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. Author manuscript; available in PMC: 2015 Mar 19.
Published in final edited form as: Acta Physiol (Oxf). 2014 Mar 13;211(1):240–248. doi: 10.1111/apha.12257

Leptin into the ventrolateral medulla facilitates chemorespiratory response in leptin-deficient (ob/ob) mice

M Bassi 1, W I Furuya 1, J V Menani 1, D S A Colombari 1, J M do Carmo 2, A A da Silva 2, J E Hall 2, T S Moreira 3, I C Wenker 4, D K Mulkey 4, E Colombari 1
PMCID: PMC4365783  NIHMSID: NIHMS670797  PMID: 24521430

Abstract

Aim

Leptin, an adipocyte-derived hormone, is suggested to participate in the central control of breathing. We hypothesized that leptin may facilitate ventilatory responses to chemoreflex activation by acting on respiratory nuclei of the ventrolateral medulla. The baseline ventilation and the ventilatory responses to CO2 were evaluated before and after daily injections of leptin into the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG) for 3 days in obese leptin-deficient (ob/ob) mice.

Methods

Male ob/ob mice (40–45 g, n = 7 per group) received daily microinjections of vehicle or leptin (1 μg per 100 nL) for 3 days into the RTN/pFRG. Respiratory responses to CO2 were measured by whole-body plethysmography.

Results

Unilateral microinjection of leptin into the RTN/pFRG in ob/ob mice increased baseline ventilation (VE) from 1447 ± 96 to 2405 ± 174 mL min−1 kg−1 by increasing tidal volume (VT) from 6.4 ± 0.4 to 9.1 ± 0.8 mL kg−1 (P < 0.05). Leptin also enhanced ventilatory responses to 7% CO2 (Δ = 2172 ± 218 mL min−1 kg−1, vs. control: Δ = 1255 ± 105 mL min−1 kg−1), which was also due to increased VT (Δ = 4.71 ± 0.51 mL kg−1, vs. control: Δ = 2.27 ± 0.20 mL kg−1), without changes in respiratory frequency. Leptin treatment into the RTN/pFRG or into the surrounding areas decreased food intake (83 and 70%, respectively), without significantly changing body weight.

Conclusion

The present results suggest that leptin acting in the respiratory nuclei of the ventrolateral medulla improves baseline VE and VT and facilitates respiratory responses to hypercapnia in ob/ob mice.

Keywords: breathing, central chemoreception, leptin, obesity, ventrolateral medulla

Leptin is an adipocyte-derived hormone that acts in the central nervous system to regulate energy homeostasis (Grill 2006) and sympathetic activity (Rahmouni & Morgan 2007, Hall et al. 2010). In addition, previous studies demonstrated that leptin is also involved in the control of breathing (Polotsky et al. 2001, 2004, Inyushkin et al. 2009, Inyushkina et al. 2010, Malli et al. 2010, Bassi et al. 2012). Transgenic leptin-deficient (ob/ob) mice show attenuated breathing responses to CO2, suggesting that leptin deficiency impairs ventilatory function. Moreover, systemic or intracerebroventricular leptin replacement reverses the deficit of ventilation in ob/ob mice by still unknown mechanisms (Tankersley et al. 1996, O’Donnell et al. 2000, Bassi et al. 2012).

Leptin receptors are present in several hindbrain regions including the nucleus of the solitary tract (NTS), locus coeruleus (LC), rostral ventrolateral medulla (RVLM) and Bötzinger complex (BötC; Mercer et al. 1998, Elias et al. 2000, Hosoi et al. 2002, Grill & Hayes 2009, Bassi et al. 2012). The administration of leptin into the caudal portion of the NTS increases sympathetic nerve activity (Mark et al. 2009), and leptin-injected intracerebroventricularly activates pre-sympathetic neurones in the RVLM (Zhang & Felder 2004).

The activity of the respiratory system is controlled by a long column of cells extending laterally in the brainstem from the dorsolateral pons to the caudal ventrolateral medulla (Nattie 1999, Feldman et al. 2003, Guyenet et al. 2005, Moreira et al. 2006). The retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG) in the medulla is considered a primary site for central chemoreception and also receives inputs from the carotid body providing important facilitatory signals for respiration (Guyenet et al. 2008). Acting in the RTN/pFRG or in other nuclei of the ventrolateral medulla involved in the control of breathing like Pre-Bötzinger (pre-Böt) or Bötzinger complex (BötC), leptin might influence breathing control and modulate chemoreception. However, to our knowledge, no previous studies tested the effects of leptin injected into the ventral areas of the medulla in controlling breathing and ventilatory responses to hypercapnia.

In this study, we investigated whether leptin microinjections chronically into the ventrolateral medulla, including the RTN/pFRG, would improve respiratory activity in ob/ob mice under resting condition and during central chemoreflex activation by hypercapnia.

Materials and methods

The experimental procedures and protocols used in this study were approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center and followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Animals

Male C57BL/6J-Lepob leptin-deficient (ob/ob) mice from the Jackson Laboratory (Ann Arbor, MI, USA) between 7 and 9 weeks of age were used in these studies.

Surgery for brain cannula implantation

The ob/ob mice were anesthetized with ketamine (80 mg kg−1 of body weight) and xylazine (7 mg kg−1 of body weight) and immobilized in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA). Under aseptic conditions, a unilateral stainless steel cannula (14 mm × 27 G) was implanted in the RTN/pFRG using the following stereotaxic coordinates: 1.3 mm caudal to lambda, 4.5 mm below the dura mater and 1.0 mm from the midline (Paxinos & Franklin 2001). The guide cannula was anchored to the skull with stainless steel screws and dental acrylic resin. A metal stylet was inserted into seal the guide cannulas when not in use. At the end of the surgery, the animals received a subcutaneous injection of the analgesic/anti-inflammatory Ketoflex (cetoprofeno 1%, 0.01 mL per animal) and were allowed to recover for 6 days before daily microinjections of leptin or vehicle, and ventilatory measurements were performed.

Leptin microinjections into the RTN/pFRG region

Leptin (1 μg per 100 nL) from National Hormone & Peptide Program (Torrance, CA, USA) or the vehicle (100 nL of phosphate buffer saline, PBS, pH = 7.4) was administered into the RTN/pFRG region of ob/ob mice once a day for three consecutive days. The injections were performed between 9:00 and 10:00 am under anaesthesia (isoflurane 1–3%) to avoid stress to the animals using a Hamilton syringe (1 μL) coupled with a PE-10 tube connected to a needle (15.7 mm × 33 G). This dose of leptin was chosen based on previous studies examining the acute effects of leptin on cardiovascular function (Harris et al. 1998, Rahmouni & Morgan 2007).

Ventilation measurements

Pulmonary ventilation (VE) was measured using whole-body plethysmography as previously described (Malan 1973, Bassi et al. 2012). Briefly, mice were acclimatized to the plethysmography chamber (700 mL) at room temperature (25 °C) for 1 h. The ports for gas exit or entrance in the chamber were closed to produce an internal constant volume. The chamber temperature was <1 °C above room temperature (approx. 25 °C) with non-significant variations over the period of time (5 min) that all measurements were made. Rectal temperature was constant, and no significant changes were observed between groups (leptin treated: 37.0 ± 0.2 °C and vehicle treated: 37.3 ± 0.2 °C). Then, signals of breathing frequency (fR) and tidal volume (VT) were measured by changes in the pressure inside the chamber due to both temperature and humidification changes in the inspired/expired gases. A spirometer (model ML141; AD Instruments, Colorado Springs CO, USA) was used for measurements, and the signals were analysed using POWERLAB software (Ad Instruments, Colorado Springs, CO, USA). The system was calibrated with injections of 0.2 mL of room air with the animal inside the plethysmography chamber. VE was calculated as the product of fR and VT.

Experimental protocol

After recovery from surgery (6 days), each mouse was acclimatized inside the plethysmography chamber for VE measurements. Baseline VE was measured during room air conditions (21% O2) followed by hypercapnia produced by switching the room air for a gas mixture containing 21% O2 plus 4 or 7% CO2. The mice were exposed to hypercapnia for 5–10 min before measurements were obtained during one additional minute at the end of hypercapnia. The VE measurements were made before treatment (control period), on the 3rd day of leptin or vehicle treatment and again 4 days after the treatment was stopped (recovery period). Food intake and body weight were measured daily. At the end of the recovery period, the brains were removed for histological analysis.

Histology

At the end of the tests, mice received injections of 2% Evans Blue solution into the RTN/pFRG region in the same volume used for drug injections. They were then deeply anesthetized with sodium thiopental (80 mg kg−1 of body weight) and perfused transcardially with PBS pH 7.4 followed by 4% paraformaldehyde (Sigma-Aldrich, Saint Louis, MO, USA). Brains were removed, fixed in 4% paraformoldehyde (24 h) and submerged in 30% sucrose for cryoprotection. Frozen coronal section 50 μm was cut and stained with Giemsa and analysed by light microscopy to confirm the injection sites into the RTN/pFRG region.

Statistical analysis

The results are presented as mean ± SEM. Two-way repeated measures ANOVA followed by Bonferroni’s post hoc test was used for comparisons, using GRAPHPAD PRISM 5 software (GraphPad Software, San Diego, CA, USA). Statistical significance was accepted at the level of P < 0.05.

Results

Histological analysis

Typical sites of leptin microinjections into the ventrolateral medulla are shown in the Figure 1a,b. The centre of the injections was typically located into the RTN/pFRG, medial and ventral to the facial nucleus, a region that contains the highest density of CO2-sensitive neurones that control cardiorespiratory responses (Takakura et al. 2006, 2011). The histological analysis showed that some mice received leptin injections outside the RTN/pFRG in sites more lateral and dorsal to the CO2-sensitive region (Fig. 1c). The results of these mice were also analysed and compared with the results of leptin injections specifically into the RTN/pFRG.

Figure 1.

Figure 1

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(a) Photomicrograph showing a typical site of injection into the ventrolateral medulla (RTN/pFRG) in one of the mice tested. (b,c) Diagrams showing the sites of leptin microinjections inside (b) or outside (c) the RTN/pFRG region at different coronal levels (from top to bottom: Bregma −6.24, −6.36 and −6.48 mm, respectively). sp5, spinal trigeminal tract; 7N, facial nucleus; Rob, raphe obscurus nucleus; Rpy, parapyramidal region; RVL/C1, rostral ventrolateral and adrenergic C1 nuclei; Rpa, raphe pallidus nucleus; 4V, 4th ventricle. pFRG, parafacial respiratory group; RTN, retrotrapezoid nucleus.

Baseline ventilation in leptin-deficient ob/ob mice treated with leptin injections into the RTN/pFRG region

At the end of 3 day treatment, microinjections of leptin (1 μg per 100 nL day−1) into the RTN/pFRG in leptin-deficient ob/ob mice increased baseline VT (9.1 ± 0.8 mL kg−1 vs. vehicle: 6.5 ± 0.4 mL kg−1) [F1,29 = 4.23; P < 0.05] and baseline VE (2406 ± 174 mL min−1 kg−1, vs. vehicle: 1581 ± 159 mL min−1 kg−1) [F1,29 = 6.62; P < 0.02], without changing fR (Fig. 2, Table 1). The increased baseline VT and VE produced by leptin injections into the RTN/pFRG region returned to control values 4 days after stopping leptin treatment (Fig. 2, Table 1).

Figure 2.

Figure 2

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Baseline respiratory frequency (fR), tidal volume (VT) and ventilation (VE) in control period (before leptin), after 3 days of leptin (n = 7) or PBS vehicle (n = 5) injections into the RTN/pFRG and after 4-day recovery period. α = P < 0.05 compared with control and recovery period, and also compared with PBS injections. pFRG, parafacial respiratory group and RTN, retrotrapezoid nucleus.

Table 1.

Respiratory frequency, tidal volume and ventilation in leptin-deficient ob/ob mice treated for 3 days with PBS or leptin injected into the RTN/pFRG or outside the RTN/pFRG in control condition and 4 or 7% CO2

PBS
Leptin inside RTN/pFRG
Leptin outside RTN/pFRG
Baseline 4% CO2 7% CO2 Baseline 4% CO2 7% CO2 Baseline 4% CO2 7% CO2
CTL
 fR 248 ± 18 281 ± 8 310 ± 3 225 ± 9 281 ± 8 310 ±3 240 ± 11 293 ± 11 300 ± 22
 VT 6.7 ± 0.4 7.7 ± 0.3 8.5 ± 0.4 6.5 ± 0.4 7.7 ± 0.3 8.5 ± 0.4 5.9 ± 0.6 6.6 ± 0.6 8.1 ± 0.5
 VE 1658 ± 107 2114 ± 100 2582 ± 111 1447 ± 96 2114 ± 100 2583 ± 112 1422 ± 178 1934 ± 229 2407 ± 212
3 days
 fR 246 ± 15 276 ± 13 306 ± 8 273 ± 6 316 ± 11 340 ±8 240 ± 31 308 ± 5 345 ± 10
 VT 6.5 ± 0.6 7.7 ± 0.3 8.5 ± 0.4 9.1 ± 0.8α 10.9 ± 1.0α 13.7 ± 0.9α 6.9 ± 0.5β 8.1 ± 0.58β 10.7 ± 0.7αβ
 VE 1581±159 2109 ± 59 2592 ± 128 2406 ± 174α 3261 ± 290α 4584 ± 344α 1780 ± 145β 2574 ± 118β 3659 ± 132αβ
REC
 fR 226 ± 24 284 ± 24 324 ± 17 242 ± 13 396 ± 13 326 ± 10 255 ± 16 305 ± 5 325 ± 3
 VT 6.1 ± 0.8 6.9 ± 1.0 7.9 ± 0.9 6.6 ± 0.4 8.1 ± 0.7 9.4 ± 0.5 6.2 ± 0.6 7.1 ± 0.9 7.6 ± 0.4
 VE 1362 ± 192 1967 ± 325 2554 ± 327 1552 ± 75 2202 ± 99 2949 ± 95 1578 ± 184 2174 ± 261 2712 ± 262
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Values are mean ± SE. fR, respiratory frequency (breaths min−1), VT, tidal volume (mL kg−1) and VE, pulmonary ventilation (mL min−1 kg−1) in control period (CTL), after 3 days of PBS (n = 5) or leptin treatment inside (n = 7) or outside (n = 4) the RTN/pFRG and in recovery period (4 days after stopping leptin or vehicle treatment).

α

P < 0.05 compared with control and recovery periods and also compared with PBS injections;

β

P < 0.05 compared with leptin treatment inside the RTN/pFRG region.

Ventilatory responses to 4 and 7% CO2 in leptin-deficient ob/ob mice treated with leptin injections into the RTN/pFRG

Leptin (1 μg per 100 nL day−1) injected for 3 days into the RTN/pFRG region of leptin-deficient ob/ob mice markedly increased the ventilatory responses to 4 and 7% CO2 (Fig. 3, Table 1). Leptin facilitated 4 and 7% CO2-induced increase in VT (Δ = 2.37 ± 0.57 and 4.71 ± 0.51 mL kg−1, respectively) and VE (Δ = 1091 ± 166 and 2172 ± 218 mL min−1 kg−1, respectively) compared with CO2-induced increase in VT (Δ = 0.91 ± 0.29 and 2.41 ± 0.54 mL kg−1, respectively) and VE (Δ = 483 ± 101 and 1292 ± 291 mL min−1 kg−1, respectively) in PBS-treated mice (Fig. 3). There was a significant difference in VT comparing leptin and PBS-treated mice exposed to 4% [F1,29 = 8.08; P < 0.01) and 7% CO2 [F1,29 = 18.5; P < 0.0002)] and also in VE to 4% [F1,29 = 12.3; P < 0.002) and 7% CO2 [F1,29 = 11.9; P < 0.002; Fig. 3a,b). The slopes of the VE/CO2 curve responses for leptin- and PBS-treated mice were also significantly different (leptin: 325 ± 49 vs. vehicle: 157 ± 37, P < 0.01; Fig. 3c).

Figure 3.

Figure 3

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(a,b) Changes in respiratory frequency (fR), tidal volume (VT) and ventilation (VE) in response to (a) 4% CO2 or (b) 7% CO2 during control period, after 3 days of leptin (n = 7) or PBS vehicle (n = 5) injections into the RTN/pFRG region and after 4-day recovery period. (c) VE/CO2 curve responses after 3 days of leptin or vehicle treatment into the RTN/pFRG in ob/ob mice. α = P < 0.05 compared with control, recovery period or PBS injections. pFRG, parafacial respiratory group and RTN, retrotrapezoid nucleus.

The increases in VT and VE produced by 4 and 7% CO2 after 3-day leptin treatment were also facilitated compared with the increases in VT (Δ = 1.09 ± 0.19 and 2.27 ± 0.20 mL kg−1, respectively) and VE (Δ = 488 ± 53 and 1255 ± 105 mL min−1 kg−1, respectively) produced by 4 and 7% CO2 in the control period in the same group of mice (Fig. 3, Table 1). The increased ventilatory response to CO2 in leptin-treated mice returned to control values 4 days after stopping leptin treatment (Fig. 3, Table 1). Leptin injections into the RTN/pFRG region did not affect the increases in fR produced by CO2 (Fig. 3, Table 1).

Baseline ventilation and ventilatory responses to 4 and 7% CO2 in leptin-deficient ob/ob mice treated with leptin injections in sites outside the RTN/pFRG region

Leptin (1 μg per 100 nL day−1) injected for 3 days in sites outside the RTN/pFRG did not significantly change baseline ventilation and the ventilatory responses to 4% CO2 when compared to control period (before leptin) or vehicle PBS treatment (Table 1). However, leptin injected outside the RTN/pFRG increased VT (10.7 ± 0.7 mL kg−1, vs. control period: 8.1 ± 0.5 mL kg−1) and VE (3659 ± 132 mL kg−1 min−1, vs. control period: 2407 ± 212 mL min−1 kg−1) under 7% CO2. Nevertheless, the increased VE response to 7% CO2 in mice that received leptin in the surrounding areas was less pronounced than the response observed in mice treated with leptin into the RTN/pFRG region (3659 ± 132 mL min−1 kg−1, vs. leptin inside: 4584 ± 344 mL min−1 kg−1) [F1,27 = 7.26; P < 0.01].

Daily food intake and body weight in leptin-deficient ob/ob mice treated with leptin injections into the RTN/pFRG region or in the surrounding areas

Leptin microinjections into the RTN/pFRG or in the areas outside the RTN/pFRG similarly reduced daily food intake. Food intake was reduced from 5.2 ± 0.5 to 0.9 ± 0.2 g per 24 h (reduction of approx. 83%) in the mice that received injection of leptin into the RTN/pFRG and from 5.3 ± 0.4 to 1.7 ± 0.3 in those that received injections of leptin in sites outside the RTN/pFRG (reduction of approx. 70%). However, probably due to the short-term period of treatment, no significant changes in the body weight were observed comparing the weight of leptin-treated mice with that of vehicle-treated mice (37.6 ± 2.5 g and 35.1 ± 2.2 g vs. vehicle: 39.1 ± 2.9 g).

Discussion

The present study shows that leptin microinjected into the RTN/pFRG for three consecutive days increased baseline ventilation and the ventilatory responses to 4 and 7% CO2 in obese leptin-deficient ob/ob mice. Although the injections of leptin in sites outside the RTN/pFRG also increased the ventilatory responses to CO2, in this case, the effects were less intense. These results suggest that leptin acting in the respiratory nuclei of the ventrolateral medulla, including RTN/pFRG, improves baseline ventilation and facilitates the ventilatory responses to CO2 in leptin-deficient ob/ob mice.

Leptin injected into the RTN/pFRG or in sites outside the RTN/pFRG similarly reduced food intake in obese leptin-deficient ob/ob mice (around 83 and 70%, respectively). Therefore, the reduction in food intake produced by the injections of leptin into the RTN/pFRG cannot be considered a consequence of the action of leptin specifically into the RTN/pFRG. Probably at the dose tested, leptin spread from the RTN/pFRG to other areas to affect the mechanisms involved in the control of food intake. Even the small amount of leptin that reached other brain areas was apparently enough to affect food intake. In spite of the reduction in food intake, the injections of leptin did not significantly change body weight, which supports previous suggestion that leptin acting centrally may improve ventilatory response to hypercapnia by mechanisms not dependent on changes in body weight (Bassi et al. 2012).

Although modest, the increase in the ventilatory response to 7% CO2 when leptin was injected outside the RTN/pFRG suggests that leptin spreading to areas outside the RTN/pFRG may also produce respiratory effects. From the RTN/pFRG, leptin might spread to the surrounding areas that express leptin receptors like the Botzinger complex and the RVLM (Barnes et al. 2010, Bassi et al. 2012). Reaching these areas even in a small concentration, leptin might produce part of the ventilatory effects reported in the present study. Therefore, with the present results, it is not possible to exclude the action of leptin on other ventrolateral medullary areas outside RTN/pFRG to produce at least part of the respiratory responses in obese leptin-deficient ob/ob mice. In addition, although the present results suggest that leptin may facilitate respiratory responses also acting in the RTN/pFRG, it is necessary to consider that no study showed the presence of leptin receptors within the RTN/pFRG (Scott et al. 2009, Barnes et al. 2010), and more studies are necessary to solve these controversies. Previous studies have suggested that leptin acting centrally modulates respiratory function (Inyushkin et al. 2009, Inyushkina et al. 2010, Bassi et al. 2012); however, to our knowledge, the present study is the first to demonstrate that leptin may act at the level of ventrolateral medulla to facilitate ventilatory responses to central chemoreflex activation.

A previous study showed that intracerebroventricular leptin administration also required few days to improve ventilatory responses to CO2 in ob/ob mice (Bassi et al. 2012). Although the mechanisms by which leptin modulates respiratory function are not well understood, the current and previous data (Bassi et al. 2012) are consistent with the idea that these effects may involve changes in gene expression and protein synthesis, thus requiring chronic treatment of at least 3 days to have the effects. Also consistent with this possibility, preliminary slice-patch experiments showed that chemosensitive RTN/pFRG neurones and astrocytes elicited no change in membrane potential in response to acute exposure to leptin (data not shown). However, in this case, it is also necessary to consider that there is no study showing the presence of leptin receptors within the RTN/pFRG (Scott et al. 2009, Barnes et al. 2010).

The baseline VE values reported in the present study for obese leptin-deficient ob/ob mice (1528 mL kg−1 min−1) are similar to baseline VE values reported in the literature for obese ob/ob mice (1383 mL kg−1 min−1) or lean wild-type mice (1969 mL kg−1 min−1 when corrected to express the data using the same unit employed in the present study; O’Donnell et al. 1999) or to our previous data (1806 mL kg−1 min−1; Bassi et al. 2012) in obese ob/ob mice. However, it is important to note that although improved, the ventilation during 7% CO2 in leptin-deficient ob/ob mice treated with leptin injected into the RTN/pFRG region (4584 ± 344 mL kg−1 min−1, present results) or i.c.v. (6156 ± 312 mL kg−1 min−1, Bassi et al. 2012) was still reduced compared with wild-type mice in the same condition (7747 ± 337 mL kg−1 min−1; Bassi et al. 2012).

It is important to consider that chronic leptin administration may also affect metabolic rate due to changes in O2 consumption (VO2) and/or CO2 production (VCO2), which might affect chemorespiratory responses. Previous studies have shown controversial results about leptin effects on respiratory quotient (RQ = CO2/VO2), such as a reduction in RQ due to increased VO2 without changes in VCO2 (O’Donnell et al. 1999) or due to decreased VCO2 (Hogberg et al. 2006). According to these previous studies, leptin produces no increase in VCO2. For this reason, it has been suggested that the stimulatory effects of leptin on ventilation may occur due to its action on central mechanisms that control respiratory system and not due to metabolic changes like increases in VCO2. In the present study, we did not evaluate the metabolic rate during leptin administration into the RTN/pFRG, which limits any interpretations of the leptin effects on metabolic rates and possible metabolic modulation of ventilation. Further studies are necessary to better clarify this point.

Multiple mechanisms may contribute to chemoreception at the level of the ventral medulla. Among them, a population of glutamatergic neurones pH sensitive projects to respiratory centres (Nattie & Li 1995, Mulkey et al. 2004, Abbott et al. 2009). Leptin may facilitate the activity of these glutamatergic neurones in the ventral medulla as suggested for glutamatergic neurones located in the hippocampus and NTS (Williams et al. 2007, Guo et al. 2012). In addition to the glutamatergic neurones, it has been suggested that glia cells in the ventral medulla, including the RTN, may also contribute to chemoreception (Gourine et al. 2005, Mulkey et al. 2006). Previous studies have shown that hypercapnia evokes discrete release of adenosine 5′-triphosphate (ATP) by astrocytes to stimulate respiratory output (Gourine et al. 2005, Wenker et al. 2010). Chronic central leptin administration activates astrocytes (García-Cáceres et al. 2011) by mechanisms involving calcium signalling and ATP release (Tu et al. 2008, Hsuchou et al. 2009). Thus, it is possible to speculate that leptin may affect chemorespiratory response to hypercapnia by altering calcium and ATP signalling in glia cells located in the ventral medulla. Future studies should investigate the action of leptin on these mechanisms to produce respiratory effects.

In conclusion, the present results suggest that leptin improves baseline respiratory function and ventilatory responses to hypercapnia in ob/ob mice by acting in the respiratory nuclei of the ventrolateral medulla, including the RTN/pFRG. Additional studies are necessary to further examine the complex mechanisms and specific brain cells that mediate the actions of leptin on respiratory function.

Acknowledgments

The authors are indebted to FAPESP, CNPq and the National Heart, Lung and Blood Institute grant (PO1 HL 51971) and HL104101 (DKM) for financial support.

Footnotes

Conflict of interest

We have no any actual or potential conflict of interest.

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