1 CNRS UMR 5018, Paul Sabatier University, Toulouse, France
2 CNRS UMR 5543, Victor Segalen University, Bordeaux, France
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ABSTRACT |
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The hypothalamus contributes to the control of energy homeostasis by integrating metabolic information and eliciting adaptive responses. The arcuate nucleus (ARC) plays a pivotal role in this control (13). Indeed, ARC neurons release neuromodulators of food intake (4) and are located just above the median eminence, a highly vascularized structure with many capillary loops and fenestrated endothelial cells (5). This location allows this nucleus to easily integrate peripheral signals of body energy status into changes in neuronal activity (6, 7).
The role of glucose as a signal that informs the hypothalamus about body energy status has been investigated by different groups (8). Several studies demonstrated that an intracarotid injection of glucose at a concentration that did not modify peripheral glycemia induced c-fos activation in hypothalamic nuclei such as the ARC and triggered a rapid and transient insulin secretion, indicating that the brain senses and regulates glucose homeostasis (9, 10). Moreover, it has been shown that an intravenous injection of glucose modified electrical activity of neurons located in the lateral hypothalamus or in the ventromedial hypothalamus (VMH), which contains both the ARC and the ventromedian nucleus (VMN) (11, 12). Since that time, glucosensing neurons whose electrical activity may be modified by changes in extracellular glucose concentration have been identified by patch-clamp on hypothalamic slices (7, 1316). Glucose-excited or -inhibited neurons increase or decrease their electrical activity, respectively, as extracellular glucose concentration increases (16).
Whereas the mechanism whereby glucose-inhibited neurons sense glucose is unclear, glucose-excited neurons would use the ATP-sensitive K+ (KATP) channels to respond to glucose (17). This is similar to pancreatic ß-cells, where hyperglycemia is detected via a system involving the glucose transporter GLUT2 coupled to glucokinase and KATP channels (18). KATP channels are widely distributed throughout the brain (19) and are present in VMN and ARC glucose-excited neurons (2022). The glucose-induced activation of these glucose-excited neurons was mimicked by sulfonylureas (KATP channel blockers) and correlated with a decrease of K+ conductance (7, 1316). However, both in pancreatic ß-cells and in hypothalamus, a KATP channelindependent mechanism underlying glucose effect has been suggested (2326).
Extracellular brain glucose concentration seems to be lower than that of plasma glucose (27). However, the location of the ARC near the median eminence makes it likely that glucose concentrations in this region may approach plasma levels. ARC glucosensing neurons have predominantly been characterized using large steps in glucose concentration from 0 to 10 or 20 mmol/l (1315). However the brain would never be exposed to such a single-step increase in glucose concentration but rather would see more gradual changes above and below some steady-state level. Below 5 mmol/l, the sensitivity of ARC glucosensing neurons to glucose has been examined in detail (7). Above 5 mmol/l, the presence of glucosensing neurons has been shown (22, 28, 29), but the underlying mechanism has been poorly investigated. Thus, the aims of the present study were to characterize in ARC neurons 1) their biophysical properties and 2) the intracellular mechanisms in response to an increase in extracellular glucose concentration from 5 to 20 mmol/l.
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RESEARCH DESIGN AND METHODS |
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Some experiments used homozygous KATP channeldeficient mice (Kir6.2/) that were generated by disruption of the Kir6.2 gene (30). Slices from 19- to 22-day-old male Kir6.2/ mice were prepared.
Patch-clamp recordings.
Slices were transferred into a recording chamber on the stage of an upright microscope (Nikon), immobilized by a nylon grid, and perfused at 23 ml/min with the extracellular medium. The ARC was identified by using the mouse brain stereotaxic atlas (31). ARC neurons were visualized using a x60 water immersion objective (Nikon) under infrared differential interference contrast illumination and an infrared video camera (Hamamatsu Photonics).
Borosilicate pipettes (57 M; GC150F-10, Phymep) were filled with an internal solution that contained (in mmol/l) 130 K-gluconate, 0.1 EGTA, 1 CaCl2, 10 HEPES, 5 Mg-ATP, and 0.4 Na-GTP (pH and osmolarity adjusted at 7.25 and 290 mOsmol/l, respectively) for whole-cell recordings or with filtered extracellular medium for cell-attached recordings. Recordings were made using an Axopatch 1D amplifier (Axon Instruments), digitized using the Digidata 1320A interface, and acquired using pClamp 8.2 software (Axon Instruments). The amplifier filter was set at 5 kHz for whole-cell current-clamp recordings and at 2 kHz for whole-cell voltage-clamp or cell-attached recordings. Pipettes and cell capacitances were fully compensated. Junction potential was calculated using pClamp 8.2 and corrected off-line.
Drugs and glucose application.
Glucosensing neurons were identified using extracellular medium that contained 20 mmol/l D-glucose and 0 mmol/l sucrose (300310 mOsmol/l [pH 7.4]).
D-Glucose, cesium chloride, tolbutamide, and diazoxide were purchased from Sigma; pinacidil was purchased from Tocris; and tetrodotoxin (TTX) was purchased from Latoxan. All (except for the D-glucose) were prepared as concentrated stock solution and stored at 80°C. When drugs (diazoxide, pinacidil, and tolbutamide) were prepared in DMSO, the final concentration of solvent was always kept below 0.001. Drugs that were diluted in oxygenated extracellular medium were delivered at 2 ml/min by a multibarrel gravity-feed system (ALA Scientific Instruments, Sega Electronique) positioned at 2 mm from the ARC. Drug wash-in occurred within 2 and 10 s as measured by application of high potassium solution.
Histochemistry protocol.
The location of recorded cells was determined via diffusion of neurobiotin (0.2% in the whole-cell pipette solution; Vector Laboratories) into the cytosol. After recording, brain slices were fixed in 4% paraformaldehyde/0.2% picric acid in PBS for 90 min at room temperature. Slices were then cryoprotected in 20% sucrose for at least 12 h at 4°C and frozen at 60°C in isopentane (Prolabo). Slices (300 µm) were then cut into 16-µm serial sections using a cryostat (Leica) and collected on microscope slides (Menzel-Glaser). After quenching peroxidase activity with 0.1% H2O2 for 20 min at room temperature, sections were incubated in PBS/0.3% triton X100 (Sigma)/3% normal goat serum (Sigma) for 30 min at room temperature. Neurobiotin was then detected by incubation in streptavidin-peroxidase conjugate (1:4,000; Jackson Laboratories) for 90 min at room temperature. Afterward, peroxidase activity was revealed by diaminobenzidine (DakoCytomation). After hematoxylin counterstaining (Shandon) and alcohol dehydration, sections were cleared in Bioclear (MicroStain [D-limonene]; Micron) and examined under a transmitted-light microscope (Leica).
Data analysis and representation.
Recordings were analyzed using pClamp 8.2 and Clampfit 8.2 software (Axon Instruments) and plotted with Origin 5.0 (Microcal Software). Data were shown as mean ± SE, and differences between groups were analyzed by paired or unpaired Students t test, with P < 0.05 taken as significant.
In illustrations of current-clamp experiments, resting membrane potential was systematically indicated by a dotted line and mentioned on the right of each trace. All action potentials were truncated at 10 mV. For long recordings (in the case of glucose responses), data were acquired and shown in episodic mode with 10-s sweeps.
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RESULTS |
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Triggered hyperpolarizing current injections were performed to investigate membrane properties of ARC neurons. Five distinct electrical phenotypes thus could be distinguished. Types A, B, and C neurons similar to those described by Burdakov and Ashcroft (32) were identified. Twenty-five percent of ARC neurons (79 of 308) immediately resumed normal firing after hyperpolarizing current injection and corresponded to type A neurons (Fig. 2A); 34% (106 of 308; type B neurons) showed a rebound depolarization at the end of hyperpolarizing current injections (Fig. 2B), and 5% (15 of 308; type C neurons) displayed a rebound hyperpolarization that caused them to resume firing with a delay of 742 ± 115 ms (Fig. 2C). However, two other electrical phenotypes were also identified: 9% (27 of 308; type D neurons) showed a "sag" in membrane potential (Fig. 2D), and 26% (81 of 308; type E neurons) showed a "sag" and a rebound depolarization (Fig. 2E). Voltage-clamp steps from 70 to 150 mV on both of these neuron subtypes revealed a hyperpolarizing-activated inward (Ih) current (Fig. 3A, right). Both "sag" in membrane potential and Ih current were reversibly blocked by external Cs+ (4 mmol/l) application (Fig. 3B). This pharmacological approach confirmed the presence of Ih current in types D and E ARC neurons.
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Two types of glucose responses were recorded: 19% of neurons (26 of 135) responded by a depolarization (3.6 ± 0.3 mV) and increased firing rate from 2.1 ± 0.3 to 4.5 ± 0.5 Hz (Fig. 4A, left). These glucosensing neurons had a mean membrane capacitance of 40 ± 4.5 pF and input resistance ranging from 200 to 1,300 M. Conversely, 7% of ARC neurons (9 of 135) were inhibited, i.e., they were hyperpolarized by 4.6 ± 0.9 mV, and their firing rate decreased from 2.1 ± 0.6 to 0.6 ± 0.3 Hz in response to the 5- to 20-mmol/l glucose increase (Fig. 4B, left). These neurons had a mean membrane capacitance of 24.2 ± 3.4 pF and input resistance ranging from 430 to 1,700 M
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Unfortunately, glucosensing neurons could not be identified a priori on the basis of their firing pattern at resting potential or membrane responses to hyperpolarizing current injection (Fig. 4C). In view of the low percentage of HGI neurons in the ARC, we decided to focus on the HGE neurons for the rest of the study.
Glucose detection by HGE neurons.
To determine whether the glucose-induced excitation was direct or due to presynaptic inputs, the effect of a 5- to 20-mmol/l glucose increase was investigated when action potentials and thus synaptic transmission were blocked by TTX. The excitatory effect of the 5- to 20-mmol/l glucose increase persisted in the presence of TTX in all HGE neurons tested (6 of 6; Fig. 5A), without any significant modification in glucose-induced depolarization (20 mmol/l glucose, 3.4 ± 0.5 mV vs. 20 mmol/l glucose + TTX, 3.6 ± 0.2 mV; n = 6; P > 0.05; Fig. 5B). These results suggest that HGE neurons directly detect an increase of glucose concentration from 5 to 20 mmol/l.
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The reversal potential of glucose response was different from EK in our solutions (96 mV) and did not correspond to any other equilibrium potential. Thus, these results suggest that a 5- to 20-mmol/l glucose increase induced depolarization of HGE neurons through the opening of a nonselective cationic conductance.
State of KATP channels at 5 mmol/l glucose concentration.
The usual hypothesis for glucose-induced neuron excitation involves KATP channel closure. Because our results were not in agreement with closure of K+ channels, it was important to ascertain their presence and state (opened or closed) in ARC neurons in our experimental conditions. Therefore, we tested the effect of KATP channel openers (pinacidil and diazoxide) and blockers (tolbutamide) on glucose-insensitive and HGE neurons with an extracellular glucose concentration of 5 mmol/l.
Application of pinacidil and diazoxide (both at 250 µmol/l) inhibited 8 of 10 glucose-insensitive neurons (Fig. 7A) and 5 of 5 HGE neurons (Fig. 7B and C, ). KATP channel openers hyperpolarized ARC neurons by
9 mV (Fig. 7C) with decrease of firing rate at
97% (Fig. 7B). Tolbutamide (250 µmol/l) reversed this inhibition and depolarized ARC neurons by
3 mV with an increase of action potential frequency of 17% above basal activity. Nevertheless, the magnitude of tolbutamide response was much smaller than in response to pinacidil and diazoxide (Fig. 7B and C). Cell-attached recordings confirmed whole-cell results (data not shown). Taken together, these results suggest that 1) most ARC neurons have functional KATP channels and 2) these channels are closer to the closed than the open state in 5 mmol/l glucose.
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DISCUSSION |
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Whole-cell recordings revealed the presence of numerous spontaneously active ARC neurons and important presynaptic excitatory and/or inhibitory inputs as previously described (33, 34). Investigation of membrane properties revealed five distinct phenotypes in response to hyperpolarizing current injection: type A neurons, which immediately resume firing at the end of current injection; type B neurons, which exhibit rebound depolarization; type C neurons, which display a rebound hyperpolarization; type D neurons, which exhibit a "sag" in membrane potential; and type E neurons, which display a "sag" in membrane potential and a rebound depolarization. Types A, B, and C have already been described in the ARC, but the presence of an Ih current underlying the "sag" in membrane potential (in neuron types D and E) was more controversial (22, 32, 35). Our results in the anterior ARC are consistent with those of Poulains and Ibrahims groups describing the presence of "sag" and/or Ih current in ARC. This disagreement with the data from Burdakov and Ashcroft may be because they evaluated the posterior ARC (32, 36). The finding of Ih channel expression in ARC reinforces our results (37). The Ih current in anterior ARC neurons may control neuronal activity 1) by determining resting membrane potential, 2) by regulating the response to hyperpolarization such as during arrival of inhibitory synaptic potentials that are numerous in ARC (33, 34, and unpublished personal observations), and 3) by contributing to "pacemaker" activity also present in ARC (35, 38, and our study).
The second part of the present study clearly demonstrates the presence of ARC glucosensing neurons that respond to increased extracellular glucose concentration from 5 to 20 mmol/l. These data are very important because previous studies have evaluated ARC glucosensing neurons mainly in response to large nonphysiological steps in extracellular glucose (i.e., from 0 to 10 or 20 mmol/l) or to a glucose level <5 mmol/l (7, 14, 15). However, there is a paucity of data regarding the responses of ARC glucosensing neurons as glucose levels are raised above 5 mmol/l. This is because the majority of studies measuring brain glucose levels suggest that, in general, they are 30% that of plasma glucose, ranging from 0.1 to 4.5 mmol/l for hypo- to hyperglycemic states, with a set point at
2.5 mmol/l (27, 39, 40). However, actual glucose levels in the ARC are more controversial because of its proximity to the median eminence, where the blood-brain barrier is "leaky" (5). Thus, it is reasonable to hypothesize that extracellular glucose concentration in the ARC may approximate plasma level and exceed 4.5 mmol/l. Moreover, like in the pancreatic ß-cells, glucokinase plays a role in central nervous system glucosensing (41) and is functional mainly in the range >5 mmol/l (42). Therefore, for these reasons, we used a basal glucose concentration of 5 mmol/l and an increase to 20 mmol/l. With this glucose step, we recorded glucose-insensitive neurons and neurons that can be either excited or inhibited. By analogy to the definitions of Song et al. (16), for our higher glucose concentrations, these last two neuron subpopulations were called HGE and HGI neurons.
To our knowledge, this is the first study clearly investigating the proportion of neurons that are sensitive to high-glucose changes in mouse ARC. Compared with Yang et al. (28, 29), who used the same glucose step in rat VMH, the proportion of glucosensing neurons in the ARC is similar for HGE but lower for HGI neurons. The proportion found in the present study is equivalent to that reported by Wang et al. (7) for glucose-excited and -inhibited neurons in the rat ARC using extracellular glucose changes from 2.5 to 0.1 or 5 mmol/l. Because we did not evaluate glucose levels <5 mmol/l, we cannot conclude whether these glucose-excited and -inhibited neurons may correspond to HGE and HGI neurons. Nevertheless, we can speculate that glucose-excited and HGE neurons form two subpopulations. Indeed, HGE neurons show a large change in firing rate in response to a 5- to 20-mmol/l glucose step, whereas action potential frequency of glucose-excited neurons plateaus above 2.5 mmol/l (7). Glucose-inhibited and HGI ARC neurons also seem to be different cells. Indeed, HGI neurons present spontaneous action potentials at 5 mmol/l glucose, whereas glucose-inhibited neurons are mostly quiescent above 2.5 mmol/l glucose (V.H. Routh, personal communication).
We next investigated the mechanism involved in high-glucose detection by HGE neurons. Inhibition of synaptic transmission by TTX does not modify HGE neuron response, thus suggesting a direct glucose detection. Similar results were also found by Wang et al. (7) for rat ARC glucose-excited neurons. However, in the VMN, detection of a glucose increase above 2.5 mmol/l is due to presynaptic inputs (16). Thus, HGE and HGI neurons may provide the presynaptic regulation of VMN neurons in response to high glucose described by Song et al. (16). These data are consistent with those concerning the neuropeptide Y and pro-opiomelanocortin neuronal networks (1, 2), which suggest that the ARC may be the first nucleus to detect peripheral metabolic signals and then relay this information to other hypothalamic nuclei such as VMN or PVN (paraventricular nucleus).
Over the past 15 years, it has been demonstrated that KATP channel closure is a key step in cerebral glucosensing (13, 28, 43). Recent studies, using extracellular glucose changes at 2.5 mmol/l, show that glucose-induced activation of VMN or ARC glucose-excited neurons is correlated to a decrease of K+ conductance (7, 16). Moreover, Miki et al. (44) failed to detect glucosensing neurons in the VMN of KATP channeldeficient mice. These results point out that the glucose sensitivity, at least at
2.5 mmol/l, depends on KATP channel activity. However, for glucose concentrations >5 mmol/l, we show a different mechanism for glucose activation of HGE neurons: first, the glucose-induced depolarization was correlated to a significant decrease of input resistance, indicating an increase of membrane conductance. Moreover, our results and others (19, 22) have demonstrated that KATP channels were present and functional in ARC neurons. However, in glucose-insensitive neurons as well as in HGE neurons, these KATP channels were mostly closed at 5 mmol/l extracellular glucose concentration. This is consistent with the work of Wang et al. (7), which shows that the decrease in KATP channel current in response to increase from 0.1 to 10 mmol/l plateaus above 2.5 mmol/l. Finally, we identified ARC HGE neurons in Kir6.2 knockout mice. The number and the responses of these HGE neurons were similar to those obtained in wild-type mice. These results agree with other studies suggesting a likely additional mechanism independent of KATP channels, as it has been demonstrated in pancreatic ß-cells (2325). Indeed, the presence of KATP channels is not entirely correlated to glucose-induced neuronal response (21). Furthermore, an increase in glucose concentration from 3 to 15 mmol/l did not significantly modify intracellular ATP levels in VMH neurons (45). Finally, the KATP channel opener diazoxide blocked only the 5 to 20 mmol/l glucose effect in a minority of glucose-excited neurons in rat VMH, clearly calling into question the importance of KATP channels. In this later study, the authors suggested that, instead of ATP, the key metabolic signal would be NADH (26, 28). Taken together, these data give clear evidence that, at glucose concentrations >5 mmol/l, a KATP channelindependent mechanism mediates glucose-induced excitation of ARC HGE neurons.
Current- and voltage-clamp recordings suggested that a nonselective cationic conductance mediates depolarization of HGE neurons. Indeed, we showed that the glucose-activated conductance reversed at a potential close to 20 mV, which is different from the K+ equilibrium potential. A similar conductance has been described for orexin and leptin in hypothalamic neurons (6, 46). It is tempting to speculate that this cationic conductance may be a common target for different metabolic signals.
In conclusion, we show that ARC neurons are capable of sensing increased extracellular glucose levels >5 mmol/l. These neurons may play a role in regulation of hyperglycemia. Activation of HGE and HGI neurons could lead to modulation of autonomic nervous system activity and, as a consequence, peripheral glucose homeostasis regulation (47). The second important point of our results is the demonstration of a new, additional KATP channelindependent mechanism in glucose detection. This feature argues for similarity between ARC and pancreatic glucosensing. The nature of the cationic conductance and the mechanisms by which high-glucose concentration activates it remain to be determined. However, this pathway may represent a novel brain "molecular glucosensor."
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ACKNOWLEDGMENTS |
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We are indebted to J.-M. Lerme for involvement in mice care and very grateful to G. Portolan and M. Belliure for technical help in immunohistochemistry. We thank Dr. V. Routh for helpful discussions and critical reading of the final manuscript. We are grateful to Dr. S. Seino for providing Kir6.2 null mice.
Address correspondence and reprint requests to Dr. Anne Lorsignol, UMR 5018 CNRS-UPS, IFR 31, CHU Rangueil, 1 Avenue Jean Poulhès, 31403 Toulouse, France. E-mail: anne.lorsignol{at}toulouse.inserm.fr
Received for publication June 8, 2004 and accepted in revised form July 30, 2004
ARC, arcuate nucleus; HGE, high glucose excited; HGI, high glucose inhibited; KATP channel, ATP-sensitive K+ channel; TTX, tetrodotoxin; VMH, ventromedial hypothalamus; VMN, ventromedian nucleus
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REFERENCES |
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