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A Time Differential Staining Technique Coupled with Full Bilateral Gill Denervation to Study Ionocytes in Fish

Branchial ionocytes (ICs) are the functional units for ionic regulation in fish. In adults, they are found on the filamental and lamellar epithelia of the ...

University of Ottawa

The presence of an interlamellar cell mass (ILCM) on the gills of goldfish significantly decreases the functional lamellar surface area and increases the diffusion distance for gas transfer and thus may impose a serious challenge for the transfer of respiratory gases (O₂ and CO₂). Here we tested the hypothesis that the presence of the ILCM in goldfish acclimated to 7°C impedes the uptake of O2 and excretion of CO₂. While Pa(O₂) remained unaltered, the baseline values of Pa(CO)₂ were significantly higher in goldfish at 7°C with ILCM present (5.55 ± 0.54 mmHg; mean ± SEM) than in goldfish at 25°C without the ILCM (3.98 ± 0.18 mmHg). Carbonic anhydrase (CA) injections relieved the apparent diffusion limitation imposed by the presence of the ILCM on CO₂ excretion (Pw(CO₂) levels dropped to 3.07 ± 0.32 mmHg). Interestingly, the exposure of fish to acute hypoxia evoked similar changes in Pa(O₂) at the two acclimation temperatures. Ethanol (EtOH) exposure was also used as a tool to further investigate the potential effects of the ILCM on branchial solute transfer. The results showed that the ILCM does not impede EtOH uptake in 7°C goldfish. Overall, the results of this study demonstrate that the remodelling of the goldfish gill associated with acclimation to 7°C water, while increasing Pw(CO₂) , has minimal impact on branchial O2 transfer.

Respiratory responses to hypoxia or hypercapnia in goldfish (Carassius auratus) experiencing gill remodelling.

Goldfish acclimated to cold water (e.g. 7°C) experience a marked reduction in functional lamellar surface area owing to the proliferation of an interlamellar cell mass (ILCM), a phenomenon termed gill remodelling. The goal of the present study was to assess the consequences of the reduced functional surface area on the capacity of goldfish to excrete ammonia. Despite the expected impact of ambient temperature on functional surface area, fish acclimated to 7°C and 25°C exhibited similar rates of ammonia excretion (J(net,amm)); the Q₁₀ values for fed and starved fish were 1.07 and 1.20, respectively. To control for possible temperature-related differences in rates of endogenous ammonia production, J(net,amm) was determined at the two acclimation temperatures after loading fish with 1.12 μmol g₋₁ of NH₄Cl. In the 3 h post-injection period, J(net,amm) was elevated to a greater extent in the 25°C fish. To estimate the potential contribution of increased ventilation and cardiac output to ammonia clearance in the warmer fish, the ammonia loading experiment was repeated on the 7°C fish immediately after they were exercised to exhaustion. The rate of excretion of ammonia was significantly increased in the exercised 7°C fish (presumably experiencing increased ventilation and cardiac output for at least some of the measurement period) suggesting that differences in external and internal convection may at least partially explain the enhanced capacity of the 25°C fish to clear the ammonia load. To more specifically assess the contribution of the different functional surface areas on the differing rates of ammonia clearance at the two acclimation temperatures, the 7°C fish were exposed for 7 days to hypoxia (P(O₂)=10 mmHg=1.33 kPa), a treatment known to cause the disappearance of the ILCM. The results demonstrated that the hypoxia-associated loss of the ILCM was accompanied by a significant increase in the rate of ammonia clearance in the 7°C fish when returned to normoxic conditions. To determine whether compensatory changes in the ammonia transporting proteins might be contributing to sustaining J(net,amm) under conditions of reduced functional lamellar surface area, the relative expression and branchial distribution of four Rh proteins were assessed by western blotting and immunocytochemistry. Although the relative expression of the Rh proteins was unaffected by acclimation temperature, there did appear to be a change in the spatial distribution of Rhag, Rhbg and Rhcg1. Specifically, these three Rh proteins (and to a lesser extent Rhcg2) appeared to localize in cells on the outer edge of the ILCM that were enriched with Na(+)/K(+)-ATPase. Thus, we suggest that despite the impediment to ammonia excretion imposed by the ILCM, goldfish acclimated to 7°C are able to sustain normal rates of excretion owing to the redistribution of ammonia transporting cells.

The consequences of reversible gill remodelling on ammonia excretion in goldfish (Carassius auratus).

At temperatures below 15°C the gill lamellae of goldfish (Carassius auratus) are largely covered by an interlamellar cell mass (ILCM) which decreases the functional surface area of the gill. The presence of the ILCM in goldfish acclimated to cold water conceivably could lead to a covering of the neuroepithelial cells (NECs), which are believed to be important for sensing ambient O₂ and CO₂ levels. In this study we tested the hypothesis that goldfish with covered lamellae (and presumably fewer NECs exposed to the water) exhibit a decreased capacity to hyperventilate in response to hypoxic stimuli. Measurements of ventilation amplitude and frequency were performed during exposure to acute hypoxia (P(w(O₂))=30 mmHg) or following injections of the O₂ chemoreceptor stimulant NaCN into the buccal cavity or caudal vein of fish acclimated to 25°C (uncovered lamellae) or 7°C (covered lamellae) to stimulate predominantly the externally or internally oriented NECs, respectively. The results demonstrated no significant differences in the response to hypoxia, with each group exhibiting similar percentage increases in ventilation amplitude (90-91%) and frequency (34-43%). Similarly, with the exception of a rightward shift of the ventilation frequency dose-response in the fish acclimated to 7°C, there were no significant differences between the two groups of fish in the ED(50) values. These findings suggest that goldfish with covered lamellae retain the capacity to sense external hypoxic stimuli. Using immunohistochemistry to identify serotonin-enriched NECs, it was demonstrated that the presence of the ILCM results in the NECs being redistributed towards the distal regions of the lamellae. In 25°C-acclimated fish, the NECs were distributed evenly along the length of the lamellae with 53±3% of them in the distal half, whereas in fish acclimated to 7°C, 83±5% of the NECs were confined to the distal half. Using the neuronal marker antibody ZN-12, it was demonstrated that the NECs at the distal edges of the lamellae are innervated by nerve fibres. Thus, it is hypothesised that the capacity to sense external hypoxic stimuli in goldfish acclimated to cold water is maintained despite the increasing coverage of the gill epithelial surfaces because of a redistribution of innervated NECs to the exposed distal regions of the lamellae.

The control of breathing in goldfish (Carassius auratus) experiencing thermally induced gill remodelling.

Acclimation of crucian carp and goldfish to temperatures below 15°C causes covering of the gill lamellae by a mass of cells termed the interlamellar cell mass (ILCM). Here we explore the cues underlying gill remodeling (removal or growth of an ILCM) and specifically test the hypotheses that 1) depletion of internal O(2) stores in the absence of any change in external O(2) status can trigger the removal of the ILCM in goldfish acclimated to 7°C, 2) exposing fish acclimated to 25°C to an abundance of O(2) (hyperoxia) can reverse the gill remodeling (i.e., cause the covering of lamellae by an expansion of the ILCM), and 3) neuroepithelial cells (NECs) are involved in signaling the shedding of the ILCM. Hypoxemia induced by phenylhydrazine (anemia) or 5% CO caused a decrease in the ILCM from 80% to 23% and 35%, respectively. Hyperoxia exposure at 25°C caused an increase to 67% of total ILCM and a smaller decrease in the size of the ILCM when fish were transferred from 7 to 25°C. Daily sodium cyanide injections were used to stimulate NECs; this treatment led to a significant decrease in the ILCM. Thus, the three major conclusions of this study are 1) that gill remodeling can occur during periods of internal hypoxemia, 2) that O(2) supply and demand may be a significant driving force shaping gill remodeling in goldfish, and 3) the NECs may play a role in triggering the shedding of the ILCM during hypoxia.

The interactive effects of hypoxemia, hyperoxia, and temperature on the gill morphology of goldfish (Carassius auratus).

Gill remodeling in goldfish (Carassius auratus) is accomplished by the appearance or retraction of a mass of cells (termed the interlamellar cell mass or ILCM) between adjacent lamellae. Given the presumed effects of gill remodeling on diffusing capacity, the goals of the current study were (1) to determine the consequences of increased aerobic O(2) demand (swimming) on gill remodelling and (2) to assess the consequences of the presence or absence of the ILCM on aerobic swimming capacity. Fish acclimated to 7 °C exhibited a marked increase in the ILCM which occupied, on average, 70.0 ± 4.1% of the total interlamellar channel area in comparison to an average ILCM area of only 28.3 ± 0.9% in fish acclimated to 25 °C. Incrementally increasing swimming velocity in fish at 7 °C to achieve a maximum aerobic swimming speed (U (CRIT)) within approximately 3 h resulted in a marked loss of the ILCM area to 44.8 ± 3.5%. Fish acclimated to 7 °C were subjected to 35 min swimming trials at 30, 60 or 80% U (CRIT) revealing that significant loss of the ILCM occurred at swimming speeds exceeding 60% U (CRIT). Prior exposure of cold water-acclimated fish to hypoxia to induce shedding of the ILCM did not affect swimming performance when assessed under normoxic conditions (control fish U (CRIT) = 2.34 ± 0.30 body lengths s(-1); previously hypoxic fish U (CRIT) = 2.99 ± 0.14 body lengths s(-1)) or the capacity to raise rates of O(2) consumption with increasing swimming speeds. Because shedding of ILCM during U (CRIT) trials complicated the interpretation of experiments designed to evaluate the impact of the ILCM on swimming performance, additional experiments using a more rapid 'ramp' protocol were performed to generate swimming scores. Neither prior hypoxia exposure nor a previous swim to U (CRIT) (both protocols are known to cause loss of the ILCM) affected swimming scores (the total distance swum during ramp U (CRIT) trials). However, partitioning all data based on the extent of ILCM coverage upon cessation of the swimming trial revealed that fish with less than 40% ILCM coverage exhibited a significantly greater swimming score (539 ± 86 m) than fish with greater than 50% ILCM coverage (285 ± 70 m). Thus, while loss of the ILCM at swimming speeds exceeding 60% U (CRIT) confounds the interpretation of experiments designed to assess the impact of the ILCM on swimming performance, we suggest that the shedding of the ILCM, in itself, coupled with improved swimming scores in fish exhibiting low ILCM coverage (<40%), provide evidence that the ILCM in goldfish acclimated to cold water (7 °C) is indeed an impediment to aerobic swimming capacity.

The interactive effects of exercise and gill remodeling in goldfish (Carassius auratus).

The presence of an interlamellar cell mass (ILCM) on the gills of goldfish acclimated to 7°C leads to preferential distribution of branchial ionocytes to the distal edges of the ILCM, where they are likely to remain in contact with the water and hence remain functional. Upon exposure to hypoxia, the ILCM retracts, and the ionocytes become localized to the lamellar surfaces and on the filament epithelium, owing to their migration and the differentiation of new ionocytes from progenitor cells. Here we demonstrate that the majority of the ionocytes receive neuronal innervation, which led us to assess the consequences of ionocyte migration and differentiation during hypoxic gill remodelling on the pattern and extent of ionocyte neuronal innervation. Normoxic 7°C goldfish (ILCM present) possessed significantly greater numbers of ionocytes/mm(2) (951.2 ± 94.3) than their 25°C conspecifics (ILCM absent; 363.1 ± 49.6) but a statistically lower percentage of innervated ionocytes (83.1% ± 1.0% compared with 87.8% ± 1.3%). After 1 week of exposure of goldfish to hypoxia, the pool of branchial ionocytes was composed largely of pre-existing migrating cells (555.6 ± 38.1/mm(2)) and to a lesser extent newly formed ionocytes (226.7 ± 15.1/mm(2)). The percentage of new (relative to pre-existing) ionocytes remained relatively constant (at ∼30%) after 1 or 2 weeks of normoxic recovery. After hypoxia, pre-existing ionocytes expressed a greater percentage of innervation than newly formed ionocytes in all treatment groups; however, their percentage innervation steadily decreased over 2 weeks of normoxic recovery.

Effects of hypoxia-induced gill remodelling on the innervation and distribution of ionocytes in the gill of goldfish, Carassius auratus.

In this study we investigated the role of heme oxygenase-1 (HO-1) in modulating the hypoxic and hyperoxic ventilatory responses of goldfish (Carassius auratus) acclimated to 7 and 25°C. HO-1 was present in the neuroepithelial cells (NECs; putative branchial O2 chemoreceptors) of fish acclimated to 7°C only. Hypoxia exposure increased gill HO-1 activity in 7°C fish (14.0±1.4 to 42.5±3.2pmolbilirubinmin(-1)mgprotein(-1)). Inhibition of HO-1 activity with zinc protophorphyrin IX (ZnPPIX) increased the ventilation frequency response to acute hypoxia (30mmHg); frequency increased from 48.3±5.1 to 137.4±16.0 breaths per min (BPM) in hypoxic 7°C fish treated with ZnPPIX compared to 46.2±4.2 to 77.9±5.3 BPM in control fish. Unlike in the control (untreated) 7°C fish exposed to hyperoxia, fish injected with ZnPPIX did not significantly decrease breathing frequency. Inhibiting HO-1 activity was without effect on the hypoxic or hyperoxic ventilatory responses of fish acclimated to 25°C. Based on these observations, we suggest that HO-1 plays an inhibitory role in regulating breathing frequency but only in goldfish acclimated to 7°C.

Velislava Tzaneva

Department of Biology

University of Ottawa

A Time Differential Staining Technique Coupled with Full Bilateral Gill Denervation to Study Ionocytes in Fish