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Department of Physiology and Pharmacology (N.I., M.A.B., J.Q., O.K.R., M.J.K.), The Vollum Institute (J.L.S., M.J.L.), and Department of Behavioral Neuroscience (M.J.L.), Oregon National Primate Research Center (O.K.R.), Oregon Health & Science University, Portland, Oregon 97239-3098; and Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (M.R.), Consejo Nacional de Investigaciones Científicas y Técnicas, and Department of Biology, School of Sciences, University of Buenos Aires, Buenos Aires, Argentina
Address all correspondence and requests for reprints to: Martin J. Kelly, Ph.D., Department of Physiology and Pharmacology, L334, Oregon Health Sciences University, 3181 Southwest Sam Jackson Park Road, Portland, Oregon 97239-3098. E-mail: kellym{at}ohsu.edu.
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-aminobutyric acidB receptor agonist baclofen (40 µM) caused an outward current (21.6 ± 4.0 pA) that reversed at EK+ in these same neurons. The ATP-sensitive potassium channel opener diazoxide also induced an outward K+ current (maximum of 18.7 ± 2.2 pA) in the majority (92%) of POMC neurons with an EC50 of 61 µM. The response to diazoxide was blocked by the sulfonylurea tolbutamide, indicating that the POMC neurons express both Kir6.2 and sulfonylurea receptor 1 channel subunits, which was verified using single cell RT-PCR. This pharmacological and molecular profile suggested that POMC neurons might be sensitive to metabolic inhibition, and indeed, we found that their firing rate varied with changes in glucose concentrations. Therefore, it appears that POMC neurons may function as an integrator of metabolic cues and synaptic input for controlling homeostasis in the mammal. |
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-MSH, have been associated with many physiological functions including reproduction, metabolic homeostasis, stress responses, and natural reward (4, 5, 6). At the cellular level, the opioid peptide ß-END has been shown to postsynaptically modulate the excitability of local -aminobutyric acid (GABA) and dopamine neurons (7, 8, 9). In addition, GnRH, oxytocin, and vasopressin neurosecretory (8) neurons are inhibited by ß-END (10, 11, 12, 13). The other putative neurotransmitter of POMC neurons, MSH, has been shown to modulate synaptic input to paraventricular neurons that are thought to be involved in the regulation of metabolic homeostasis (14).
The opioid peptide ß-END modulates target neurons through a G protein-coupled receptor. The µ-opioid receptor is Gi/o-coupled to either the activation of an inwardly-rectifying K+ channel (Kir3.1–3.4, GIRKs), the inhibition of adenylyl cyclase, or the inhibition of Ca2+ channels (15). The GIRK-type subfamily of inwardly rectifying K+ channels comprises four different channel subtypes, all of which are expressed in the rat hypothalamus (16). Hypothalamic neurons are inhibited through a µ-opioid receptor that is coupled to GIRKs (7, 12, 17, 18, 19).
ATP-sensitive potassium (KATP) channels are another member of the inwardly-rectifying K+ channel family (20). They are heteromultimeric complexes of sulfonylurea receptors (SUR; the regulatory subunit) and inwardly rectifying K+ channel (Kir6.1–6.2) subunits (21, 22). These channel complexes couple membrane excitability to cellular metabolism by directly sensing and integrating intracellular changes in the concentration of nucleotides (23). The Kir6.2 plus SUR1 channel complex is activated by diazoxide and by metabolic inhibition and is blocked with high affinity by sulfonylureas such as glibenclamide and tolbutamide (23). Sulfonylurea binding and electrophysiological studies have characterized neuronal KATP channels in a variety of neurons. For example, Kir6.2 is widely distributed in rat brain and is present in neurons expressing tyrosine hydroxylase, neuropeptide Y, and glutamic acid decarboxylase (24).
In the rat hypothalamus, the adipocyte hormone leptin hyperpolarizes glucose-responsive, ventromedial nucleus neurons via activation of a K+ current that is blocked by tolbutamide (25). Also, insulin hyperpolarizes glucose-responsive neurons via a tolbutamide-sensitive K+ current (26). More recent studies have shown that these glucose-responsive neurons express Kir6.2 and SUR1 transcripts (27), which renders them diazoxide and tolbutamide sensitive. Previous findings suggest that the G protein-coupled receptors such as the dopamine D2, GABAB, and somatostatin SST5 receptors are coupled to both GIRKs and Kir6.2 in pancreatic ß-cells and substantia nigra neurons (28, 29). However, the relationship between expression of GIRKs and Kir6.2 channel subtypes in hypothalamic neurons is not known. Because POMC neurons are so critical for regulating homeostasis and motivated behaviors in the mammal (6, 30), we hypothesized that POMC neurons would respond to activators of GIRKs and Kir6.2 channels and serve as integrators of both synaptic and hormonal (metabolic) input. We used a transgenic mouse model in which we could visualize enhanced green fluorescent protein (EGFP)-labeled POMC neurons and measure the direct effects of the µ-opioid agonist DAMGO, the KATP channel opener diazoxide and metabolic inhibition.
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Electrophysiology
Female POMC-EGFP transgenic mice (14–21 d) were selectively bred in-house, and maintained under the conditions described above. On the day of experiment, the mice were anesthetized with halothane, decapitated, the brain rapidly removed from the skull and a block containing the hypothalamus immediately dissected. (The trunk blood was collected, and serum estrogen levels determined by chromatography and subsequent RIA by Oregon National Primate Research Center. Serum estrogen levels in these immature female mice were 5.9 ± 0.8 pg/ml, which were significantly below castrate levels of adult females.) The hypothalamic block was submerged in cold (4 C) oxygenated (95% O2, 5% CO2) artificial cerebrospinal fluid (aCSF) with low Ca2+ containing the following constituents, in mM: NaCl, 124; KCl, 5; NaHCO3, 26; NaH2PO4, 2.6; dextrose, 10; HEPES, 10; MgSO4, 2; CaCl2, 1. Coronal slices (300 µm) through the caudal-rostral extent of the arcuate nucleus were cut with a vibratome during which time (20 min) the slices were bathed in aCSF with low Ca2+ at 4 C. The arcuate slices were then transferred to an auxiliary chamber where they were kept at room temperature (25 C) in aCSF with normal Ca2+ (2 mM) until recording (recovery 
1.5 h), at which time a single slice was transferred to the recording chamber. Once in the recording chamber, the slices were kept viable by continually perfusing with warm (35 C), oxygenated normal aCSF at 1.5 ml/min.
For imaging and recording, slices were viewed with a Zeiss Axioskop outfitted for fluorescence (fluorescein isothiocyanate filter) and infrared differential interference contrast videomicroscopy. After visualizing fluorescent POMC-EGFP neurons with the 5x objective, a 40x water immersion objective was used for infrared differential interference contrast imaging to visualize neurons for whole-cell patch clamp recording. Microelectrodes (resistances 3–6 M
) were fabricated from borosilicate glass pipettes (1.5 mm OD) and filled with an internal solution (pH 7.30) containing the following constituents, in mM: K-gluconate, 128; NaCl, 10; MgCl2, 2; EGTA, 11; HEPES, 10; ATP, 1; GTP, 0.25. Standard whole-cell voltage clamp procedures were followed using an Axopatch 200A amplifier (2-kHz lowpass filter, Axon Instruments, Union City, CA) as previously described (32). Signals were digitized with a Digidata 1200, and analyzed using pClamp 7.0 software (Axon Instruments). The liquid junctional potential of -10 mV was corrected in the data analysis. Current and voltage traces were also recorded on a analog chart recorder (Gould Instruments, Valley View, OH).
Following formation of a greater than 1 G seal, intracellular access was achieved by suction, followed by perfusion with 1 µM tetrodotoxin (TTX, Alomone Labs, Jerusalem, Israel) for at least 4–6 min to block spontaneous firing and action potential-generated synaptic potentials. All the responses to agonists and antagonists were measured in voltage clamp (Vhold = -60 mV) with the exception of the glucose experiments. The access resistance was checked before and after each drug treatment, and only those cells that showed less than 10% change in access resistance throughout the recording were included in this study. The access varied from 20–30 M (
= 24.4 ± 0.8 M), which ensured adequate voltage clamp of this slow outward K+ current and minimal rundown during pharmacological testing due to rapid dialysis of intracellular (second messenger) constituents.
For the cell attached recordings, the patch pipettes were filled with the external solution (aCSF), and a loose seal (100 M) was formed on the identified POMC neurons to measure spontaneous activity in current clamp. After a stable baseline was established after several minutes of recording, the glucose concentration was rapidly decreased from 10–5 mM. The firing activity was measured over a minute period after stabilization and compared with the firing rate measured 1 min before the change in the glucose concentration. Only cells that showed a full recovery were used to calculate the change in firing frequency with altered glucose concentrations.
Drug application
Following generation of a control current-voltage plot in the presence of TTX, drugs were perfused until a steady-state outward current was obtained. Diazoxide (7-chloro-3-methyl-2H-1,2,4-benzo-thiadiazin 1,1-dioxide) and tolbutamide (Sigma, St. Louis, MO) were dissolved in dimethylsulfoxide 99.5% to a stock concentration of 300 mM and 100 mM, respectively. Perfusion of aCSF containing 0.1–0.3% dimethylsulfoxide (vehicle controls) had no effect on the cells. Naloxone (Sigma) and DAMGO (D-Ala2, N-Me-Phe4, Gly-ol5-enkephalin; Peninsula Laboratories, Inc., Belmont, CA) were dissolved in Milli-Q H2O to a stock concentration of 1 mM. Baclofen (Sigma) was dissolved in 0.1 N HCl to a concentration of 40 mM. Aliquots of the stock solutions were stored appropriately until needed. Final drug concentrations were made up in 10 ml volumes and perfused at 1.5 ml/min. On the average, it took 2–5 min to reach a steady-state outward current with DAMGO, baclofen, or diazoxide. The drug-induced change in conductance was determined by subtracting the pre- from the postdrug current/voltage (I/V) slopes. Composite dose-response curves were generated from the following logistic equation fitted by computer (Origin 4.1, Microcal) to the data: 
Imax = 100 · ([agonist]n/([agonist]n + EC50n)), where Imax is the maximum outward current for a given agonist, EC50 represents the agonist potency, and n is the Hill slope.
The pharmacodynamics sometimes were reevaluated after the drug wash-out in the presence of antagonists. Estimates of the Ki for antagonists were derived from the logistic equation fitted by computer (SigmaPlot 2000, Jandel Scientific) to the data: Imax = 100 · ([agonist]n/([agonist]n + (EC50n · (1 + ([antagonist]n/Kin))))).
Acutely dispersed neurons
For these experiments, we prepared hypothalamic slices from adult Topeka guinea pigs using the same procedures as for the preparation of mouse hypothalamic slices (see above). The 300-µm coronal hypothalamic slices were cut on a vibratome from caudal to rostral and placed in an auxiliary chamber containing oxygenated, normal aCSF. The slices were allowed to recover for 1–2 h in the chamber before dispersion. The arcuate nucleus of the hypothalamus was microdissected and incubated in 2–3 ml of Hanks’ balanced salt solution [HBSS (in mM): CaCl2 ,1.26; MgSO4,1; KCl, 5.37; KH2PO4, 0.44; NaCl,136.89; Na2HPO4, 0.34; D-glucose, 5.55; HEPES,15 in diethylpyrocarbonate (DEPC)-treated water, pH 7.3, 300 mOsm] containing 1 mg/ml protease XIV (Sigma) for approximately 15 min at 37 C. The tissue was then washed four times in one volume low calcium aCSF and two times in HBSS. The cells were isolated by trituration with flame-polished pasteur pipettes, dispersed on a 35-mm Petri dish and continuously perfused with HBSS at a rate of 1.5 ml/min. Cells were visualized using an inverted microscope, and individual neurons were patched and harvested into the patch pipette by applying negative pressure. The content of the pipette was expelled into a siliconized microcentrifuge tube containing 5 µl of the following solution: 0.5 µl of 10x buffer (100 mM Tris-HCl, 500 mM KCl, 1% Triton X-100; Promega Corp., Madison, WI), 15 U RNasin (Promega Corp.), 0.5 µl 100 mM dithiothreitol, and DEPC-treated water.
Tissue total RNA purification
Hypothalamic tissue was homogenized and total RNA extracted using the RNeasy kit (QIAGEN, Valencia, CA) according to the manufacturer’s protocol. Total RNA was treated with deoxyribonuclease I, which was then inactivated and removed using DNA-free reagents as described by the manufacturer (Ambion, Inc., Austin, TX). The RNA was diluted and used as a positive (+ reverse transcriptase [+RT]) or negative (-RT) control for the PCRs.
RT-PCR of single cells and tissue RNA
The harvested cell solution and 25 ng of hypothalamic total RNA in 1 µl were denatured for 5 min at 65 C then cooled on ice for 5 min. Single-stranded cDNA was synthesized from cellular RNA by adding 50 U murine leukemia virus RT (Applied Biosystems, Foster City, CA), 1.5 µl 10x buffer, 2 mM MgCl2, 0.2 µM deoxynucleotide triphosphate, 15 U RNasin, 10 mM dithiothreitol, 100 ng random hexamers, and DEPC-treated water to a final volume of 20 µl. Cells and tissue RNA used as negative controls, were processed as described above but without RT. The reaction mixtures were incubated at 42 C for 60 min, denatured at 99 C for 5 min, and cooled on ice for 5 min.
PCR was performed using 2–3 µl of cDNA template from each RT reaction in a 30-µl PCR volume containing: 3 µl 10x buffer, 2.4 µl MgCl2 (2 mM final concentration for POMC, Kir6.1, and SUR1) or 4.8 µl MgCl2 (4 mM final concentration for Kir6.2), 0.2 mM deoxynucleotide triphosphate, 0.2 µM forward and reverse primers, 2 U Taq DNA polymerase (Promega Corp.), and 0.22 µg TaqStart Antibody (CLONTECH Laboratories, Inc., Palo Alto, CA). Taq DNA polymerase and TaqStart Antibody were combined and incubated at room temperature for 5 min, the remainder of the reaction contents were added to the tube and incubated at 94 C for 2 min. Then, each reaction went through 60 cycles of amplification according to the following protocols: 94 C, 45 sec (denaturation); 60 C, 45 sec (annealing); 72 C, 1 min 10 sec (elongation), with a final 72 C extension for 5 min (POMC, Kir6.1, and SUR1) or 94 C, 45 sec (denaturation); 68 C, 1 min (annealing and elongation combined), with a final 72 C extension for 5 min (Kir6.2). Ten microliters of the PCR products were visualized with ethidium bromide on a 1.5% agarose gel.
The primers used were as follows: guinea pig POMC; 344-bp product (accession no. S78260), forward primer (bases 40–60) 5'-CTGGCCTTGCTGCTTCAGAT-3'; reverse primer (bases 383–363) 5'-ATGGAGTAGGAGCGCTTGTC-3'. Guinea pig Kir6.2; 398-bp product (accession no. AF183920), forward primer (bases 1608–1627) 5'-GCCCGCTTTGTGTCCAAGAA-3'; reverse primer (bases 2005–1985) 5'-CCCAGCATGATGGCGTTGAT-3'. Guinea pig SUR1; 238-bp product (accession no. AF183921), forward primer (bases 1325–1345) 5'-GCCACGGCTTCCATCGACAT-3'; reverse primer (bases 1562–1542) 5'-CGCTGGCAGGTCACTTGTCT-3'. Guinea pig Kir6.1; 220-bp product (accession no. AF183918), forward primer (bases 379–399) 5'-GGACATCTACGCTTACATGG-3'; reverse primer (bases 598–578) 5'-GACAGCGTTGATGATCAGAC-3'. Guinea pig glyceraldehyde-3 phosphate dehydrogenase (GAPDH); 212-bp product (accession no. CPU51572), forward primer (bases 123–143)5'CATCCACTGGTGCTGCCAAG-3'; reverse primer (bases 334–314) 5'-GTCCTCGGTGTAGCCCAAGA-3'. Primers were synthesized by Invitrogen (Carlsbad, CA), and the optimum PCR conditions for each primer pair was established in preliminary experiments. The PCR product from single cells for each primer pair was sequenced in our core facilities.
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. Moreover, the majority (68%) of female mouse POMC neurons exhibited the same endogenous conductances that we have described in female guinea pig POMC neurons under low steroid (ovariectomized) conditions, which included expression of a hyperpolarization-activated, cation current (Ih) and a transient outward K+ current (IA; Fig. 1
). Hence, the passive membrane properties of mouse POMC neurons labeled with EGFP are similar to what we have reported for guinea pig and rat POMC neurons identified post hoc by immunocytochemistry (17, 18). Therefore, we do not think that the EGFP expression in POMC neurons altered the physiological properties of these neurons.
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). Moreover, the nonselective opioid receptor antagonist naloxone attenuated the response to DAMGO (Fig. 2). A concentration-response relationship was generated from 54 neurons, and the majority of POMC neurons were tested with a single concentration of DAMGO. In addition, a small number of POMC neurons were tested with two concentrations of DAMGO, a lower concentration followed by a higher concentration of agonist to establish a maximum (outward current) response within a given cell. A logistics equation fit to the data points yielded an EC50 of 102.8 nM (Fig. 3).
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). This Ki for naloxone inhibition of µ-opioid response is similar to what we and others have published for naloxone antagonism of µ-opioid receptor-mediated responses in the central nervous system (33, 34).
GABAB receptor agonist baclofen activates GIRK
We have shown that guinea pig POMC neurons are inhibited by both DAMGO and the GABAB receptor agonist baclofen via activation of GIRKs (35). Therefore, we tested mouse POMC neurons to see if they would show a similar response to the GABAB receptor agonist baclofen. Indeed, all of the mouse POMC cells that were sensitive to DAMGO responded to the GABAB receptor agonist baclofen (40 µM) with an outward current (21.6 ± 4.0 pA) that reversed near EK+ (Ebaclofen = -80.7 ± 7.2 mV, n = 8) and with an increase in slope conductance of 1.8 ± 0.5 nS (Fig. 4). Therefore, it appears that the GABAB receptor is similarly coupled to activation of GIRK as the µ-opioid receptor in mouse POMC neurons. Further elucidation of the GABAB-mediated response was not undertaken because we have extensively characterized this response in guinea pig and rat hypothalamic neurons (18, 35, 36, 37, 38).
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). Moreover, the SUR1 selective drug tolbutamide antagonized the actions of diazoxide at equimolar concentrations (n = 12, Fig. 4). A concentration-response relationship for diazoxide was generated, using a single concentration of diazoxide to test each POMC neuron. A logistics equation fit to the data yielded an EC50 of 61.3 µM (Fig. 6). We did not see any evidence for desensitization of the diazoxide response even at concentrations of the drug that gave a maximum response (300–1000 µM).
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). In fact, 9 of 14 cells showed an additive response that was not significantly different from the theoretical maximum based on both channels (GIRK and Kir6.2) being activated (27.7 ± 2.8 pA vs. 31.5 ± 2.2 pA). The additive response did not depend on the order of drug application. The other five cells of this subpopulation showed an additive response to perfusion of DAMGO followed by diazoxide (n = 3) or diazoxide followed by DAMGO (n = 2) that was less than the theoretical maximum additive response (13.8 ± 0.7 pA vs. 31.5 ± 1.2 pA). POMC neurons that responded to both drugs were not localized to any particular region (rostral vs. caudal) of the mediobasal hypothalamus.
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). A third subpopulation showed a robust diazoxide-induced outward current (18.2 ± 1.1 pA, n = 5) without any further effect of DAMGO (Fig. 7C). Again, the order of drug perfusion did not make any difference in terms of the responses, and the responses did not appear to be region specific. In addition, we did not see a specific effect of tolbutamide to block the DAMGO-activated GIRK (n = 5) or the opioid receptor antagonist naloxone to block the diazoxide response (n = 2) in POMC neurons. Therefore, there appeared to be three distinct subpopulations of mouse POMC neurons. One population responded to both µ-opioid receptor activation by DAMGO, coupling to GIRK, and KATP channel activation by diazoxide. Another population responded to µ-opioid activation only; and finally, a smaller population of POMC neurons responded to the KATP channel opener diazoxide only.
Although there is evidence for activation of GIRK and KATP channels by G protein-coupled receptors (28, 29), we did not see any evidence of a direct activation of KATP channels by DAMGO. However, over a longer time period there could be a change in activity of KATP channels due to Gi/o inhibition of adenylyl cyclase activity.
Expression of Kir6.2 and SUR1 transcripts in POMC neurons
The electrophysiological data on the potency of diazoxide to induce an outward K+ current in POMC cells suggested that POMC cells express Kir6.2 subunits. Also, the sensitivity of this response to tolbutamide antagonism suggested that SUR1 is the sulfonylurea receptor subunit within the KATP channel. To define the molecular composition of the KATP channel directly, we measured the expression of transcripts using single cell RT-PCR. These experiments were carried out in dispersed guinea pig POMC neurons in which we have measured an equivalent pharmacological response to diazoxide and tolbutamide and have specific primers for Kir6.1, Kir6.2, SUR1, and POMC. The PCR product from single cells for each primer was sequenced and found to be specific. In addition, the specificity of the single cell PCR products had been verified in preliminary experiments using real-time PCR (39). Based on previous electrophysiological recordings from the caudal mediobasal hypothalamus (17, 36, 40), we predicted that POMC neurons make up about 20% of the population. Indeed, in our analysis of 20 arcuate neurons that were dispersed, patched and then harvested for RT-PCR, four POMC neurons were identified, three of which expressed Kir6.2 and SUR1 (Fig. 8). Although adjacent neurons also expressed Kir6.1, Kir6.2 and SUR1 transcripts, Kir6.2 plus SUR1 appear to be the predominant transcripts expressed in arcuate neurons (Fig. 8), which agrees with our pharmacological profile for the KATP channel in these neurons.
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). The spontaneous firing rate returned to control levels in all of the POMC neurons; however, the time course of recovery from metabolic inhibition varied among POMC neurons. Therefore, although the changes in the firing rate of POMC neurons may be due in part to altered synaptic input, the direct expression of KATP channels and the sensitivity to glucose suggests that POMC neurons are glucose-responsive neurons (i.e. they increase their firing rate in response to increases in glucose concentrations).
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Discussion |
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Acting through Gi/o-coupled µ-opioid receptors, ß-END can inhibit its target neurons through activation of GIRK, inhibition of adenylyl cyclase or inhibition of Ca2+ channels (15). At the cellular level, µ-opioid receptor agonists have been shown to modulate the excitability of dopamine neurons (7), GnRH neurons (10, 12), oxytocin and vasopressin neurons (13) and finally local GABA neurons (37). In addition, we have demonstrated that mediobasal hypothalamic POMC neurons are similarly self-inhibited through a µ-opioid autoreceptor that is coupled to GIRK activation (17). In fact, the µ-opioid receptor agonist DAMGO is more potent (EC50 60 nM) but equally efficacious in hyperpolarizing female guinea pig POMC neurons (40) as mouse POMC neurons. However, the present findings indicate that DAMGO is more potent to hyperpolarize POMC neurons (102 nM) vs. other arcuate neurons (315 nM) in the C57BL/6J strain of mice (19).
The GABAB receptor is also coupled to GIRK channels in guinea pig POMC neurons (35), and we have found a similar coupling in mouse POMC neurons. This indicates that GABA input from local arcuate GABA/NPY neurons would also provide a powerful inhibitory tone onto these POMC neurons via GABAB receptors (18, 31, 36), which is thought to play an important role in inhibiting POMC neurons during activation of feeding circuits (43).
On the other hand, KATP channels couple membrane excitability to cellular metabolism by directly sensing and integrating intracellular concentration changes of nucleotides such as ATP and ADP (20). Sulfonylurea binding and electrophysiological studies have characterized neuronal KATP channels with different properties in a variety of cell types, including hippocampal and midbrain neurons. Kir6.2 is widely distributed in rat brain, and is present in neurons expressing tyrosine hydroxylase, NPY and glutamic acid decarboxylase (24, 27, 39). Presently, our electrophysiological findings together with the single cell RT-PCR indicate that POMC neurons also express Kir6.2. In fact, the EC50 for diazoxide is similar to what has been reported for pancreatic ß-cells (44) and rat hippocampal pyramidal neurons (51 µM; Ref. 45). Based on the sensitivity to tolbutamide and single cell RT-PCR data, hypothalamic POMC neurons also appear to express SUR1. Liss and colleagues (46, 47) have shown that GABAergic neurons in the substantia nigra pars recticularis show a similar KATP channel subunit profile, with Kir6.2 and SUR1 coexpression detected in the majority of the neurons. In all cases, Kir6.2 plus SUR1-KATP channels are activated by diazoxide and by metabolic inhibition, and are blocked with high affinity by sulfonylureas such as tolbutamide. In addition, we have found that another SUR1-selective antagonist, glipizide, potently blocks the diazoxide response in POMC neurons (unpublished observations).
One of the classical feeding centers of the hypothalamus is the ventromedial nucleus in which resides glucose-responsive neurons, i.e. neurons that increase their firing in response to elevations in blood glucose levels (48, 49, 50). Based on recent single cell RT-PCR experiments, ventromedial glucose-responsive neurons express Kir6.2 plus SUR1 subunits (27). Therefore, it appears that glucose-responsive neurons can transduce, via the KATP channel, changes in extracellular glucose levels to changes in neuronal excitability. Based on our single cell RT-PCR results, arcuate guinea pig POMC neurons also express this same compliment of KATP channel subunits. In preliminary experiments with specific primers to mouse Kir6.2, we have found that the majority of dispersed mouse POMC-EGFP neurons express Kir6.2. However, the expression of Kir6.2 plus SUR1 is necessary but not sufficient for sensing changes in glucose (41). Therefore, it is important that we have found that a small reduction in extracelluar concentrations of glucose significantly inhibited POMC cell firing, whereas adjacent non-POMC neurons were not affected even though some of these may express Kir6.2 and SUR1 mRNA. It is also known that POMC neurons express glucokinase, which is a necessary enzyme for glucose-sensing cells (51). So, in addition to activation by leptin (31, 52), POMC neurons appear to be glucose responsive.
It is not surprising that we have identified three subpopulations of POMC neurons based on their reponse to µ-opioid agonists and KATP channel openers. In addition to their role in energy homeostasis (43, 53, 54), POMC neurons have multiple other functions including regulating reproduction (12, 55), parturition (13), fluid balance (56), stress responses (4, 6), and natural reward (5, 30, 57). Because of their involvement in all these different functions, POMC neurons are thought to be the "command" neurons of the hypothalamus. Perhaps part of the diversity in the POMC neurons lies in the differential processing of POMC to -MSH, which is prominent in feeding circuits, and to ß-END, which is involved in modulating reproduction, stress and natural rewards. In addition, POMC neurons are located in the arcuate and periarcuate region including the median eminence, which is outside the blood brain barrier. Therefore, they are in a strategic location for sensing humoral signals and translating these signals into neural activity.
For example, we have found that POMC neurons respond rapidly to estrogen, which serves to inhibit GnRH neurons during the negative feedback phase of the ovulatory cycle (12, 58). Perhaps another subset of POMC neurons respond rapidly to changing levels of leptin and/or glucose to translate these metabolic cues into neural signals. Interestingly, earlier studies in the female rat demonstrated that glucose-responsive neurons in the ventromedial nucleus were also sensitive to the acute actions of estrogen, which depolarized these cells via a cAMP-dependent pathway (59). Because we know that estrogen is anorexic (60) and activates POMC neurons in the female guinea pig via protein kinase A (34), the steroid may synergize with leptin and glucose to inhibit feeding in the female. Electrophysiology experiments are currently underway to measure the response of mouse POMC neurons to estrogen since there are profound gender differences in the control of metabolic homeostasis (60).
In summary, we have found that POMC neurons express a unique complement of inwardly rectifying K+ channels (GIRKs, Kir6.2) and SUR proteins that allow them to respond to metabolic (e.g. glucose) changes and synaptic input (e.g. GABA/NPY neurons). In addition, we know that the synaptic input to POMC neurons can be modulated by gonadal steroids and that leptin can directly excite these cells through activation of a cation-selective current. Therefore, these POMC neurons are uniquely situated to respond to ascending sensory input, metabolic changes and hormonal fluctuations that enable them to integrate multiple inputs and serve as the command neurons of the hypothalamus to maintain homeostasis in the mammal.
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Footnotes |
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Abbreviations: aCSF, Artificial cerebrospinal fluid; DAMGO, D-Ala2, N-Me-Phe4, Gly-ol5-enkephalin; DEPC, diethylpyrocarbonate; EGFP, enhanced green fluorescent protein; EK+, equilibrium potential; ß-END, ß-endorphin; GABA, -aminobutyric acid; GIRK, G protein-coupled inwardly rectifying K+ channel; HBSS, Hanks’ balanced salt solution; IA, a transient outward K+ current; Ih, hyperpolarization-activated, cation current; I/V, current/voltage; KATP, ATP-sensitive potassium; Kir, inwardly rectifying K+ channels; MBH, mediobasal hypothalamus; POMC, proopiomelanocortin; RT, reverse transcriptase; SUR, sulfonylurea receptors; TTX, tetrodotoxin.
Received October 7, 2002.
Accepted for publication December 20, 2002.
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References |
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-aminobutyric acid neurons: pharmacology, estrogen sensitivity and relevance to the control of the reproductive axis. J Pharmacol Exp Ther 299:21–30This article has been cited by other articles:
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 V. E. Cotero and V. H. Routh Insulin blunts the response of glucose-excited neurons in the ventrolateral-ventromedial hypothalamic nucleus to decreased glucose Am J Physiol Endocrinol Metab, May 1, 2009; 296(5): E1101 - E1109. [Abstract] [Full Text] [PDF]
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 K. W Williams, M. M Scott, and J. K Elmquist From observation to experimentation: leptin action in the mediobasal hypothalamus Am. J. Clinical Nutrition, March 1, 2009; 89(3): 985S - 990S. [Abstract] [Full Text] [PDF]
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A. Guyon, M. P. Tardy, C. Rovere, J.-L. Nahon, J. Barhanin, and F. Lesage Glucose Inhibition Persists in Hypothalamic Neurons Lacking Tandem-Pore K+ Channels J. Neurosci., February 25, 2009; 29(8): 2528 - 2533. [Abstract] [Full Text] [PDF]
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 J. Xu, M. A. Kirigiti, M. A. Cowley, K. L. Grove, and M. S. Smith Suppression of Basal Spontaneous Gonadotropin-Releasing Hormone Neuronal Activity during Lactation: Role of Inhibitory Effects of Neuropeptide Y Endocrinology, January 1, 2009; 150(1): 333 - 340. [Abstract] [Full Text] [PDF]
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 J. A. Gonzalez, F. Reimann, and D. Burdakov Dissociation between sensing and metabolism of glucose in sugar sensing neurones J. Physiol., January 1, 2009; 587(1): 41 - 48. [Abstract] [Full Text] [PDF]
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W. Huang, M. Acosta-Martinez, T. H. Horton, and J. E. Levine Fasting-induced suppression of LH secretion does not require activation of ATP-sensitive potassium channels Am J Physiol Endocrinol Metab, December 1, 2008; 295(6): E1439 - E1446. [Abstract] [Full Text] [PDF]
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 D. A. Sandoval, D. Bagnol, S. C. Woods, D. A. D'Alessio, and R. J. Seeley Arcuate Glucagon-Like Peptide 1 Receptors Regulate Glucose Homeostasis but Not Food Intake Diabetes, August 1, 2008; 57(8): 2046 - 2054. [Abstract] [Full Text] [PDF]
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 X. Ma, L. Zubcevic, and F. M. Ashcroft Glucose regulates the effects of leptin on hypothalamic POMC neurons PNAS, July 15, 2008; 105(28): 9811 - 9816. [Abstract] [Full Text] [PDF]
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W. Huang, M. Acosta-Martinez, and J. E. Levine Ovarian Steroids Stimulate Adenosine Triphosphate-Sensitive Potassium (KATP) Channel Subunit Gene Expression and Confer Responsiveness of the Gonadotropin-Releasing Hormone Pulse Generator to KATP Channel Modulation Endocrinology, May 1, 2008; 149(5): 2423 - 2432. [Abstract] [Full Text] [PDF]
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E. Rother, A. C. Konner, and J. C. Bruning Neurocircuits integrating hormone and nutrient signaling in control of glucose metabolism Am J Physiol Endocrinol Metab, May 1, 2008; 294(5): E810 - E816. [Abstract] [Full Text] [PDF]
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C. Zhang, T. A. Roepke, M. J. Kelly, and O. K. Ronnekleiv Kisspeptin Depolarizes Gonadotropin-Releasing Hormone Neurons through Activation of TRPC-Like Cationic Channels J. Neurosci., April 23, 2008; 28(17): 4423 - 4434. [Abstract] [Full Text] [PDF]
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C. Zhang, M. A. Bosch, J. E. Levine, O. K. Ronnekleiv, and M. J. Kelly Gonadotropin-Releasing Hormone Neurons Express KATP Channels That Are Regulated by Estrogen and Responsive to Glucose and Metabolic Inhibition J. Neurosci., September 19, 2007; 27(38): 10153 - 10164. [Abstract] [Full Text] [PDF]
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B. E. Levin Why some of us get fat and what we can do about it J. Physiol., September 1, 2007; 583(2): 425 - 430. [Abstract] [Full Text] [PDF]
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M. Acosta-Martinez and J. E. Levine Regulation of KATP channel subunit gene expression by hyperglycemia in the mediobasal hypothalamus of female rats Am J Physiol Endocrinol Metab, June 1, 2007; 292(6): E1801 - E1807. [Abstract] [Full Text] [PDF]
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X. Fioramonti, S. Contie, Z. Song, V. H. Routh, A. Lorsignol, and L. Penicaud Characterization of Glucosensing Neuron Subpopulations in the Arcuate Nucleus: Integration in Neuropeptide Y and Pro-Opio Melanocortin Networks? Diabetes, May 1, 2007; 56(5): 1219 - 1227. [Abstract] [Full Text] [PDF]
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P. D. Mountjoy and G. A. Rutter Glucose sensing by hypothalamic neurones and pancreatic islet cells: AMPle evidence for common mechanisms? Exp Physiol, March 1, 2007; 92(2): 311 - 319. [Abstract] [Full Text] [PDF]
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M. A. Smith, K. Hisadome, H. Al-Qassab, H. Heffron, D. J. Withers, and M. L. J. Ashford Melanocortins and agouti-related protein modulate the excitability of two arcuate nucleus neuron populations by alteration of resting potassium conductances J. Physiol., January 15, 2007; 578(2): 425 - 438. [Abstract] [Full Text] [PDF]
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 B. E Levin Metabolic imprinting: critical impact of the perinatal environment on the regulation of energy homeostasis Phil Trans R Soc B, July 29, 2006; 361(1471): 1107 - 1121. [Abstract] [Full Text] [PDF]
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 B. S. Muhlhausler, C. L. Adam, P. A. Findlay, J. A. Duffield, and I. C. McMillen Increased maternal nutrition alters development of the appetite-regulating network in the brain FASEB J, June 1, 2006; 20(8): 1257 - 1259. [Abstract] [Full Text] [PDF]
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L. Huo, H. J. Grill, and C. Bjorbaek Divergent Regulation of Proopiomelanocortin Neurons by Leptin in the Nucleus of the Solitary Tract and in the Arcuate Hypothalamic Nucleus Diabetes, March 1, 2006; 55(3): 567 - 573. [Abstract] [Full Text] [PDF]
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C. Fekete, P. S. Singru, E. Sanchez, S. Sarkar, M. A. Christoffolete, R. S. Riberio, W. M. Rand, C. H. Emerson, A. C. Bianco, and R. M. Lechan Differential Effects of Central Leptin, Insulin, or Glucose Administration during Fasting on the Hypothalamic-Pituitary-Thyroid Axis and Feeding-Related Neurons in the Arcuate Nucleus Endocrinology, January 1, 2006; 147(1): 520 - 529. [Abstract] [Full Text] [PDF]
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D. Burdakov, S. M Luckman, and A. Verkhratsky Glucose-sensing neurons of the hypothalamus Phil Trans R Soc B, December 29, 2005; 360(1464): 2227 - 2235. [Abstract] [Full Text] [PDF]
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C. Acuna-Goycolea and A. N. van den Pol Peptide YY3-36 Inhibits Both Anorexigenic Proopiomelanocortin and Orexigenic Neuropeptide Y Neurons: Implications for Hypothalamic Regulation of Energy Homeostasis J. Neurosci., November 9, 2005; 25(45): 10510 - 10519. [Abstract] [Full Text] [PDF]
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S. L. Tang, V. Tran, and E. J. Wagner Sex Differences in the Cannabinoid Modulation of an A-Type K+ Current in Neurons of the Mammalian Hypothalamus J Neurophysiol, October 1, 2005; 94(4): 2983 - 2986. [Abstract] [Full Text] [PDF]
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B. S. Muhlhausler, C. L. Adam, E. M. Marrocco, P. A. Findlay, C. T. Roberts, J. R. McFarlane, K. G. Kauter, and I. C. McMillen Impact of glucose infusion on the structural and functional characteristics of adipose tissue and on hypothalamic gene expression for appetite regulatory neuropeptides in the sheep fetus during late gestation J. Physiol., May 15, 2005; 565(1): 185 - 195. [Abstract] [Full Text] [PDF]
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S. M. Appleyard, T. W. Bailey, M. W. Doyle, Y.-H. Jin, J. L. Smart, M. J. Low, and M. C. Andresen Proopiomelanocortin Neurons in Nucleus Tractus Solitarius Are Activated by Visceral Afferents: Regulation by Cholecystokinin and Opioids J. Neurosci., April 6, 2005; 25(14): 3578 - 3585. [Abstract] [Full Text] [PDF]
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X. Fioramonti, A. Lorsignol, A. Taupignon, and L. Penicaud A New ATP-Sensitive K+ Channel-Independent Mechanism Is Involved in Glucose-Excited Neurons of Mouse Arcuate Nucleus Diabetes, November 1, 2004; 53(11): 2767 - 2775. [Abstract] [Full Text] [PDF]
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B. E. Levin, V. H. Routh, L. Kang, N. M. Sanders, and A. A. Dunn-Meynell Neuronal Glucosensing: What Do We Know After 50 Years? Diabetes, October 1, 2004; 53(10): 2521 - 2528. [Abstract] [Full Text] [PDF]
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D. K. Sindelar, L. Ste. Marie, G. I. Miura, R. D. Palmiter, J. E. McMinn, G. J. Morton, and M. W. Schwartz Neuropeptide Y Is Required for Hyperphagic Feeding in Response to Neuroglucopenia Endocrinology, July 1, 2004; 145(7): 3363 - 3368. [Abstract] [Full Text] [PDF]
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A. Sahu Minireview: A Hypothalamic Role in Energy Balance with Special Emphasis on Leptin Endocrinology, June 1, 2004; 145(6): 2613 - 2620. [Abstract] [Full Text] [PDF]
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S. T. Hentges, M. Nishiyama, L. S. Overstreet, M. Stenzel-Poore, J. T. Williams, and M. J. Low GABA Release from Proopiomelanocortin Neurons J. Neurosci., February 18, 2004; 24(7): 1578 - 1583. [Abstract] [Full Text] [PDF]
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) and the steady-state (
) currents were measured.
