The crucian carp (Carassius carassius) tolerates anoxia for days to months depending on temperature. During episodes of stress, heat shock proteins (HSPs) are important for limiting cellular damage, mainly by ensuring protein function. Accordingly, we hypothesized that anoxia would change the expression of HSPs and that this response would be temperature dependent. Real-time RT-PCR was used to investigate the effects of 1 and 7 days anoxia (A1 and A7) on the expression of HSP70a, HSP70b, HSC70, HSP90, and HSP30 in the brain and heart of 8 degrees C- and 13 degrees C-acclimated crucian carp. In general, the expression of all HSPs changed in response to anoxia, although varying in size and direction, and with organ and temperature. HSP70a expression increased drastically (approximately 10-fold) in A7 brains and hearts at 13 degrees C but not at 8 degrees C. HSC70 and HSP90 expression decreased in A7 brains (by 60-70%), but not in A7 hearts. HSC70 expression increased in A1 brains and hearts at both temperatures (by 60-160%), and HSP30 expression decreased in A7 brains and hearts at both temperatures (by 50-80%). Notably, normoxic fish showed 7- and 11-fold higher HSP70a expression in the brain and heart at 8 degrees C compared with 13 degrees C. This difference disappeared during anoxia, suggesting that cold may function as a cue for preconditioning the crucian carp's HSP70a expression to the approaching anoxic winter period.

The crucian carp, Carassius carassius, survives months without oxygen. During anoxia it needs to keep energy expenditure low, particularly in the brain, with its high rate of ATP use related to neuronal activity. This could be accomplished by reducing neuronal excitability through altered expression of genes involved in excitatory neurotransmission. Through cloning and the use of a recently developed real-time RT-PCR approach, with an external RNA control for normalization, we investigated the effect of 1 and 7 days of anoxia (12 degrees C) on the expression of 29 genes, including 8 3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunits, 6 N-methyl-d-aspartate (NMDA) receptor subunits, 7 voltage-gated sodium and calcium channels, 4 glutamate transporters, and 4 genes involved in NMDA receptor-mediated neuroplasticity. The subunits of the majority of the gene families had expression profiles similar to those observed in the mammalian brain and showed remarkably stable expression during anoxia. This suggests that the genes may have similar functions in crucian carp and mammals, and that the excitatory abilities of the crucian carp brain are retained during anoxia. Although the data generally argue against profound neural depression ("channel arrest"), NMDA receptor subunit (NR) expression showed features that could mediate reduced neural excitability. Primarily, the NR2 subunit expression, which was dominated by NR2B and NR2D, resembled that seen in hypoxia-tolerant neonatal rats, and decreased anoxic expression of NR1, NR2C, and NR3A indicated reduced numbers of functional NMDA receptors. We also report the full-length sequence of crucian carp NR1 mRNA and a novel NR1 splice cassette introducing an N-glycosylation site into the extracellular S1S2 domain.

Many teleost species respond with fright reactions to olfactory cues from injured skin of conspecifics, but they may also display responses to skin extracts of heterospecific fish. In the present study, we exposed crucian carp to skin extracts of conspecifics and three cross-order species of fish (brown trout, pike, and perch). Behavioural experiments showed that conspecific skin extracts induced fright reactions in crucian carp; extracts of brown trout induced such behaviour less frequently, while extracts of perch and pike were poor inducers of fright responses. The olfactory bulb is chemo-topically organized, and different sub-sets of neurons respond to functionally related odorants that mediate distinct behaviours. Accordingly, behavioural responses to an alarm signal should be reflected by activation of the neurons mediating fright reaction. Extracellular recordings from single units in the olfactory bulb showed that the relay neurons activated by conspecific skin extracts were also activated by extracts of brown trout, whereas extracts of perch and pike less frequently activated these units. Thus, the difference in behavioural responses matched the differences in the neural responses, indicating that skin extracts of heterospecific fish are more likely to induce fright behaviour when the responding sub-set of olfactory neurons is similar to the sub-set responding to conspecific extracts. Our results suggest that responses to injured heterospecific fish rely on chemical resemblance between odorants from heterospecific and conspecific skin, and need not be based on any form of associative learning.