Growth-related and antennular amputation-induced changes in the olfactory centers of crayfish brain

Abstract

Freshwater crayfish increase in size throughout their lives, and this growth is accompanied by an increase in the length of the appendages and number of mechanoreceptive and chemoreceptive sensilla on them. We find that in the Australian freshwater crayfish Cherax destructor, neuropil volumes of the olfactory centers increase linearly with body size over the entire size range of animals found in their natural habitat. The number of cell somata of two groups of interneurons associated with the olfactory centers (projection neurons and small local neurons) also increases linearly with the size of the animals. In contrast, axon counts of interneurons that represent a nonolfactory input to the olfactory centers show that these reach a total number in the very early adult stages that then remains constant regardless of the size of the animal. Only the axon diameter of these interneurons increases linearly with body size. Amputation of the antennule and olfactory sensilla reduces the number of projection and local interneurons on the amputated side. No change in the size of the olfactory centers occurs on the unamputated side. Amputation of the olfactory receptor neurons in crayfish therefore leads not only to a degeneration of the receptor cell endings in the olfactory lobe but also to a trans-synaptic response in which the number of higher order neurons decreases. Reconstitution of the antennule and olfactory receptor neurons in small adult crayfish is accompanied by the reestablishment of the normal number of interneurons and neuropil volume in the olfactory centers.

Fig. 1.

Volume of the accessory and olfactory lobes compared with increasing body size (carapace length). Each point represents the mean of the volume of the left and right accessory lobes (•) and olfactory lobes (○) from one individual. A, Individuals ranging from 70% embryos to the POI day 4. The accessory lobes are smaller than the olfactory lobes. B, Individuals ranging from POII to ADI day 3. The accessory lobe is equal in size to the olfactory lobe at the POII and becomes larger than the olfactory lobe during the late POII and the molt to ADI.C, ADII–ADIV: at ADII the accessory lobe volume is approximately 3.5 times larger than the olfactory lobe and from this point on the ratio of 3.5:1 is maintained throughout life.D, Individuals with carapace length from 1 to 6 cm. Theline drawn through the points represents the calculated linear regression (accessory lobe, r²= 0.7332, p < 0.0001; olfactory lobe,r² = 0.9238, p < 0.0001). E, Combined data over the entire range of animal sizes showing the rapid change in the ratio between the accessory (AL) and olfactory (OL) lobes in the early stages of development and the maintenance of a relatively constant size ratio after ADII (arrow).

Fig. 2.

The relationship of the number of aesthetasc sensilla on the antennule to body size ranging from the first appearance of the sensilla at POII (3 mm carapace length) to adults with a carapace length of 6 cm. Despite the shedding that occurs in animals after they have reached a carapace length of 1 cm, there is a linear increase in the number of sensilla with the increase in body size.

Fig. 3.

Changes in the size and number of cell somata of local interneurons (○) and projection neurons (•) in clusters 9 and 10. A, The mean diameter of these cells increases rapidly from POI to the ADIV stage when animals reach a carapace length of 1 cm. Cell soma diameter remains stable at ∼8 μm for the rest of the animal’s life. B, The volume of both cell soma clusters increases linearly with body size over the life of the animal.C, Cell number, calculated by dividing cell volume (including the intercellular space) into the cluster volume, shows the linear increase in cell number from juvenile to the largest adult, which has ∼200,000 projection neurons and ∼5000 local interneurons.

Fig. 4.

Light micrograph of a median sagittal section through the deutocerebral commissure of an adult animal. Dorsal is at the top of the figure; anterior is to the left. The commissure contains two separate bundles of axons with different diameters (DC 1 and DC 2), separated from neighboring neuropil by glial cells (G) and bounded on the dorsal side by the finer fibers of the olfactory globular tract (OGT). Scale bar, 25 μm.

Fig. 5.

Electron micrographs of median sagittal sections through the deutocerebral commissures of crayfish at stages POI (A), POII (B), ADI (C), and ADII (D). The sections are all oriented with dorsal at the top of the figures; anterior is to the left. A, Axons in the commissures of POI animals do not fall into two classes. B, C, In POII, some smaller profiles can be recognized along the posterior ventral edge of the commissure (arrow) that appear as a very small bundle in ADI (arrow, C). D, Two axon bundles (DC1 and DC2), like those in commissures of large adults, appear in ADII. Scale bars, 5 μm.

Fig. 6.

The change in axon diameter with growth. Axons in POI and POII are treated here as large axons. Both small and large axons increase in diameter throughout the growth of the animal over the range of carapace length from 3 to 6 cm and in proportion to the change in the body size. A calculated regression line (not shown) indicates that this extends back to the smallest adult stages (large axons,r² = 0.9917, p < 0.0001; small axons, r² = 0.9802,p = 0.0001).

Fig. 7.

Axon number in the deutocerebral commissure of postembryonic, immature, and mature adults. Each point is the mean of all counts. •, Large axons; ○, small axons; ▴, totals of large and small axons. Total axon counts are shown for POI, POII, and ADI because the axons within the commissures during these stages are all nearly the same size. Total axon number increases through stages POI to ADII and stabilizes by the time the animals have a carapace length of ∼3.0 cm (ADIII). From this time on (ADIV, ADV, and ADVI) there is no increase in the number of large or small axons. The large-diameter axon number stabilizes by stage ADII, so that the increase in the overall total is caused by the addition of small-diameter axons to the commissure.

Fig. 8.

Reduction in olfactory and accessory lobe volume and local interneuron and projection neuron cell number in five adult animals (histograms 1–5) caused by amputation of one antennule. The time between amputation and killing of animals for brain measurement was the same for all five animals. Reconstituted stumps were removed after each molt. Histogram 6 shows the means of normal left/right variation in 38 unamputated control animals. (White columns = accessory lobe volume;light gray = olfactory lobe volume; dark gray = small local neurons; black = projection neurons). Olfactory lobe volume on the amputated side is reduced by ∼70% of the control side in all animals; accessory lobes are reduced by only ∼10% and lie within the normal variation in size between the lobes in an unamputated control animal. Local interneurons on the amputated side are reduced by ∼30%, and the projection neurons on the amputated side are reduced by ∼20%.

Fig. 9.

BrdU-labeled projection neurons (PN) and local interneurons (LN) in 10 μm wax sections taken horizontally through the brain. Only the left side of the brain is shown in which the projection neuron cluster is on the left and the local interneuron cluster is on the right.A, ADI animal. B, Animal with a carapace length of 4.0 cm. Scale bars: A, 50 μm;B, 100 μm.

Fig. 10.

TUNEL profiles among the cell somata of the projection neurons, revealed by the TUNEL method and fluorescence microscopy. Only intensely fluorescing profiles were counted. Scale bar, 20 μm.

Fig. 11.

A, Counts of TUNEL profiles in 35 animals in which the number of profiles on the amputated side (•) is plotted with those on the unamputated side (○) in the same animal. The size of the animals ranged from POII to ADI, and the time between amputation and killing of animals ranged from 1 to 17 d. The data are pooled here and show that in each individual more TUNEL profiles were counted on the amputated side than on the control side.B, The ratio of the profiles on the amputated side to the unamputated side for a series of animals that were all subjected to amputation at the ADI stage and then killed at intervals thereafter is shown on the abscissa.

Fig. 12.

Stages in the reconstitution of an antennule of an animal in which one antennule was amputated at the POI stage. The drawings show the reconstituted and control antennules in the ADI (A), ADII (B), and ADIII (C) stages. Scale bar, 0.5 mm.

Fig. 13.

Recovery of the olfactory lobe after amputation of the antennule at POI. Olfactory lobe volume is reduced by 40% after the first molt and continues to decrease between the POII and ADI. Recovery begins during ADI and is complete by ADV.