Fossorial Damaraland mole-rats (Fukomys damarensis) mount a robust hypoxic metabolic response (HMR) but a blunted hypoxic ventilatory response (HVR) to acute hypoxia. Although these reflex physiological responses have been described previously, the underlying signalling pathways are entirely unknown. Of particular interest are contributions from γ-aminobutyric acid (GABA), which is the primary inhibitory neurotransmitter in the nervous system of most adult mammals, and adenosine, the accumulation of which increases during hypoxia as a breakdown product of ATP. Therefore, we hypothesized that GABAergic and/or adenosinergic signalling contributes to the blunted HVR and robust HMR in Damaraland mole-rats. To test this hypothesis, we injected adult animals with saline alone (controls), or 100 mg kg−1 aminophylline or 1 mg kg−1 bicuculline, to block adenosine or GABAA receptors, respectively. We then used respirometry, plethysmography and thermal RFID probes to non-invasively measure metabolic, ventilator and thermoregulatory responses, respectively, to acute hypoxia (1 h in 5 or 7% O2) in awake and freely behaving animals. We found that bicuculline had relatively minor effects on metabolism and thermoregulation but sensitized ventilation such that the HVR became manifest at 7% instead of 5% O2 and was greater in magnitude. Aminophylline increased metabolic rate, ventilation and body temperature in normoxia, and augmented the HMR and HVR. Taken together, these findings indicate that adenosinergic and GABAergic signalling play important roles in mediating the robust HMR and blunted HVR in Damaraland mole-rats.

The initial physiological reflex response of most adult mammals to inhalation of hypoxic gas is to increase ventilation (Pamenter and Powell, 2016; Powell et al., 1998). This is referred to as the hypoxic ventilatory response (HVR) and is one of the primary mechanisms through which O2 supply to the body can be increased in hypoxic environments. In most adult mammals, the HVR is biphasic, consisting of a sharp increase in ventilation following the initial detection of inhaled hypoxia, followed by a decrease after several minutes of sustained hypoxic exposure to a new baseline level that remains above normoxic ventilation. Neonatal mammals also exhibit a biphasic HVR; however, neonatal ventilation is often reduced below normoxic levels during the second phase of the HVR (Mortola et al., 1989), which likely contributes to energy savings in hypoxia.

The signalling mechanisms that regulate the adult mammalian HVR are well understood in non-fossorial mammals. In most such species, excitatory glutamatergic signalling mediates the first phase of the HVR and also underlies the increase in ventilatory tone during the second phase, whereas inhibitory γ-aminobutyric acid (GABAergic) signalling plays a central role in the decline of ventilation in the second phase of the HVR, relative to the initial peak of the first phase (Pamenter and Powell, 2016; Teppema and Dahan, 2010). In neonates, the glutamatergic system is not fully developed, and instead, the GABAergic system dominates neurotransmission (Wong-Riley et al., 2019), resulting in a net inhibitory shift of the neonatal HVR. A similar system appears to regulate the HVR of eusocial naked mole-rats, which are among the most hypoxia-tolerant mammals and putatively experience intermittent hypoxia in their crowded underground burrows (Buffenstein et al., 2022; Chung et al., 2016; Pamenter, 2022; Pamenter et al., 2019, 2018). Specifically, and unlike in all other adult mammals studied (but similar to neonates from non-fossorial mammalian species), modulation of glutamate receptors has no impact on the HVR of naked mole-rats exposed to 7% O2 (Dzal et al., 2019). Instead, antagonism of GABA receptors abrogates the HVR in this species (Chung et al., 2016), suggesting a central role for GABA receptors in the control of the HVR in a fossorial mammal.

Conversely, few studies have explored the control of metabolic responses to hypoxia in adult mammals, presumably because a robust hypoxic metabolic response (HMR) is not a commonly observed strategy in hypoxia-intolerant adult mammals, which have received the lion's share of research attention in this field. As a result, the mechanisms that regulate the HMR are poorly understood, and knowledge in this area is particularly lacking in fossorial species. However, this question has also recently received some attention in naked mole-rats: in response to hypoxia, naked mole-rats drastically reduce their metabolic rate (Pamenter et al., 2015, 2019), in part owing to a rapid decrease in thermogenesis (Cheng et al., 2021; Kirby et al., 2018; Vandewint et al., 2019). Unlike in other adult mammals, the naked mole-rat HMR is mediated by inhibitory signaling, with adenosine playing a key role (Chung et al., 2016; Dzal et al., 2019; Pamenter, 2022; Pamenter et al., 2015). Adenosine is a breakdown product of ATP hydrolysis and the concentration of adenosine in the blood increases during periods of hypoxia in most species (Buck, 2004; Phillis et al., 1992; Saito et al., 1999); therefore, it is a promising candidate to regulate physiological responses to hypoxia.

Damaraland mole-rats (Fukomys damarensis) are close phylogenetic cousins of naked mole-rats, are the only other eusocial mammal, and also live in large, densely populated underground colonies (Thomas et al., 2016). The physiological response of Damaraland mole-rats to acute hypoxia has recently been described and is like that of naked mole-rats: they have a blunted HVR and a robust HMR (Ivy et al., 2020). Therefore, Damaraland mole-rats may also have similar control pathways underlying these responses. Unfortunately, nothing is known about the signalling mechanisms that control physiological responses to hypoxia in this species.

Given the role for GABAergic and adenosinergic signalling in mediating metabolic and ventilatory adaptations to hypoxia in naked mole-rats, and the generally inhibitory tone of physiological responses to acute hypoxia in Damaraland mole-rats, we hypothesized that the same signalling pathways regulate the hypoxic metabolic and ventilatory responses in Damaraland mole-rats. To test this hypothesis, we measured metabolism, ventilation and thermoregulation in freely behaving Damaraland mole-rats exposed to normoxia (21% O2) and then acute hypoxia (1 h in 7 or 5% O2), with and without intraperitoneal injections of the adenosine receptor antagonist aminophylline or the GABAA receptor antagonist bicuculline.

List of symbols and abbreviations

     
  • ACR

    air convection requirement

  •  
  • fR

    breathing frequency

  •  
  • GABA

    gamma-amino butyric acid

  •  
  • HMR

    hypoxic metabolic response

  •  
  • HVR

    hypoxic ventilatory response

  •  
  • RER

    respiratory exchange ratio

  •  
  • Tb

    body temperature

  •  
  • E

    minute ventilation

  •  
  • CO2

    carbon dioxide production

  •  
  • O2

    oxygen consumption rate

  •  
  • VT

    tidal volume

Animals

Damaraland mole-rats [Fukomys damarensis (Ogilby 1838)] were bred at the University of Ottawa group-housed in interconnected multi-cage systems at 22°C in 21% O2 and 0.04% carbon dioxide (CO2) and 50% humidity with a 12 h:12 h light:dark cycle. Animals were fed fresh tubers, vegetables, fruit and Pronutro cereal supplement ad libitum. The age of our experimental animals was selected based on a growth study of Damaraland mole-rats, which reported that this species reaches 90% of their full size by 600 days post-birth (Thorley and Clutton-Brock, 2019). Neural dimorphisms exist dependent on breeding status such that breeding animals have greater development in certain areas of the brain relative to subordinates of both sexes (Anyan et al., 2011), which may have unpredictable impacts on the neural control of physiological responses to hypoxia. As such, the queen and breeding male from our colony were omitted from this study.

Experimental ethics and design

All experimental procedures were approved by the University of Ottawa's Animal Care Committee (protocol no. 2535) and conducted in accordance with the Animals for Research Act and other regulations of the Canadian Council on Animal Care. Experiments were performed during the daylight portion of the animals' daily light:dark cycle, and the experimental time within this window was randomized for each individual animal and experimental protocol to remove any bias induced by circadian rhythms. Briefly, 10 adult subordinate Damaraland mole-rats (5 males, 5 females, age 1.5–3 years; 208.1±12.3 g, mean±s.e.m.) were subjected to each of five experimental conditions, consisting of exposure to 1 h of normoxia followed by intraperitoneal injections of: saline alone (controls), or aminophylline (to block adenosine receptors; 100 mg kg−1) (Barros et al., 2006; Martin, 1946; Pamenter et al., 2015) or bicuculine (to block GABAA receptors; 1 mg kg−1) (Beart and Johnston, 1972; Chung et al., 2016; Curtis et al., 1970a,b). Animals were then replaced in normoxic conditions for 1 h to assess the impact of the drug on the physiological parameters of interest, and were finally then exposed to either 7 or 5% O2 for 1 h in random order (protocols 1 and 2, respectively). The doses and half-lives of both drugs are sufficient to ensure efficacy in vivo for at least 3 h post-injection (Gale and Casu, 1981; Lohmann and Miech, 1976; Ueno et al., 1997). Aminophylline had major effects on metabolic rate (see Results), and animals treated with this drug were unable to tolerate exposure to 5% O2. Therefore, the impact of aminophylline in hypoxia was only tested in 7% O2 and the sample size for this dataset was reduced relative to the other experimental groups owing to animal deaths in pilot trials in 5% O2. Saline experiments in both 7 and 5% O2 are also presented in Devereaux et al. (2023). Animals were not fasted prior to experimental trials and all animals were exposed to all six experimental paradigms (3× injected treatments in two different hypoxic conditions) in random order. Animals were permitted a minimum of 1 week of rest between experiments to reduce any confounding effects of previous hypoxic exposures.

For each experiment, animals were individually placed unrestrained in a 1.0 l Plexiglass experimental chamber held at 22°C in normoxia (21% O2/0.04% CO2) until O2 consumption reached steady state for a minimum of 20 min. For the purposes of these experiments, we defined steady state as a period where the concentration of O2 in the excurrent air did not fluctuate by more than 0.2% for a minimum of 15 min. Following this normoxic control period, animals were removed from the experimental chamber and injected intraperitoneally with either saline or one of the two drugs. Drugs were diluted to inject animals with a standard bolus of 2.5 µl g−1 of body mass and drug dosages were based on previous experiments performed on naked mole-rats in our lab, which were in turn based on studies in other small rodents (see references above). Dosages were updated for the Damaraland mole-rats on an as-needed basis. Animals must have been awake and not exhibiting signs of physical stress (e.g. haunching, scratching or licking injection site, irregular or strained breathing patterns, etc.) post-injection before they were subjected to experimentation.

After injection, animals were placed back into normoxia until O2 consumption reached steady state. This was done to control for any effects the drugs may have had on metabolic or ventilatory parameters while the animals were still breathing normoxic air, and to account for stress following the injection and related handling. Finally, O2 was lowered to 7% or 5%, taking 15–30 min to achieve the new hypoxic equilibrium in the experimental chamber and until metabolic parameters once again reached steady state. Our lab has previously conducted experiments in naked mole-rats using these same drug treatments in a 7% O2 hypoxic exposure. Therefore, we chose this O2 level for our experiments to permit simple comparison between the two species. However, in preliminary studies we did not observe a ventilatory response in 7% O2 (protocol 1). Therefore, we repeated all experiments at a deeper level of hypoxia (5% O2; protocol 2) to better assess the effect of each drug on ventilation. Although Damaraland mole-rats have been studied in as low as 3% O2, pilot trials determined this was only tolerable for a maximum of 35 min with saline, and for less time in certain drug trials. Thus, 5% O2 was determined to be the lowest safe O2 level for the animals and we did not test deeper levels of hypoxia. Each experimental stage lasted approximately 1 h.

To maximize the sensitivity and resolution of metabolic rate measurements, gases were continuously supplied at 0.4 l min−1. As a result, animal-induced changes in the concentrations of O2 and CO2 would have altered the gas composition in the experimental chamber, reducing O2 by ∼0.4–2.0% and increasing CO2 by ∼0.1–1.0%, depending on their metabolic activity. However, standard practices in this field are based largely on studies in hypoxia-intolerant animals, such as mice, dogs and rats, which are considerably more sensitive to smaller changes in atmospheric O2 and CO2. Conversely, Damaraland mole-rats live at mild altitude in nature, in which atmospheric air is ∼18.5% O2, and their metabolic and ventilatory responses to hypoxia and hypercapnia are not activated until well beyond 19% O2 or 1% CO2 (Ivy et al., 2020; Zhang and Pamenter, 2019a,b). Therefore, it is unlikely that this affected our evaluation of the regulation of the HVR and HMR in this species.

Respirometry

During experimentation, the animal chamber was sealed and continuously ventilated with gas mixtures, set to the desired fractional gas composition by calibrated rotameters (Praxair, Mississauga, ON, Canada). Incurrent air was bubbled in distilled H2O prior to entering the animal chamber. Excurrent air passed through an RH-300 Water Vapour Pressure Analyzer to measure humidity, and the average incurrent gas humidity was 94.5±3.1%, which is consistent with previous experiments using similar equipment in our laboratory (Clayson et al., 2020). Excurrent gas passed through a desiccant medium before entering the O2 and CO2 analyzers. The final 10-min period of stable activity from each experimental stage was used for data analysis. To determine metabolic rate, eqns 10.6 and 10.7 from Lighton (2008) were used to calculate the rate of O2 consumption (O2, ml min−1 kg−1) and the rate of CO2 production (CO2, ml min−1 kg−1), respectively:
formula
(1)
formula
(2)

In these equations, FRi is the incurrent flow rate (ml min−1), FiO2 and FiCO2 are the fractional concentrations of incurrent O2 and CO2 of dry gas, respectively, and FeO2 and FeCO2 are the fractional concentrations of excurrent O2 and CO2 from the experimental chamber, respectively (Lighton, 2008). Respiratory exchange ratios (RERs) were calculated as CO2/O2.

Thermal measurements

Body temperature (Tb, °C) was recorded non-invasively every 10 min throughout all experiments using an RFID microchip reader (Allflex USA Inc., Dallas, TX, USA) to scan previously implanted subcutaneous RFID microchips (Destron Fearing) along the back flank of the animal. Chamber temperature was recorded during each inflow period using a custom thermocouple.

Whole-body plethysmography

Attached to the experimental chamber was an identical second chamber that acted as a reference chamber. Continuous monitoring by a differential pressure transducer connected between the two chambers amplified small pressure fluctuations in the experimental chamber, allowing us to detect and measure breaths at 1000 Hz. Before each trial, the transducer was calibrated by injecting six known volumes of air (0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 ml) 10 times into the experimental chamber. Injections were performed with continuous airflow through the pressure-sealed system at the same respiratory frequency (fR, breaths min−1) the animal was observed to have under normoxic/normocapnic conditions. To calculate tidal volume (VT, ml kg−1) and fR, five breath sets consisting of a minimum of 10 consecutive and clearly defined breaths within the same 10 min period as that used for metabolic rate calculations were analyzed. The Drorbaugh and Fenn (1955) equation was used to calculate VT:
formula
(3)

The average oscillation height was taken from each breath set, representing the average total pressure deflection of a breath (Pm). Pcal (V) and Vcal (µl) are the pressure deflection and volume of a known calibrated volume, respectively. The average oscillation height of each calibration set was plotted against its respective volume and used to create a linear relationship. The point on this line representing 0.2 ml was chosen as Pcal and Vcal. TA is the body temperature of the animal (K) and TC is the temperature of the chamber (K), both recorded at the end of the 10 min period. PB is the barometric pressure in the lab (mmHg) as measured by the O2 analyzer. PA is the vapour pressure of water at the animal's body temperature (mmHg) and PC is the partial pressure of water vapour (mmHg) in the incurrent gas stream (Drorbaugh and Fenn, 1955). PA was calculated using relative humidity (%) of excurrent air, animal temperature (°C) and barometric pressure (kPa). PC used relative humidity (%) of incurrent air, chamber temperature (°C) and barometric pressure (kPa). The same breath samples were used to calculate fR. Minute ventilation (E) was calculated as the product of fR and VT. The air convection requirements (ACRs) for O2 and CO2 were calculated as the quotient of E and O2 or CO2, respectively.

Analysis

Ventilatory and metabolic data were collected using LabChart software (ADInstruments, Colorado Springs, CO, USA) and analysed in PowerLab (ADInstruments). Statistical analysis was performed using commercial software (Prism v.9.2.0, GraphPad Software Inc., La Jolla, CA, USA). Responses to hypoxia and changes mediated by aminophylline or bicuculline administration were not different between sexes and so these data were pooled in our analysis. P<0.05 was considered the threshold for statistical significance. Data are presented in box and whisker plots. Aminophylline and bicuculline target pathways that do not directly interact, and which were not inherently linked by our hypothesis; therefore, we compared the results of drug trials directly with saline controls only and not with each other. Statistical significance was evaluated using a two-way repeated-measures analysis of variance (RM-ANOVA) to test for interactions between normoxia and hypoxia (7 or 5% O2), or drug treatments. Tukey's or Šidák's multiple comparisons tests were performed on each dependent variable to determine significance. Statistical significance between hypoxia-mediated changes in each variable were analyzed using Student’s t-tests.

Note that for all datasets, statistical analysis of control (saline) experiments is presented in the companion paper to this study (Devereaux et al., 2023) and is not duplicated here.

Metabolism and thermoregulation are minimally impacted by GABAA receptor antagonism

First, we evaluated the effect of bicuculline on O2 and CO2 as indicators of metabolic rate (Fig. 1A–F). Bicuculline had mixed effects on these variables in normoxia, but no effect in hypoxia. Specifically, both variables increased following drug injection during normoxia in protocol 1 (F1,13=31.04, P<0.0001 and F1,13=5.655, P=0.0034 for O2 and CO2, respectively; Fig. 1A,D), but not in protocol 2 (F1,16=0.2459, P=0.6267 and F1,17=0.5099, P=0.2389 for O2 and CO2, respectively; Fig. 1B,E), despite these experiments being conducted in random order and in the same animals. However, bicuculline-mediated changes during normoxia in protocol 1 were primarily due to lower average metabolic rates and reduced interindividual variability during normoxia in saline-treated animals in this group, which were not replicated in saline-treated animals in protocol 2. Similarly, bicuculline injection had a minimal effect on the HMR, with the changes in O2 and CO2 being largely similar between saline and drug treatment groups, except for the comparison between saline and drug in protocol 1 (F1,29=7.281, P=0.0115 and F1,35=0.02522, P=0.8947 for ΔO2 and ΔCO2, respectively; Fig. 1C,F).

Fig. 1.

Damaraland mole-rat metabolism and thermoregulation are minimally regulated by GABAergic signalling. (A,B,D,E,G,H,J,K) Summaries of the rates of O2 consumption (O2; A,B), the rates of CO2 production (CO2; C,D), respiratory exchange ratios (RERs; G,H) and body temperatures (Tb; J,K) from Damaraland mole-rats exposed to 21% O2, before and after intraperitoneal injections of saline alone (white bars and open symbols), or the γ-amino butyric acid type A (GABAA) receptor antagonist bicuculline (1 mg kg−1; dissolved in saline; pink bars and closed symbols), and subsequent exposure to acute hypoxia (7 or 5% O2; 1 h). Data are presented as box and whisker plots of min–max with individual replicates shown from n=7–10 biological replicates per dataset. (C,F,I,L) Summaries of the hypoxia-mediated change in O2 (C), CO2 (E), RER (I) and Tb (L) from animals treated as in A and B. Summary data are means±s.e.m. Different letters indicate significance as determined using a two-way ANOVA or mixed-effects model with Tukey's post hoc tests, P<0.05.

Fig. 1.

Damaraland mole-rat metabolism and thermoregulation are minimally regulated by GABAergic signalling. (A,B,D,E,G,H,J,K) Summaries of the rates of O2 consumption (O2; A,B), the rates of CO2 production (CO2; C,D), respiratory exchange ratios (RERs; G,H) and body temperatures (Tb; J,K) from Damaraland mole-rats exposed to 21% O2, before and after intraperitoneal injections of saline alone (white bars and open symbols), or the γ-amino butyric acid type A (GABAA) receptor antagonist bicuculline (1 mg kg−1; dissolved in saline; pink bars and closed symbols), and subsequent exposure to acute hypoxia (7 or 5% O2; 1 h). Data are presented as box and whisker plots of min–max with individual replicates shown from n=7–10 biological replicates per dataset. (C,F,I,L) Summaries of the hypoxia-mediated change in O2 (C), CO2 (E), RER (I) and Tb (L) from animals treated as in A and B. Summary data are means±s.e.m. Different letters indicate significance as determined using a two-way ANOVA or mixed-effects model with Tukey's post hoc tests, P<0.05.

We also calculated RERs to indirectly measure metabolic fuel substrate use (Fig. 1G–I). The RER was tightly maintained in normoxia, and following bicuculine injection, with the means from these experimental groups ranging from 0.8 to 0.85, indicating a mix of lipid and carbohydrate fuel use. The RER in bicuculline-treated animals breathing 5 or 7% O2 was not statistically different from measurements from saline-treated animals breathing hypoxic gas mixtures (F1,15=4.4944, P=0.0511 and F1,16=0.1594, P=0.6950 for 7 and 5% O2, respectively; Fig. 1G,H), and was similarly elevated relative to normoxic measurements in the same animals (P=0.0296 and 0.0146 for 7 and 5% O2, respectively). The magnitude of change of the RER was not affected by drug treatment (Fig. 1I).

Next, we measured Tb to gain insight into thermoregulation during hypoxia. We found that bicuculline injection had no effect on Tb during normoxia and did not alter the hypoxic decrease in Tb in either experimental protocol (F1,16=0.2870, P=0.5995 and F1,17=3.489, P=0.1728 for 7 and 5% O2, respectively; Fig. 1J,K). The magnitude of change of Tb was not affected by drug treatment (Fig. 1L).

GABAA receptor antagonism has opposing effects on breathing in normoxia versus hypoxia and abolishes the hypoxic change in the ACR

To evaluate the role of GABAA receptors in regulating Damaraland mole-rat breathing, we next measured changes in E and its component parameters (fR and VT; Fig. 2). This analysis revealed that bicuculline had mixed effects on breathing in normoxia and more consistent effects in hypoxia. In normoxia, bicuculline increased baseline E and fR in protocol 1 (F1,16=61.53, P<0.0001 and F1,17=3.896, P=0.8256 for E in 7 and 5% O2, respectively; and F1,16=51.02, P=0.009 and F1,17=9.239, P=0.0073 for fR in 7 and 5% O2, respectively; Fig. 2A,B,D,E), but only fR in protocol 2. Conversely, VT was not affected by bicuculline in normoxia (F1,16=3.556, P=0.5625 and F1,17=4.116, P=0.9135 in 7 and 5% O2, respectively; Fig. 2G,H).

Fig. 2.

GABAA receptor antagonism regulates ventilation in hypoxia but not normoxia. (A,B,D,E,G,H) Summaries of minute ventilation (E; A,B), breathing frequency (fR; D,E) and tidal volume (VT; G,H) from Damaraland mole-rats exposed to 21% O2, before and after intraperitoneal injections of saline alone (white bars and open symbols), or the GABAA receptor antagonist bicuculline (1 mg kg−1; dissolved in saline; pink bars and closed symbols), and subsequent exposure to acute hypoxia (7 or 5% O2; 1 h). Data are presented as box and whisker plots of min–max with individual replicates shown from n=7–10 biological replicates per dataset. (C,F,I) Summaries of the hypoxia-mediated change in E (C), fR (E) and VT from animals treated as in A and B. Summary data are means±s.e.m. Different letters indicate significance as determined using a two-way ANOVA or mixed-effects model with Tukey's post hoc tests, P<0.05.

Fig. 2.

GABAA receptor antagonism regulates ventilation in hypoxia but not normoxia. (A,B,D,E,G,H) Summaries of minute ventilation (E; A,B), breathing frequency (fR; D,E) and tidal volume (VT; G,H) from Damaraland mole-rats exposed to 21% O2, before and after intraperitoneal injections of saline alone (white bars and open symbols), or the GABAA receptor antagonist bicuculline (1 mg kg−1; dissolved in saline; pink bars and closed symbols), and subsequent exposure to acute hypoxia (7 or 5% O2; 1 h). Data are presented as box and whisker plots of min–max with individual replicates shown from n=7–10 biological replicates per dataset. (C,F,I) Summaries of the hypoxia-mediated change in E (C), fR (E) and VT from animals treated as in A and B. Summary data are means±s.e.m. Different letters indicate significance as determined using a two-way ANOVA or mixed-effects model with Tukey's post hoc tests, P<0.05.

In protocol 1, bicuculline reduced E by ∼60% (P<0.0001; Fig. 2A). This change was driven by 60–70% reductions in both fR and VT (P<0.0001 for both; Fig. 2D,G). Conversely, in protocol 2, there was a trend in bicuculline-treated animals such that all ventilatory variables tended to decrease relative to saline-treated animals breathing the same hypoxic gas; however, this difference only reached significance for VT (P=0.0349; Fig. 2G). Furthermore, in bicuculline-treated animals breathing 5% O2 (protocol 2), the HVR was enhanced such that E, fR and VT were all decreased by ∼10–20% beyond the change observed in animals breathing 7% O2 in protocol 1.

As a result of these effects, bicuculline caused ventilatory changes to manifest in a shallower level of hypoxia, as evidenced by the appearance of a robust HVR in E, fR and VT in animals treated with bicuculline and breathing 7% O2, which was not apparent in saline-treated animals breathing the same hypoxic gas (F1,31=27.71, P<0.0001, F1,31=50.53, P<0.001, and F1,31=12.48, P=0.0012, for E, fR and VT, respectively; Fig. 2C,F,I). In protocol 2, during which the natural HVR is apparent, bicuculline enhanced the HVR for both E and fR, but not VT (P=0.0132, 0.006 and 0.3911, respectively).

Finally, bicuculline treatment had no effect on either ACR in normoxia (P=0.8845 and >0.9999 for ACRO2 and ACRCO2, respectively, in protocol 1, and 0.8116 and 0.9586 for ACRO2 and ACRCO2, respectively, in protocol 2; Fig. 3A,B). Conversely, the inhibitory effect of bicuculline on breathing, combined with the natural HMR of this species, resulted in the abolishment of the hypoxia-driven change in ACRO2 and ACRCO2 in both experimental protocols (F1,26=21.36, P=0.0002 and F1,26=14.25, P=0.0026 for ACRO2 and ACRCO2, respectively, in protocol 1, and F1,16=13.43, P=0.0348 and F1,16=7.7, P=0.0129 for ACRO2 and ACRCO2, respectively, in protocol 2).

Fig. 3.

GABAA receptor antagonism prevents the hypoxic ventilatory response. (A,B,D,E) Summaries of the air convection requirements of O2 (ACRO2; A,B) and CO2 (ACRCO2; D,E) from Damaraland mole-rats exposed to 21% O2, before and after intraperitoneal injections of saline alone (white bars and open symbols), or the GABAA receptor antagonist bicuculline (1 mg kg−1; dissolved in saline; pink bars and closed symbols), and subsequent exposure to acute hypoxia (7 or 5% O2; 1 h). Data are presented as box and whiskers plots of min–max with individual replicates shown from n=7–10 biological replicates per dataset. (C,F) Summaries of the hypoxia-mediated change in ACRO2 (C) and ACRCO2 (F) from animals treated as in A and B. Summary data are means±s.e.m. Different letters indicate significance as determined using a 2-way ANOVA or mixed-effects model with Tukey's post hoc tests, P<0.05.

Fig. 3.

GABAA receptor antagonism prevents the hypoxic ventilatory response. (A,B,D,E) Summaries of the air convection requirements of O2 (ACRO2; A,B) and CO2 (ACRCO2; D,E) from Damaraland mole-rats exposed to 21% O2, before and after intraperitoneal injections of saline alone (white bars and open symbols), or the GABAA receptor antagonist bicuculline (1 mg kg−1; dissolved in saline; pink bars and closed symbols), and subsequent exposure to acute hypoxia (7 or 5% O2; 1 h). Data are presented as box and whiskers plots of min–max with individual replicates shown from n=7–10 biological replicates per dataset. (C,F) Summaries of the hypoxia-mediated change in ACRO2 (C) and ACRCO2 (F) from animals treated as in A and B. Summary data are means±s.e.m. Different letters indicate significance as determined using a 2-way ANOVA or mixed-effects model with Tukey's post hoc tests, P<0.05.

Adenosine receptor antagonism increases metabolic rate in normoxia and reduces the hypoxia-mediated decreases in metabolic rate and thermogenesis

Next, we evaluated the impact of aminophylline on O2 and CO2 (Fig. 4). Note that aminophylline treatment in 5% O2 was lethal to some animals and so we only completed the 7% O2 (protocol 1) dataset in this experimental group. Aminophylline had a significant effect on both variables (F1,14=26.43, P=0.0002 and F1,14=26.24, P=0.0002 for O2 and CO2, respectively). Specifically, inhibition of adenosine receptors increased both O2 and CO2 by ∼2-fold in normoxia relative to saline-treated animals (P<0.0001 for both variables). In aminophylline-treated animals breathing 7% O2, both O2 and CO2 returned to pre-drug baseline levels, as compared with the ∼55–65% hypoxia-mediated reductions in these variables in saline-treated animals (P=0.0022 and 0.0016 for O2 and CO2, respectively). Nonetheless, the magnitude of change of both O2 and CO2 following the onset of hypoxia was greater in aminophylline-treated animals than saline-treated animals because of the drug-mediated elevation in these variables in normoxia (Fig. 4B,D). Despite these changes in O2 and CO2 with aminophylline treatment, the ratio between these variables (i.e. the RER) was tightly maintained in both normoxia and hypoxia and was not significantly different from saline-treated animals in the same conditions (Fig. 4E). Similarly, the magnitude of the hypoxia-mediated change in the RER was not affected by aminophylline (Fig. 4F).

Fig. 4.

Adenosine receptor antagonism reduces the hypoxic metabolic response and abolishes the hypoxia-mediated decrease in body temperature. (A,C,E,G) Summaries of the rate of O2 consumption (O2; A), the rate of CO2 production (CO2; C), respiratory exchange ratios (RERs; E) and body temperatures (Tb; G) from Damaraland mole-rats exposed to 21% O2, before and after injection of saline (white bars and open symbols) or the general adenosine receptor antagonist aminophylline (100 mg kg−1; dissolved in saline; blue bars and closed symbols), and subsequently exposed to acute hypoxia (7% O2 for 1 h). Data are presented as box and whisker plots of min–max with individual replicates shown from n=7–10 biological replicates per dataset. Different letters indicate significance as determined using a two-way ANOVA or mixed-effects model with Tukey's post hoc tests, P<0.05. (B,D,F,H) Summaries of the hypoxia-mediated change in O2 (B), CO2 (D), RER (F) and Tb (H) from animals treated as in A. Summary data are means±s.e.m. Asterisks indicate significant difference from saline controls as determined using paired Student’s t-tests, P<0.05.

Fig. 4.

Adenosine receptor antagonism reduces the hypoxic metabolic response and abolishes the hypoxia-mediated decrease in body temperature. (A,C,E,G) Summaries of the rate of O2 consumption (O2; A), the rate of CO2 production (CO2; C), respiratory exchange ratios (RERs; E) and body temperatures (Tb; G) from Damaraland mole-rats exposed to 21% O2, before and after injection of saline (white bars and open symbols) or the general adenosine receptor antagonist aminophylline (100 mg kg−1; dissolved in saline; blue bars and closed symbols), and subsequently exposed to acute hypoxia (7% O2 for 1 h). Data are presented as box and whisker plots of min–max with individual replicates shown from n=7–10 biological replicates per dataset. Different letters indicate significance as determined using a two-way ANOVA or mixed-effects model with Tukey's post hoc tests, P<0.05. (B,D,F,H) Summaries of the hypoxia-mediated change in O2 (B), CO2 (D), RER (F) and Tb (H) from animals treated as in A. Summary data are means±s.e.m. Asterisks indicate significant difference from saline controls as determined using paired Student’s t-tests, P<0.05.

Aminophylline injection had similar and significant impacts on Tb (F1,14=13.19, P=0.0027; Fig. 4G,H). Specifically, aminophylline increased Tb by∼1.5°C in normoxia (P=0.0021). In hypoxia, the Tb of aminophylline-treated animals returned to pre-drug baseline levels. However, because of the drug-mediated increase in Tb in normoxia, there was still a net decrease in Tb following the transition to hypoxia in aminophylline-treated animals (Fig. 4H), although the magnitude of this hypoxic change was decreased relative to saline-treated animals.

Aminophylline increases ventilation in normoxia and abolishes the relative hypoxic ventilatory response

Aminophylline markedly increases all ventilatory variables in normoxia, with E and VT each increasing 2- to 3-fold and fR increasing mildly (F1,14=34.37, P<0.0001 for E, F1,14=7.74, P=0.0004 for fR, and F1,14=45.14, P<0.0001 for VT; Fig. 5). These changes were largely reversed during the subsequent hypoxia exposure (P=0.7266, 0.3967 and 0.3202 versus normoxia, respectively). As with metabolism, the magnitude of change in all ventilatory parameters was greater in aminophylline-treated animals than in animals treated with saline, but this was due to the large increase in ventilation that occurs with drug treatment in normoxia, and which is subsequently reversed during hypoxia.

Fig. 5.

Adenosine receptor antagonism induces hyperventilation in normoxia but not hypoxia. (A,C,E) Summaries of minute ventilation (E; A), breathing frequency (fR; C) and tidal volume (VT; E) from Damaraland mole-rats exposed to 21% O2, before and after injection of saline (white bars and open symbols) or aminophylline (100 mg kg−1; dissolved in saline; blue bars and closed symbols), and subsequently exposed to acute hypoxia (7% O2 for 1 h). Data are presented as box and whisker plots of min–max with individual replicates shown from n=7–10 biological replicates per dataset. Different letters indicate significance as determined using a two-way ANOVA or mixed-effects model with Tukey's post hoc tests, P<0.05. (B,D,F) Summaries of the hypoxia-mediated change in E (B), fR (D) and VT (F) from animals treated as in A. Summary data are means±s.e.m. Asterisks indicate significant difference from saline controls as determined using paired Student’s t-tests, P<0.05.

Fig. 5.

Adenosine receptor antagonism induces hyperventilation in normoxia but not hypoxia. (A,C,E) Summaries of minute ventilation (E; A), breathing frequency (fR; C) and tidal volume (VT; E) from Damaraland mole-rats exposed to 21% O2, before and after injection of saline (white bars and open symbols) or aminophylline (100 mg kg−1; dissolved in saline; blue bars and closed symbols), and subsequently exposed to acute hypoxia (7% O2 for 1 h). Data are presented as box and whisker plots of min–max with individual replicates shown from n=7–10 biological replicates per dataset. Different letters indicate significance as determined using a two-way ANOVA or mixed-effects model with Tukey's post hoc tests, P<0.05. (B,D,F) Summaries of the hypoxia-mediated change in E (B), fR (D) and VT (F) from animals treated as in A. Summary data are means±s.e.m. Asterisks indicate significant difference from saline controls as determined using paired Student’s t-tests, P<0.05.

Importantly, we did not observe a hypoxic ventilatory response in 7% O2 in our control experiments because this response is blunted in Damaraland mole-rats and does not manifest until ∼3–5% O2 (Ivy et al., 2020). However, as this severe level of hypoxia was lethal when applied concomitantly with aminophylline (data not shown), we could not directly test the role of adenosine on the hypoxia-mediated changes in ventilation in this species at or below 5% O2. Therefore, we instead relied upon ACRs to determine whether adenosine receptors mediate the HVR in Damaraland mole-rats in 7% O2 because these variables incorporate metabolic changes along with the HVR. Thus, the robust HMR of this species drives a relative HVR in 7% O2 despite the lack of overt change in any ventilatory variable. Aminophylline treatment had no impact on the ACRs in normoxia (P=0.4626 and 0.2409 for ACRO2 and ACRCO2, respectively; Fig. 6), indicating that the increases in metabolism, thermogenesis and ventilation following inhibition of adenosinergic signalling are coupled and balanced and that increased metabolic demand is well matched by increases in O2 delivery in normoxia. Conversely, aminophylline abolished the hypoxia-mediated increases in ACRO2 (P=0.0641 and 0.6438 versus pre- and post-injection values in the same animals; Fig. 6B) and ACRCO2 (P=0.2232 and >0.999 versus pre- and post-injection values in the same animals; Fig. 6D).

Fig. 6.

Adenosine receptor antagonism prevents the relative hypoxic ventilatory response. (A,C) Summaries of the air convection requirements of O2 (ACRO2; A) and CO2 (ACRCO2; C) from Damaraland mole-rats exposed to 21% O2, before and after injection of saline (white bars and open symbols) or aminophylline (100 mg kg−1; dissolved in saline; blue bars and closed symbols), and subsequently exposed to acute hypoxia (7% O2 for 1 h). Data are presented as box and whisker plots of min–max with individual replicates shown from n=7–10 biological replicates per dataset. Different letters indicate significance as determined using a two-way ANOVA or mixed-effects model with Tukey's post hoc tests, P<0.05. (B,D) Summaries of the hypoxia-mediated change in ACRO2 (B) and ACRCO2 (D) from animals treated as in A and C. Summary data are means±s.e.m. Asterisks indicate significant difference from saline controls as determined using paired Student’s t-tests, P<0.05.

Fig. 6.

Adenosine receptor antagonism prevents the relative hypoxic ventilatory response. (A,C) Summaries of the air convection requirements of O2 (ACRO2; A) and CO2 (ACRCO2; C) from Damaraland mole-rats exposed to 21% O2, before and after injection of saline (white bars and open symbols) or aminophylline (100 mg kg−1; dissolved in saline; blue bars and closed symbols), and subsequently exposed to acute hypoxia (7% O2 for 1 h). Data are presented as box and whisker plots of min–max with individual replicates shown from n=7–10 biological replicates per dataset. Different letters indicate significance as determined using a two-way ANOVA or mixed-effects model with Tukey's post hoc tests, P<0.05. (B,D) Summaries of the hypoxia-mediated change in ACRO2 (B) and ACRCO2 (D) from animals treated as in A and C. Summary data are means±s.e.m. Asterisks indicate significant difference from saline controls as determined using paired Student’s t-tests, P<0.05.

In the present study, we evaluated a role for GABAergic and adenosinergic signalling in regulating the hypoxic ventilatory, metabolic and thermoregulatory responses of fossorial Damaraland mole-rats to acute hypoxia. Our study yielded several important findings. First, adenosine appears to be the primary neurotransmitter regulating metabolism and thermogenesis of Damaraland mole-rats because aminophylline treatment substantially increases metabolic rate and Tb in both normoxia and hypoxia and reduces hypoxia-mediated decreases in both variables. As a result, aminophylline abolishes the relative HVR (i.e. the ACR) through interactions with metabolic but not ventilatory parameters. Conversely, GABA receptors play a lesser role in regulating metabolism or thermoregulation in normoxia or hypoxia but have significant and opposing effects on ventilation between normoxic and hypoxic treatments. Specifically, bicuculline increases breathing in normoxia but decreases breathing in hypoxia, resulting in a more sensitive HVR in these conditions. However, the net effect of bicuculline on the ACR is to prevent hypoxia-mediated changes in this variable, and thus bicuculline abolishes the relative HVR. This is intriguing and suggests that GABA plays an inhibitory role in regulating breathing in normoxia but an excitatory role in hypoxia.

Adenosinergic signalling mediates metabolism and thermogenesis

We report that Damaraland mole-rats have a robust HMR to acute hypoxia, which is partially mediated by decreased thermoregulation. These findings are also generally consistent with responses to hypoxia in numerous other small fossorial mammal species (Arieli and Ar, 1979; Arieli et al., 1977; Barros et al., 2004; Boggs et al., 1998; Frappell et al., 1992, 1994; Guppy and Withers, 1999; Tomasco et al., 2010), including several related species of African mole-rats (Devereaux and Pamenter, 2020; Ivy et al., 2020; Pamenter, 2022). Our data extends these previous findings to show that the HMR and hypoxic decrease in thermogenesis in Damaraland mole-rats are partially mediated by adenosinergic signalling. This finding differs somewhat from a previous study (Pamenter et al., 2015) in naked mole-rats, in which the hypoxic decrease in O2 was not impacted by aminophylline (although the hypoxic decrease in CO2 was halved by this drug).

In other small mammals, including both hypoxia-tolerant and -intolerant fossorial species, adenosine receptors initiate and maintain the HMR and decreases in Tb during entry into torpor. For example, activating adenosine receptors reduces metabolic rate and thermogenesis, and induces torpor in arctic ground squirrels and rats (Jinka et al., 2011; Tupone et al., 2013). In contrast, blocking adenosine receptors prevents or reverses these changes during intra-day or fasting-induced torpor in rats (Tupone et al., 2013) and mice (Iliff and Swoap, 2010, 2012; Swoap et al., 2012), and in hibernating arctic ground squirrels (Jinka et al., 2011; Olson et al., 2013).

Conversely, we demonstrated that metabolic and thermoregulatory responses to hypoxia are likely not mediated by GABAergic signalling in Damaraland mole-rats. An important caveat is that the hypoxic change in O2 is enhanced by bicuculine in protocol 1; however, this is not the case for protocol 2, and bicuculine (unlike aminophylline) does not alter the hypoxic value for O2 relative to saline treatment in either protocol. Thus, the preponderance of evidence suggests a minimal role for GABAergic signalling in mediating the HMR. This finding agrees with a previous study in naked mole-rats, in which the hypoxic decrease in O2 was not affected by bicuculline (Dzal et al., 2019).

GABAergic signalling modulates the HVR

We demonstrate that Damaraland mole-rats have a blunted HVR that does not manifest until 5% O2, which is consistent with a previous study in this species (Ivy et al., 2020), and with other fossorial species, in which the HVR is typically activated in the range of 5–12.5% O2 (Ivy et al., 2020; Tomasco et al., 2010). Blocking GABAA receptors in normoxia increases ventilatory parameters, suggesting that there is an inhibitory drag on ventilation from active GABAergic signalling in normoxia. Remarkably, our results suggest that this drag on ventilation is reversed in hypoxia and that GABAergic signalling, at least within the ventilatory control circuits, is net excitatory when O2 is limited. As a result, GABAA receptors play a role in dampening ventilation during acute hypoxia in Damaraland mole-rats and reduce the sensitivity of ventilation to hypoxia, such that blocking GABAA receptors causes the HVR to manifest at 7% instead of 5% O2 (which is the HVR threshold in saline-treated animals). Notably, however, this role is different from that in naked mole-rats, in which bicuculline prevents the hypoxic decrease in ventilation. This suggests that GABA may act at different points in the ventilatory control system and may directly modulate other inhibitory ventilatory inputs than in naked mole-rats.

The apparent reversal of GABAergic signalling is fascinating and suggests one of two possibilities: either (1) the Cl reversal potential (and thus the reversal potential of the GABAA receptors: EGABA) shifts during hypoxia in this species, rendering GABAergic signalling mildly excitatory in hypoxia, or (2) hypoxia directly activates inhibitory interneurons that synapse onto and subsequently inhibit excitatory ventilatory inputs, thereby reducing the ventilatory drive in hypoxia. Given that bicuculline increased the sensitivity of breathing to hypoxia, the former explanation is more likely.

Although EGABA is almost always hyperpolarizing relative to neuronal membrane potential in adult hypoxia-intolerant mammals, there are exceptions. For example, in isolated rat carotid body petrosal neurons, EGABA is depolarizing relative to resting membrane potential (Zhang et al., 2009). In addition, there are several examples in the nervous systems of other hypoxia- and anoxia-tolerant species in which EGABA is depolarizing (and thus initially excitatory) and/or shifts from hyperpolarizing to depolarizing with changing environments. For example, in anoxia-tolerant pond snails (Lymnaea stagnalis), EGABA shifts between an inhibitory and excitatory phenotype depending on the environmental photoperiod (Buck et al., 2017), presumably to help this animal overwinter in anoxic waters. Similarly, EGABA is depolarized relative to resting membrane potential in the cortex of anoxia-tolerant freshwater turtles (Chrysemys picta bellii), and robust GABAergic activation in anoxia or ischemia causes neurons to mildly depolarize (Buck et al., 2012; Hogg et al., 2015; Pamenter et al., 2011). A similar phenotype has also been reported in naked mole-rats, in which EGABA is depolarized by ∼10 mV relative to the neuronal membrane potential, which is like the difference between EGABA and membrane potential in turtle neurons (Zions et al., 2020). Unfortunately, there is no information available regarding EGABA in hypoxic naked mole-rat brain or in Damaraland mole-rats in any condition, but our results suggest that EGABA is also excitatory in normoxia in Damaraland mole-rats and may shift to an inhibitory phenotype in hypoxia. Further experiments are warranted to evaluate EGABA in both naked and Damaraland mole-rats in hypoxia.

In contrast, the role of adenosine receptors in mediating ventilation and the HVR in Damaraland mole-rats is less clear; aminophylline increases E in both normoxia and hypoxia but also abolishes the hypoxia-mediated increase of the ACRs. In general, this finding is consistent with other mammals, in which adenosine is known to accumulate in the blood during hypoxia and modulate the HVR (Drumm et al., 2004). For example, aminophylline reduces (but does not abolish) the second, declining component of the biphasic HVR in awake humans (Easton and Anthonisen, 1988), cats (Long and Anthonisen, 1994), rats (Maskrey and Westwood, 1994) and lambs (Koos et al., 2005), and in anaesthetized piglets (Darnall, 1985). Furthermore, in studies in which the effect of aminophylline on ventilation is examined along with its effects on metabolism, aminophylline always inhibits the HVR in addition to the HMR (Barros et al., 2006; Koos et al., 2005; Maxwell et al., 1986). Therefore, it is likely that adenosine receptor inhibition would affect hypoxic ventilation in Damaraland mole-rats if it were ethically possible (i.e. not lethal) to expose them to lower levels of O2 in combination with aminophylline treatment.

Study limitations

Bicuculline and aminophylline have clear impacts on breathing and metabolic variables in both normoxia and hypoxia, which demonstrates that these drugs, and at the concentrations we employ in our study, are efficacious in this species. Nonetheless, drugs were injected intraperitoneally and thus their specificity of action must be interpreted with caution. However, intraperitoneal injections of these pharmacological agents have concentration decay and time profiles similar to those of intravenous injections (Asheim et al., 2008), and intraperitoneal injections of both drugs used in the present study have been used previously in studies in other mammals to evaluate similar physiological and behavioural responses (e.g. Chung et al., 2016; Crisanti and Fewell, 1999; Pamenter et al., 2015; Zimmerman et al., 2003). Furthermore, aminophylline and bicuculline both cross the blood–brain barrier and can thus interact with adenosine receptors within the central nervous system (Remler and Marcussen, 1985; Veng-Pedersen et al., 1983). However, GABAA and adenosine receptors are also found outside of the central nervous system and thus our results only allow us to conclude that the effects we observed are mediated by these receptors, but not which population of receptors are involved in this regulation. It is also important to note that whereas bicuculline is primarily specific for GABAA receptors (Johnston, 2013), aminophylline is less specific. Indeed, aminophylline has many off-target effects at the cellular level, including as a weak inhibitor of some transcriptional pathways and phosphodiesterase, as well as varied impacts on inflammatory pathways and inflammatory cell survival, among others (Barnes, 2010). However, these effects are generally weak or minimal. For example, at the highest concentration of aminophylline tested, phosphodiesterase activity is inhibited by ∼14–20% (Polson et al., 1978). Furthermore, most of these off-target effects would not manifest at the physiological level within the 1–2 h period of our experiments, which should limit any confounding impact on our data.

Conclusions

In the present study, we demonstrated that GABAA and adenosine receptors contribute to the hypoxic metabolic and ventilatory responses in Damaraland mole-rats. This mechanistic phenotype is unlike that of most non-fossorial mammals studied to date, which instead rely on glutamatergic signalling to mediate the HVR (Pamenter and Powell, 2016). Regarding adenosine, our findings differ from previous studies in closely related naked mole-rats, in which adenosinergic signalling is central to the manifestation of the HVR and plays a lesser role in mediating the HMR. However, our findings are generally consistent with studies in other small rodents in which adenosine is a key mediator of hypometabolism during torpor bouts and hibernation. The mechanism via which adenosine receptors modulate metabolism has not been elucidated in any species and warrants further investigation. Further studies in other fossorial mammals are also warranted to determine whether GABAergic signalling is the predominant neurotransmission pathway regulating ventilation and the HVR in other fossorial species. In addition, research is required to understand the mechanism via which GABA receptors modulate ventilation in such species.

We thank the uOttawa veterinary technicians for their assistance in animal handling and husbandry.

Author contributions

Conceptualization: M.E.M.D., M.E.P.; Methodology: M.E.M.D., M.E.P.; Validation: M.E.M.D.; Formal analysis: M.E.M.D.; Investigation: M.E.M.D.; Resources: M.E.P.; Data curation: M.E.M.D.; Writing - original draft: M.E.P.; Writing - review & editing: M.E.M.D.; Supervision: M.E.P.; Project administration: M.E.P.; Funding acquisition: M.E.P.

Funding

This work was supported by a Natural Sciences and Engineering Research Council of Canada Discovery grant (04229) to M.E.P. Open access funding supplied by University of Ottawa. Deposited in PMC for immediate release.

Data availability

All relevant data can be found within the article.

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Competing interests

The authors declare no competing or financial interests.

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