

Results

Anatomy of the procerebrum

The procerebrum in Helix aspersa extends anteriorly from the cerebral ganglion under the olfactory and peritentacular nerves, which pass over the division between the neuropil and the neuronal cell bodies of the procerebrum. The hollow cerebral tube is connected to the lateral, ventral side of the procerebrum (Fig. 1A).

The relations of the olfactory nerve and the neuropil to the neurons in the procerebrum can be seen when the neuropil is stained with antibodies to SCP. Only a small number of neurons near the peripheral surface of the pro-cerebrum are immunoreactive to SCP antibodies, but in the neuropil there are many small immunoreactive processes. It is typical for antibodies to SCP, TPep, and FMRFamide that there is no immunoreactivity at the procerebral apex where neurons are being added (Fig. 1B for SCP; also supplemental video 2 available at http://www. biobull.org/content/supplemental/).

Neuron-free cavities in the procerebral apex connect with the cerebral tube (Fig. 2A, B). Dividing cells around these cavities and in the cerebral tube can be seen with either Sytox Green DNA fluorescence or fluorescence from TRITC that is bound to anti-phospho-histone H3 antibodies. The two methods are slightly different in that anti-phosphohistone H3 antibodies do not show mitosis in telophase (Fig. 2C, D). Unlike DNA fluorescence, which is present in all the nuclei, anti-phospho-histone H3 antibodies can show mitosis in a z-projection of a confocal stack of the procerebrum. In the second week post-hatch, a large number of cell divisions are occurring around the cavities in the apex (Fig. 2E).

The cerebral tube extends anteriorly toward the epithelium, paralleling the olfactory nerve (Fig. 2F). The wall of the cerebral tube gradually thins to a single layer of cells, which still include dividing cells (Fig. 2G), and ends with a gradual diminution of cells with no apparent connection to the epithelium (Fig. 2H). As neurons are added to the apex during growth, the connection of the cerebral tube to the procerebrum is shifted posteriorly toward the cerebral ganglion (Fig. 21).

The number of mitotic cells in the cerebral tube is greater near the procerebral apex (Fig. 3A). As the cerebral tube is displaced posteriorly toward the cerebral ganglion, cavities at the apex extend along the lateral side of the procerebrum under columnar epithelial cells and continue to communicate with the hollow cerebral tube (Fig. 3B, C). Cell division continues in the cerebral tube throughout snail growth (Fig. 3D, E).

[FIGURE 1 OMITTED]

Mitotic cells in the apex that are indicated by anti-phospho-histone H3 antibodies are countable (Fig. 4). Counts of mitotic cells in individual procerebrums decline with snail age. The results can be fitted equally well by linear regression or with a negative exponential,

[Y.sub.0][e.sup.-[alpha]t]

where [Y.sub.0] = 39 mitotic cells at hatching and [alpha] = 0.031 [day.sup.-1]. An indication of the number of neurons contributed by mitosis during the exponential growth phase can be found by integrating this negative exponential. This gives

([Y.sub.0]/[alpha]) (1-[e.sub.[alpha]t])

where the maximum number when t is large is [Y.sub.0]/[alpha] = 1248 mitotic events. Since the cell cycle length is not known, this number cannot be related to actual mitotic events except as a percentage. At 40 days post-hatch, the number of mitotic events is 71% of the maximum.

Olfactory and peritentacular nerve cells

Beginning at 30-40 days post-hatch, dividing cells are seen at the apex of the procerebrum in the olfactory and peritentacular nerves. At 14 days post-hatch, cells were seen in the olfactory nerve in one of 18 procerebrums, but in 30-day post-hatch snails, dividing cells were seen in olfactory and peritentacular nerves in 10 of 19 procerebrums. At 46 days post-hatch, all of 18 procerebrums examined showed cells in these nerves. Dividing cells in the peritentacular and olfactory nerves are seen in confocal frames in 46- and 71-day post-hatch snails (Fig. 5A, B). Similar cells in the olfactory nerve are present at the apex in a 10.6-g snail (Fig. 5C). Immunoreactivity of axons to SCP antibodies was superimposed on Sytox Green .fluorescence to show the relation of the nerves to the cells in these nerves (Fig. 5D--F). Cells from the peritentacular nerve appear to migrate directly into the procerebral apex (Fig. 5B, E), while cells from the olfactory nerve appear to migrate along the interface between the cellular part of the procereburum and its neuorpil (Fig 5A, D, F). Cells in the alfactory nerver appear in rows as they contact the procerebrum (Fig. 5A, D, F).

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

Neuronal size

As shown by the close packing of procerebral nuclei, the nuclei represent a large part of the neuronal volume in the pmcerebrum (Fig. 6A). As an indication of neuronal size, the cross-sectional area of procerebrum nuclei was measured in H. ctspersa ranging in weight from 0.03 g to 10.6g. At high magnification, areas of procerebral nuclei could be measured in optical sections of confocal frames. Not all nuclei were oriented in the same way, so the result of these measurements was the area of random sections through the nuclei. When these areas were ranked in descending order, they could be extrapolated to zero rank using a linear least-squares fit to the ranked areas (Fig. 6B). This gave an estimate of 62.2 [+ or -] 2.45 [micro] [m.sup.2] SEM (n = 8) for the mean maximum area of a nuclear cross section in the procerebrum. If maximum areas are plotted as a function of the logarithm of snail weight (Fig. 6C), the slope of the linear regression is not significantly different from zero ([F.sub.(1,7) = 0.82, P = 0.4). Therefore, the size of the nucleus does not appear to change with snail weight.

When ranked, the linear decrease of measured areas was indicative of the shape of the nucleus. If the nucleus were spherical, ranked areas of random sections through the nucleus would be fitted with a second-order polynomial instead of a straight line. In computer simulation, it was shown that randomly selected areas parallel to the long axis of a prolate spheroid--i.e., a volume formed by an ellipse rotated about its long axis--decreased linearly when ranked in descending order. This indicates that the nucleus is similar to a nonspherical irregular shape that can be approxi-mated by a prolate spheroid.

[FIGURE 5 OMITTED]

The axis lengths of a prolate spheroid can be estimated from its maximum area if a ratio of the minor axis to the major axis is assumed. For a prolate spheroid with minor axis equal to 1/2 the major axis, the nuclear volume estimated from the maximum nuclear cross-sectional area is 261 [micro] [m.sup.3]. An approximate neuronal volume of 570 [micro] [m.sup.3] was estimated by dividing the fraction of a confocal frame not occupied by nuclei (0.458]+ or -] 0.019 SEM, n = 4) into the nuclear volume. This assumes neurons are space-filling, which would give a neuron volume that is likely to be high. Time dependence of snail weight

H. aspersa weight follows the logistic equation,

W(t)=[w.sub.0][w.sub.[varies]]/[w.sub.0](1-[e.sup.-kt]) + [w.sub.[varies]][e.sup.-kt] (1)

where w(t) is the weight, [w.sub.0] is the weight when t = 0, and [w.sub.[varies]] is the maximum weight when kt is large. When kt is small, this equation reduces to exponential growth,

w(t) =[w.sub.0][e.sup.kt] (2)

The constants [w.sub.0]= 0.02 g at hatching and k = 0.0744 [+ or -] 0.0009 [day.sub.-1] SEM in equations (1) and (2) were determined experimentally for snails raised in the laboratory at room temperature (18-20 [degrees]C) over a 7-week period (Fig. 7A). Using these constants, the logistic equation and the exponential growth equation were compared (Fig. 7B).

[FIGURE 6 OMITTED]

Growth of the procerebrum

The volumes of 63 procerebrums from laboratory-raised snails were measured at four ages ranging from 1 to 46 days post-hatch. Volumes were also measured in 18 additional left and right procerebrums from nine field-collected snails of unknown age ranging in weight from 0.34 g to 10.6 g. These volumes were divided by the approximate neuronal volume of 570 [micro] [m.sup.3] to give an estimate of the number of neurons in the procerebrum.

The number N of procerebral neurons of both laboratory-raised and field-collected snails is a linear function of the logarithm of their weight,

N = a + [blog.sub.e](w) (3)

but the constants a and h are different for the two growth phases. There is a change in the slope of the regression line for field-collected snails relative to laboratory-raised snails at about 40 days or 0.4 g (Fig. 8A). From equation (3), with the appropriate value of b for each growth phase, the number of neurons added to the procerebrum for each increment of weight is inversely proportional to snail weight,

dN/dw=b/w (4)

Since the initial weight of laboratory-raised H. aspersa in equation (2) is an exponential function of time (Fig. 7A), the time-dependent number of neurons in the procerebrum during this growth phase is, from equation (3)

N=[a.sub.1] + [b.sub.1][log.sub.e]([w.sub.0])[e.sup.kt]

or

N= al +[b.sub.1][log.sub.e]([W.sub.0]) +[ b.sub.1]kt.

From this result, the time-dependent rate of neuron addition to the procerebrum during the initial exponential growth phase is constant,

dN/dt=[b.sub.1] k [approximately equal to] neurons [day.sup.-1] t<40 days. (5)

[FIGURE 7 OMITTED]

[FIGURE 8 OMITTED]

The time-dependent rate of neuron addition for field-collected H. aspersa can be calculated by using equations (1) and (3),

dN/dt = [b.sub.f]/ w dw/dt t> 40 days. (6)

The results from equations (5) and (6) for the two different values of b are shown in Fig. 8B. A larger maximum weight will extend the rate of neuron addition during the logistic growth phase to a longer time, but it will have little effect on the rate at 40 days. This idealized rate transition from laboratory-raised to field-collected snails at 40 days may be more abrupt than the transition in snails sampled from their natural environment.

Discussion

The results of this study show that neurons are added to the procerebrum of Helix aspersa throughout the life of the snail though, as in neuron addition to the mouse olfactory bulb (Pomeroy et al., 1990), neuron addition in H. aspersa declines with age. Mitosis continues after hatching in H. aspersa in both the cerebral tube and the apex of the procerebrum, but the exponentially decreasing mitotic rate in the apex cannot account for the linear increase in the number of procerebrum neurons during the exponential growth phase (Fig. 4, Eq. 5).

In Aplysia, cells are reported to migrate into the central nervous system from the ectodermal layer (Jacob, 1984), but growth of the procerebrum in H. aspersa appears to depend on an internal proliferation of neurons. Zakharov et al. (1998) suggested that during early development the cerebral tube is a source of cells that form neurons in the procerebrum of Helix lucorum, although they refer to it as a nerve on the basis of early anatomical examinations of Helix (Schmalz, 1914). As growth occurs at the apex, the cerebral tube is displaced posteriorly away from the apex. Several weeks after hatching, dividing cells begin to arrive at the procerebrum from the olfactory and peritentacular nerves. Rows of cells that appear to enter the procerebrum from the olfactory nerve suggest that they form the columns of neurons reported in the procerebrum (Zaitseva, 2000). Cells in the peritentacular nerve merge with the apex of the procerebrum when growth at the procerebral apex begins to accelerate at about 40 days post-hatch. The lack of immunoreactivity to peptidergic neurotransmitters in the apex indicates that differentiation of these cells is delayed. These results suggest that cells are migrating into the procerebrum from the peritentacular and olfactory nerves, but experiments with BrdU or other labeling methods will be necessary to confirm this.

There is a strong homology between the procerebrum of H. aspersa and the procerebrum of Limax maximus. The procerebrum of L. maximus is rotated 90[degrees] relative to that of H. aspersa and extends laterally adjacent to the cerebral ganglion instead of paralleling the olfactory nerve. As a consequence, in L. maximus the cerebral tube enters the procerebral apex at its posterior, lateral edge, near the cerebral ganglion. After hatching, a cavity that is bordered by mitotic cells and is connected to the cerebral tube extends across the apex (Fig. 9A). As in H. aspersa, cells at the apex near this cavity do not show immunoreactivity to peptidergic neurotransmitters (R. D. Longley, unpubl. obs.). This cavity is similar to that seen connected in older H. aspersa to the cerebral tube (Fig. 3B, C). In pre-hatch L. maximus, this cavity also extends along the anterior edge of the procerebrum (Fig. 9B). During pre-hatch development, mitotic cells border the length of the cavity (Fig. 9C).

[FIGURE 9 OMITTED]

For H. aspersa raised on lettuce in petri dishes in the laboratory, weight can initially be approximated by an exponential equation. The value of k found here during exponential growth of these snails is 15% less than that found for snails cultured over a 6-week period at an average temperature of 25 [degrees]C (Garcia et al., 2006) and is 26% greater than a k value found in other work by fitting a logistic equation to growth data ((Czarnolcgkiet al., 2008). During the period of exponential growth, the growth rate is relatively independent of culture conditions, although growth of older snails requires dirt, which was present in the normal environment for field-collected snails (Gomot et al., 1989).

The variability of snail weight in a clutch increases rapidly with age (San Sampelayo et al., 1990; (Czarnolcgki et al., 2008), which suggests that there is no correlation between age and weight in field-collected snails. This is especially true for the six snails collected in March, since most of the growth in the larger snails would have occurred in the previous year, and most of the growth of the smaller snails would have occurred in less favorable conditions in the winter and spring. The clear dependence on snail weight for the procerebral volumes of these field-collected snails emphasizes that it is weight and not snail age that correlates with the number of neurons in their procerebrums.

By measuring the volume of the procerebrum, it is possible to estimate how many neurons it contains. The number of neurons in the procerebrum can be described by a simple mathematical equation relating the number of neurons to snail weight, but different constants are required for the exponential growth phase and for the logistic growth phase. The abrupt change in the rate of neuron addition is not related to the different culture conditions for the two groups of snails, since the logarithmic curve for the field-collected snails cannot be reconciled with the logarithmic curve for laboratory-raised snails.

The data for laboratory-raised snails extrapolates to N = 0 when snail weight is 6.9 mg. From equation (2), this corresponds to 14 days prior to hatching, which is consistent with a 16-20-day period of embryogenesis at room temperature. The regression line for field-collected H. aspersa extrapolates when N = 0 to 53 mg, which corresponds to 11 days post-hatch. If the constant [a.sub.f] for field-collected animals is adjusted to give N = 0 prior to hatching at 6.9 mg, the number of neurons predicted in the procerebrum at hatching is two times greater than the observed result. It is more likely that this transition in the rate of neuron addition to the procerebrum at about 40 days occurs when neuronal precursors begin to be added from the olfactory and peritentacular nerves.

The timing of the transition to accelerated procerebral growth in Helix is correlated with an increase in the serotonin content in the nervous system and the development of avoidance-conditioned reactions (Zakharov and Balaban, 1987). It is a likely possibility that the continuing addition of neurons to the H. aspersa procerebrum has evolved to increase sensitivity to and recognition of odors. This appears to have occurred in the mouse, since in the mouse olfactory bulb there is both a turnover caused by neuron death and optimization of olfaction by neuron addition (Mouret et al., 2009).

It has been suggested that as in the mouse (Rochefort et al., 2002), neuron addition in the Helix and Limax procerebrums is related to odor experience (Zakharov et al., 1998; Watanabe et al., 2008), but this has not been demonstrated. Neurons from the peritentacular and olfactory nerves in laboratory-raised snails appear to be added to the procerebrum when odor experience is restricted to lettuce, but the abrupt rate increase seen in field-collected snails may be the result of a more-varied odor experience. For the snail in its natural environment, this accelerated growth of the procerebrum may enhance its ability to discriminate odors.

Acknowledgments

I thank the director of Friday Harbor Laboratories for use of the facilities during this work, and the Center for Cell Dynamics for use of the confocal microscope. This work was supported in part by Pacific Sciences Institute. I thank Margaret Longley for writing assistance.

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Received 19 May 2011; accepted 28 August 2011.

* To whom correspondence should be addressed. E-mail: rlongley@ rockisland.com

Abbreviations: a1,h1, constants for laboratory-raised Helix aspersa; ap bp constants for field collected H. aspersa; k, growth constant; w, hatching weight of H. aspersa; vv,., maximum weight of H. aspersa; SCP, small cardioactive peptide; TPep, Tritonia pedal peptide.

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Author:Longley, Roger D.
Publication:The Biological Bulletin
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Date:Oct 1, 2011
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