The data presented here show that serotonin alters the rate of adult neurogenesis in the crayfish brain, that this action is confined to the late second- and third-generation cells that reside in the LPZ of cluster 10, and that these effects are mediated, at least in part, by 5-HT1α and 5-HT2β receptors. Evidence from several different approaches contribute to these conclusions. First, serotonin increases the numbers of BrdU-labeled cells in the proliferation zones of cluster 10 (as documented previously in the lobster  and the Australian crayfish ), but does not alter the rate of BrdU incorporation among the niche precursors or their migratory daughters in the streams. Second, RT-PCR shows that 5-HT1α and 5-HT2β receptors are expressed in the brains of these animals. Third, in situ hybridization with antisense riboprobes reveals that both 5-HT1α and 5-HT2β mRNAs are found in clusters 9 and 10, the only two sites in the crayfish midbrain where adult-born neurons are incorporated. Fourth, immunolabeling with antibodies raised against P. clarkii 5-HT1α and 5-HT2β peptides demonstrates that these receptors are absent from the first-generation niche precursor cells and their daughters in the proximal and medial parts of the migratory stream. However, some of the second-generation cells in the distal stream close to the proliferation zones in clusters 9 and 10 do express these receptor proteins; it is not known, however, whether these receptors co-localize to the same cells. These receptor localization data suggest that serotonin acts directly on specific generations in the neuronal precursor lineage, rather than through an indirect pathway. An in vitro isolated niche-stream preparation is under development, which will allow a direct test of this idea. Finally, functional assays using a specific 5-HT1α agonist (QMS) increases the rate of BrdU incorporation among cells in cluster 10, while the specific 5-HT2β antagonist MMS attenuates BrdU labeling in these cells. However, QMS and MMS do not alter the number of BrdU-labeled cells found in the neurogenic niche or in the migratory streams. Additional agonists and antagonists were not tested because QMS and MMS were shown to be superior to other agents in terms of specificity and efficacy on P. clarkii 5-HT1α and 5-HT2β receptors . Therefore, in summary, both cytological and functional assays of P. clarkii indicate that serotonin acts selectively on the late second-generation neuronal precursors and their descendants in clusters 9 and 10.
Receptor labeling compared with prior studies
There are similarities and some differences between the immunocytochemical labeling for the 5-HT1crust antibody presented here, and the prior findings of Spitzer et al.  using the same antibody and crayfish species. While labeling is found in the same cell clusters and neuropils as in the earlier study, in our experiments neuropil regions (for example, olfactory and accessory lobes) were most intensely labeled, while in the prior studies cell clusters were most intensely labeled. We also found that immunoreactivity in cell clusters and neuropils was consistent, in contrast to the findings of Spitzer et al. , where the intensity of labeling with the 5-HT1crust antibody, as well as the numbers of cells in the various cell clusters, was highly variable between animals. It was suggested that the variability observed in the prior studies might be 'indicative of the physiological state of the animal' . In this regard the animals used in our assays, for unknown reasons, may have been more uniform. It is more likely, however, that these differences are due to modifications in fixation and processing methods. Spitzer et al. also observed an uneven distribution of labeling in the cytoplasm and around the periphery of irregularly shaped somata in the brain; cells showing receptor labeling in our studies were spherical or ovoid, and labeling was homogeneously distributed throughout the cytoplasm. Possible membrane-associated labeling was observed in both studies, a point that requires further investigation at the ultrastructural level.
Comparison of mRNA and protein expression patterns
The immunocytochemical localization of 5-HT1α and 5-HT2β receptor protein in the present study identifies sites of serotonin's action in the brain of the crayfish P. clarkii. In addition, the in situ hybridization results establish which cells synthesize 5-HT receptor proteins and contribute these to the neuropils and to the system generating new neurons in the adult brain. However, the most intense receptor labeling was found in neuropil regions, while 5-HT1α and 5-HT2β mRNA was predominantly localized to cell somata. For example, we observed intense labeling for the 5-HT1α protein, as well as distinct but weak labeling for the 5-HT2β receptor, in the olfactory and accessory lobes. These neuropil regions contain axonal fibers and synapses of local and projection interneurons in cell clusters 9 and 10, respectively. However, the mRNA for both 5-HT receptors is primarily found in the somata of cells in clusters 9 and 10 and not in the neuropils.
There are two possible explanations for the distinct intracellular distributions of receptor mRNA and protein. Firstly, this may suggest that there is little or no synthesis of the serotonin receptor protein at the sites where we observe the most intense immunocytochemical labeling. Instead, our data may indicate that these receptors are synthesized in the somata and transported to the synaptic terminals. To date, although local translation of membrane proteins, heat-shock proteins, anti-oxidant proteins at distal axons and their terminals has been reported [27, 28], cytoskeletal proteins remain dominant among the axonally synthesized proteins . The slow axoplasmic transport  and limited half-life of cytoskeletal proteins [30, 31] render local synthesis a more effective means to deliver these proteins to distal axons or growth cones [32, 33]. We do not know the half-life of serotonin receptor proteins in crayfish neurons, but the relatively stable lifespan (half-life >100 hours) of 5-HT1A and 5-HT2A receptors in rat  provides a reference. Secondly, therefore, the sparsity of receptor mRNA at sites where protein is most intensely labeled could be an outcome of a relatively short half-life of mRNA compared to the receptor protein. Indeed, in cultured P11 cells derived from rat pituitary tumors, 5-HT2A mRNA has an average half-life of only 70 minutes . One does not, therefore, necessarily expect to see co-localization of receptor mRNA and protein.
Serotonin transporter distribution
Our results and those of Spitzer et al.  demonstrate intense cytoplasmic labeling for the 5-HT1α and 5-HT2β receptors. Spitzer et al. have suggested that this labeling is likely to represent newly synthesized or recycled receptor. While this is quite possible, an alternative possibility is that at least some of these receptors may be functional as cytoplasmic proteins. One hypothesis is that serotonin may be taken into cells via the serotonin transporter, where it could bind to cytoplasmic receptors to initiate specific functions. It has been proposed that serotonin may act intracellularly as a growth or transcriptional regulator [36–39], although the specific actions are speculative.
To explore the possibility of a cytoplasmic role for these serotonin receptors, we localized sites of SERT labeling in the crayfish brain. Interestingly, the pattern of labeling for the serotonin transporter is similar in many regions to the distribution of receptor labeling. For instance, the medial giant cells that contain intense cytoplasmic labeling for the 5-HT2β receptor also label strongly for SERT. Likewise, cells in clusters 9, 10 and 11 contain intense labeling for both the 5-HT1α receptor and SERT, while the LPZs are weakly labeled for both 5- HT1α and SERT. These similarities between labeling for SERT and cytoplasmic 5-HT1α and 5-HT2β receptors may suggest a nontraditional role for serotonin in these neurons; serotonin may be taken into cells via the transporter, where it then activates cytoplasmic receptors. The large size of the medial giant cells makes these neurons particularly tractable for testing this hypothesis.
Functional implications of receptor distribution in the crayfish brain
The distribution of 5-HT1α and 5-HT2β receptors in somata residing in several cell clusters, as well as in many synaptic neuropils (for example, protocerebral bridge, olfactory and accessory lobes, anterior and posterior median protocerebral neuropils) suggests that serotonin mediates a variety of functions in the crustacean brain. Previous studies have implicated 5-HT1α receptors in the different degrees of social dominance in crayfish  and in diurnal rhythms in the eyestalk , functions that are likely to involve higher order brain pathways. Our studies and those of Spitzer et al.  suggest a possible role for 5-HT1α receptor in processing of olfactory information, as well as in higher order integrative functions mediated by the accessory lobes [8, 9]. Less is known about possible physiological actions of 5-HT2β receptor, although these have been localized to the processes and somata of lateral giant neurons, which are involved in the tail-flip escape response. Our present studies demonstrate the presence of 5-HT2β receptor immunoreactivity in the cytoplasm of the medial giant cells, which also are part of the tail-flip circuitry [42, 43].
The primary goal of our studies was to examine the distribution of 5-HT1α and 5-HT2β receptors in the lineage of precursor cells that is responsible for the production of neurons in the adult crayfish brain, and to relate these findings to the effects of serotonin on the cell cycle of each generation of precursors. Exposure of intact animals to serotonin increases BrdU incorporation in the LPZ, which contains the late second- and third-generation neuronal precursors, but not into the first-generation precursors or their migrating daughters. This lineage will produce neurons that will differentiate into projection neurons that innervate the olfactory and accessory lobes. Pharmacological experiments with the 5-HT1α receptor agonist QMS and the 5-HT2β receptor antagonist MMS are consistent with the effects of serotonin on neuronal precursors, and with the distribution of these receptors in the neurogenic lineage.
Serotonin and neurogenesis in crustaceans and mammals
These data are of additional interest because many features of adult neurogenesis are evolutionarily conserved. In mammalian and decapod crustacean brains, new neurons are added throughout life to the primary olfactory processing areas. The first generation neuronal precursors, which have glial properties, are located in specialized vascularized niches; their daughters migrate to sites where they will proliferate again and their progeny will differentiate into neurons [10, 44–46]. Further, the timing and rate of proliferation in the adult crustacean brain, as in the mammalian brain, are influenced by circadian signals, age, diet, environmental enrichment, nitric oxide and serotonin [11, 47].
Serotonin is a particularly potent regulator of neurogenesis during both embryonic and adult life in mammals [48–51] and crustaceans [14–16]. Depletion of brain serotonin results in a decrease in production of new neurons in the dentate gyrus and the subventricular zone of adult rats , and in clusters 9 and 10 containing local and projection interneurons in the olfactory pathway of lobsters [3, 14]. The general influence of serotonin in the regulation of neurogenesis is therefore conserved across species.
The effects of serotonin on neurogenesis are mediated via its receptors. In mammals, several receptor subtypes are involved in regulating neuronal proliferation in the subgranular zone of the hippocampus and the subventricular zone that contributes new neurons to the olfactory bulb. By various accounts, the activation of 5-HT1A and 5-HT2C receptors increases neurogenesis in the subventricular zone/olfactory bulb, while 5-HT1A, 5-HT1B, 5-HT2, 5-HT2A, and 5-HT2C receptors have been implicated in diverse effects on neuronal proliferation in the subgranular zone/hippocampus [52–58]. While it is apparent that there are differential actions of these receptors during distinct phases of neurogenesis in these brain regions, methodological disparities and contradictory results hinder clarity regarding these events. The interpretation of 5-HT receptor actions in mammalian systems also have been complicated by the complexity of the precursor cell lineage that produces adult-born neurons, the fact that multiple precursor generations coexist in neurogenic niches, and the relative scarcity of type 3 cells, which are a key population . These challenges underscore the potential value of addressing the fundamental question of lineage-dependent influences of serotonin using the crayfish model, where precursor cell generations are spatially separated and quantitative changes are easily assessed. Furthermore, the use of diverse species to address important questions about adult neurogenesis is likely to result in a broader understanding of specific issues, and of how evolutionary processes may have shaped the vertebrate/mammalian condition.
In the crayfish brain, 5-HT1α and 5-HT2β receptors first appear in the late second-generation neuronal precursors at the end of their tangential migration across the ventral surface of the brain, as they reach the proliferation zones where olfactory interneurons will proliferate and differentiate. Neither the first generation precursor cells in the niche nor their daughters in the proximal and medial parts of the migratory stream express 5-HT1α receptor. Further, the level of 5-HT1α mRNA expression in the proliferation zones appears to be lower than in the mature cluster 10 neurons. This pattern suggests that after the first expression of 5-HT1α receptor in the late second-generation precursors, receptor expression increases as their progeny differentiate into neurons. The functional assays using the 5-HT1α agonist QMS corroborate the receptor localization, as QMS upregulates BrdU incorporation only in the proliferation zones of cluster 10, not among the niche precursors or in their daughters during migration to the proliferation zones. The story with the 5-HT2β receptor appears very similar, in that 5-HT2βCrust immunoreactivity is confined to the late second- and third-generation cells in the distal migratory streams and proliferation zones of cell clusters 9 and 10, as is the action of the 5-HT2β antagonist MMS on cell proliferation in this system. This sequence of events is reminiscent of a study of the maturation of precursor cells migrating in the rostral migratory stream, which shows temporal and spatial regulation in the appearance of GABA and glutamate receptors, with GABAA receptors expressed first in the tangentially migrating class 1 cells , followed by AMPA receptors ; during the next phase of maturation, the radially migrating class 2 cells express NMDA receptors.
Another interesting aspect of the 5-HT1α receptor labeling pattern is the finding that only some of the cells in the distal migratory stream express the receptor. This variability may reflect heterogeneity in the spatiotemporal expression of the receptor in different cells, with the possible end point that all second-generation precursors will contain 5-HT1α receptor. Alternatively, other classes of 5-HT receptors that were not assessed in this study may be involved in serotonin's action on cells in the proliferation zones. Finally, it may be that only a portion of the late second-generation precursors are sensitive to serotonin. It is known that there are at least two types of local interneurons in cluster 9 expressing either the neurotransmitter orkokinin or allatostatin-like peptide . Similarly, there are two functional categories of projection neurons, those that innervate the olfactory lobe and others that innervate the accessory lobe [13, 61]. The presence or absence of serotonin receptors in the late second-generation precursors may therefore reflect distinctive chemical/hormonal sensitivities involved in diverging cell fates.