The central pathways and mediators involved in sympathoexcitatory responses to circulating leptin are not well understood, although the arcuate-paraventricular nucleus (ARC-PVN) pathway likely plays a critical role. In urethane-anesthetized rats, ipsilateral intracarotid artery (ICA) injection of murine leptin (100 μg/kg) activated most PVN neurons tested. These responses were reduced by intracerebroventricular injection of the melanocortin subtype 3 and 4 receptor (MC3/4-R) antagonist SHU-9119 (0.6 nmol). The MC3/4-R agonist MTII (0.6 nmol icv) activated PVN neurons. Some PVN neurons that were excited by ICA leptin were inhibited by local application of neuropeptide Y (NPY, 2.5 ng). ICA leptin (100 μg/kg) excited presympathetic rostral ventrolateral medulla neurons and renal sympathetic nerve activity without significant change in blood pressure or heart rate; these effects were mimicked by intracerebroventricular injection of MTII (0.6 nmol). These data provide in vivo electrophysiological evidence to support the hypothesis that circulating leptin activates the sympathetic nervous system by stimulating the release of α-melanocyte-stimulating hormone in the vicinity of PVN neurons that are inhibited by the orexogenic peptide NPY.
- sympathetic nerve activity
- extracellular recording
- neuropeptide Y
- α-melanocyte stimulating hormone
leptin is a peptide hormone that is produced by adipose tissue and acts centrally to decrease appetite and increase energy expenditure by activating the sympathetic nervous system. The central pathways mediating the sympathetic responses to leptin are still poorly understood. The paraventricular nucleus (PVN) of the hypothalamus, which has an important regulatory influence over sympathetic nerve activity, is thought to be a primary effector site for leptin's central actions (11, 14, 33). Among the proposed neuropeptide mediators of leptin's central actions are neuropeptide Y (NPY), which acts within the PVN to inhibit sympathetic nerve activity (9, 17, 21), and α-melanocyte-stimulating hormone (α-MSH), which acts within the forebrain to excite sympathetic nerve activity (18, 22). These two peptides, which are released into PVN by neurons originating in the arcuate nucleus (ARC), are likely to modulate the sympathoexcitatory responses of PVN neurons to blood-borne leptin. In the present study, we tested the hypothesis that leptin exerts its excitatory influence on sympathetic activity via release of α-MSH onto PVN neurons and that NPY acts on PVN neurons to reduce sympathetic activity. We recorded the responses of neurons in the PVN or in the rostral ventrolateral medulla (RVLM), an important medullary center for sympathetic drive, along with renal sympathetic nerve activity (RSNA) and arterial pressure (AP), to intra-arterial murine leptin injections targeting the forebrain region or to intracerebroventricular administration of a melanocortin receptor agonist and antagonist. Preliminary results of these studies have been reported in abstract form (34, 35).
MATERIALS AND METHODS
These studies were performed in accordance with the “Guiding Principles for Research Involving Animals and Human Beings” of the American Physiological Society (1). The experimental procedures were approved by the University of Iowa Institutional Animal Care and Use Committee.
All experiments were performed on adult male Sprague-Dawley rats (300-350 g; Harlan Sprague Dawley). The animals were housed in the University Animal Care Facility and exposed to a normal 12:12-h light-dark cycle.
General preparation. Rats were anesthetized with urethane (1.5 g/kg ip), and supplemental doses of urethane (0.1-0.3 g/kg ip or iv) were given when obvious increases in blood pressure and respiratory rate were observed during surgery or experimental recording. The level of anesthesia was periodically reassessed during the surgical procedures and experimental recording by examining nociceptive reflex responses and by continuously monitoring blood pressure and heart rate (HR). The left femoral artery was cannulated with PE-50 tubing filled with heparinized saline (20 U/ml) for the recording of arterial pressure, which was monitored with a Hewlett-Packard 7754A chart recorder (HP Medical Products Group, Andover, MA). The left femoral vein was cannulated with PE-20 tubing for the administration of drugs. A rostrally directed PE-20 cannula was inserted into the left common carotid artery, with the tip placed in the bifurcation for intracarotid artery (ICA) injection as previously reported (36, 37). This injection predominantly targets the ipsilateral forebrain (19) and therefore provides a means of delivering drugs to that region by the natural blood-borne route.
Animals were intubated and breathed spontaneously. Core temperature was maintained at 37 ± 0.2°C with a rectal thermometer and a temperature controller (model K-100, Baxter Healthcare, Valencia, CA). The head was fixed in a stereotaxic frame (David Kopf Instrument, Tujunga, CA).
Intracerebroventricular injection. In some animals, a 29-gauge stainless steel cannula was implanted into the left lateral cerebral ventricle [stereotaxic coordinates: 0.9-1.0 mm posterior to bregma; 1.4-1.6 mm lateral to midline; 3.2-3.3 mm ventral to dura (26)]. The intracerebroventricular position of the cannula was confirmed by the staining of all four ventricles after injection of 5 μl Pontamine sky blue at the end of the experiments.
Electrophysiological recording. A small craniotomy was made above the region of interest, and a glass micropipette was placed in the left PVN or left RVLM to record extracellular single-unit activity, using previously described techniques (36, 37). In one group of experiments, PVN recording electrode was attached to a second glass pipette (tip diameter ∼50-100 μm) for regional microinjection of NPY into PVN. The tip of the microinjection pipette was placed approximately 0.2-0.4 mm proximal to the tip of the recording electrode. The microinjection pipette was connected to a Neurophore BH-2 (Medical Systems) pressure microinjection system. Volume of injection was measured at the time of delivery by observing the movement of a meniscus in the pipette through a horizontal microscope fitted with a calibrated eyepiece micrometer. Stereotaxic coordinates were: PVN, 1.6-2.1 mm posterior to bregma, 0.3-0.5 mm from midline, and 7.0-8.0 mm ventral to dura; RVLM, 11.8-12.8 mm posterior to bregma, 1.8-2.1 mm from midline, and 8.0-10.0 mm ventral to dura (26).
Recordings of RSNA were obtained from the left renal nerve using methods previously described from our lab (31, 37). In brief, the left kidney was exposed through a flank incision. A branch of the renal nerve was dissected free from surrounding tissue and placed on bipolar silver wire recording electrodes. When an optimal signal-to-noise ratio was established, the electrode and the renal nerve were covered with silicon sealant (World Precision Instruments, Sarasota, FL). The electrodes were sutured to the back muscles. Renal nerve activity was amplified (model P511, Grass Instrument; Quincy, MA) and displayed on an oscilloscope (TDS 3014, Tektronix, Beaverton, OR). The noise level was finally determined at the end of experiment after injection of hexamethonium (30 mg/kg iv). The net value of RSNA was calculated by subtracting the background noise from the actual recorded value during the experiment.
Experimental protocols. The recording session began at least 1 h after completion of the surgical preparation. PVN and RVLM neurons were tested initially for responses to blood pressure changes induced by an intravenous bolus of phenylephrine and nitroprusside, as previously described (36, 37). RVLM neurons were also tested for correlation with the peak arterial pressure pulse.
The first series of experiments was designed to determine the effects of leptin, and of the melanocortin receptors, as putative mediators of the leptin response, on PVN neurons. Murine leptin (100 μg/kg) was injected in the left ICA, and PVN neuronal activity and arterial pressure were recorded. In some of these neurons, the melanocortin antagonist SHU-9119 was administered intracerebroventricularly after the initial response to ICA leptin was observed. Other PVN neurons were tested for the effects of the melanocortin agonist MTII, administered intracerebroventricularly; some of these neurons were tested for the effect of intracerebroventricular administration of SHU-9119 after an initial response to MTII was observed. Some PVN neurons were also tested for their response to regional microinjection of NPY and to ICA leptin. In a second series of experiments, to determine the effect of leptin and MTII on downstream sympathetic output and cardiovascular variables, we recorded the responses of RVLM neuronal activity and simultaneously recorded RSNA, AP, and HR to ICA murine leptin and intracerebroventricular MTII.
The last unit recorded in each experiment was marked with iontophoresis of pontamine sky blue for subsequent determination of recording sites.
Data acquisition and analysis. The AP signal, the rectified and integrated voltage from the renal nerve recording, the transistor-transistor logic (TTL) pulses indicating multifiber action potentials in the raw RSNA exceeding a selected voltage, and the TTL pulses indicating PVN and RVLM single-unit activity were fed into an online data-acquisition system consisting of a Cambridge Electronics Design (CED, Cambridge, UK) 1401 Plus computer interface coupled with a Gateway Pentium personal computer. HR was derived from the peak AP tracing. Mean arterial pressure (MAP), HR, RSNA, and single-unit discharge were averaged over 1-min intervals. A 3-min baseline was used as control. Since the responses to local microinjection of NPY were of shorter duration, they were analyzed over a shorter time interval (30 s) using peak responses in PVN neuronal activity and MAP. Changes in RSNA were calculated as a percent change from the baseline activity. In the representative tracings, both integrated and windowed RSNA are shown, but only the integrated activity was used in the analysis of grouped data. A net percent change in PVN or RVLM neuronal discharge rate of ±30% or more was considered a significant effect of an experimental intervention as previously reported (36). Statistical significance among multiple comparisons was determined by one-way or two-way repeated-measures ANOVA followed by post hoc Fisher's least squares difference test. Student's t-test was employed for analysis of paired or unpaired data. Values are expressed as means ± SE. P < 0.05 was considered to indicate statistical significance.
Anatomy/histology. At the conclusion of each experiment, the rat was killed with an overdose of urethane. The brain was removed and fixed in a 10% formalin solution for at least 3 days and then sectioned (40 μm) on a cryostatic microtome (OM2563, Triangle Biomedical Sciences, Durham, NC). The sections were thaw-mounted on microscope slides and then stained with 1% aqueous neutral red. The last recording site marked with Pontamine sky blue was identified with a light microscope, and the locations of other recording sites were extrapolated with respect to this reference point. Recording sites were plotted on representative schematic tracings of the PVN and RVLM, based on the rat atlas of Paxinos and Watson (26).
Drugs. NPY, phenylephrine hydrochloride, SHU-9119, and sodium nitroprusside were purchased from Sigma (St. Louis, MO). Murine leptin was generously supplied by Amgen (Thousand Oaks, CA). MTII was purchased from Phoenix Pharmaceuticals (Belmont, CA). All drugs were dissolved in artificial cerebrospinal fluid (aCSF) for ICA or intracerebroventricular injection and in saline for intravenous injection. ICA injections were given in a volume of 10-20 μl flushed by 25 μl aCSF (pH 7.5). The volume for intracerebroventricular injection was 5-10 μl. The same volume of aCSF was administered via the ICA or intracerebroventricularly as a control.
Effects of ICA murine leptin on PVN neuronal activity. Twenty-seven of 29 PVN neurons responded to ipsilateral ICA injection of murine leptin (100 μg/kg) with an increase in activity of at least 30% of baseline as previously reported (36). The onset latency of the response was 3-10 min, and the duration of the response was at least the 90 min tested. One representative PVN neuron is illustrated in Fig. 1A, showing gradual increase of neuronal firing after ICA leptin administration. The mean firing rate of neurons increased from baseline of 2.2 ± 0.3 to a peak of 4.0 ± 0.5 spikes/s 20 min after leptin injection (Fig. 1C, n = 27, P < 0.01). Two PVN neurons showed no change (<30%) in neuronal activity after ICA leptin. Mean arterial pressure (MAP) did not change significantly (Fig. 1C, from 97 ± 2 to 98 ± 2 mmHg) 20 min after leptin injection. The effects of ICA leptin were reproducible on a second injection. The same volume of aCSF injected via the ICA had no effects on PVN neuronal activity and MAP (n = 5, data not shown).
Effects of the MC3/4-R antagonist SHU-9119 on PVN neuronal responses to ICA murine leptin. Because α-MSH acting on MC4-R is a putative mechanism for the leptin-induced sympathetic response, we tested the effect of SHU-9119 on the PVN neuronal response to ICA leptin. Intracerebroventricular SHU-9119 (0.6 nmol) significantly reduced ongoing PVN neuronal responses to leptin (from 3.9 ± 0.6 to 2.2 ± 0.3 spikes/s, n = 12, P < 0.01, Fig. 1, B and C). Intracerebroventricular SHU-9119 alone had no effect on PVN neuronal activity and MAP (n = 3).
Effects of MTII on leptin-responsive PVN neurons. The melanocortin agonist MTII (0.6 nmol icv) increased PVN neuronal firing rate from a baseline of 2.0 ± 0.3 to a peak of 3.8 ± 0.6 spikes/s (n = 11, P < 0.01) within 10 min of injection (Fig. 2C) in 11 of 12 neurons tested. A typical example of PVN neuron responding to MTII is shown in Fig. 2A. The effect lasted ∼30 min. Most of these neurons (7/9) also responded to ICA murine leptin. Blood pressure did not change after intracerebroventricular MTII injection (from 101 to 104 mmHg, n = 11). The MC3/4-R antagonist SHU-9119 (0.6 nmol icv) completely abolished the PVN neuronal response to MTII (from 3.8 ± 0.4 to 2.2 ± 0.3 spikes/s, n = 7, P < 0.01, Fig. 2, B and C). The effects of MTII were reproducible on a second intracerebroventricular injection.
Effects of local NPY on PVN neuronal activity. Local microinjection of NPY (2.5 ng in 50 nl) into the PVN from a pipette attached to the recording electrode decreased firing rate of 9 of 11 PVN neurons tested from 1.8 ± 0.3 to 0.9 ± 0.3 spikes/s (n = 9, P < 0.01) with only a slight fall in MAP (from 101 ± 4 to 98 ± 5 mmHg) as shown in Fig. 3. The duration of PVN neuronal responses to local NPY lasted only several minutes. Two PVN neurons did not respond to local microinjection of NPY. Six of the nine neurons inhibited by local NPY were excited by ICA murine leptin as shown in Fig. 3. The same volume of aCSF microinjections had no effects on PVN neuronal activity and MAP (n = 3).
Effects of ICA murine leptin and intracerebroventricular MTII on simultaneously recorded RVLM neuronal activity, RSNA, AP, and HR. The above experiments suggest that leptin activates hypothalamic PVN neurons by releasing melanocortins in the PVN region. To determine whether these influences affect downstream neurons in sympathetic efferent pathways, we simultaneously recorded from RVLM neurons and the renal sympathetic outflow during ipsilateral ICA administration of murine leptin or intracerebroventricular injection of MTII.
Ipsilateral ICA administration of leptin (100 μg/kg) gradually increased RVLM neuronal activity and simultaneously increased RSNA, with only slight changes in AP and HR (Fig. 4). The peak responses occurred ∼30 min after leptin injection. The RVLM neuronal firing rate increased from baseline of 10.3 ± 1.5 to a peak of 20.0 ± 2.2 spikes/s (P < 0.01, n = 12). Two RVLM neurons showed no changes in neuronal activity after leptin injection. Most of RVLM neurons tested had pulse-related firing pattern (Fig. 4B), suggesting that they were baroreceptor-sensitive presympathetic neurons. RSNA increased 25.4 ± 1.6% from baseline (P < 0.01, n = 12). The same volume of ICA injection of aCSF (n = 5, Fig. 4C) had no effects on RVLM firing rate, RSNA, AP, or HR.
Central administration of MTII (0.6 nmol icv, n = 7) activated RVLM neurons, increasing firing rate from a baseline of 13.6 ± 2.3 to a peak of 21.6 ± 2.0 spikes/s (P < 0.01) and increased RSNA integrated voltage by 31 ± 1.8% from baseline (P < 0.01) with an onset latency of ∼2-3 min and a duration of effect of ∼30 min (Fig. 5). There were no significant changes in AP and HR, although there is a trend toward elevation in MAP (Fig. 5B).
Recording sites within PVN and RVLM regions. Figure 6 shows the locations of the single-unit recording sites in the PVN (Fig. 6A) and RVLM (Fig. 6B). The PVN neurons tested in this study were distributed mainly in the medial regions close to the third ventricle. There was no obvious clustering of neurons related to responses to administered drugs. Neurons recorded in the RVLM mostly were barosensitive and pulse related, although the specific function of these neurons are not known. Within the RVLM, there was no obvious difference in anatomic distribution between neurons activated by ICA murine leptin or intracerebroventricular MTII.
The present study demonstrates that melanocortin receptors mediate the excitatory effects of circulating murine leptin on hypothalamic PVN neurons and that NPY has an opposing effect of inhibiting the activity of PVN neurons that are excited by blood-borne leptin. The study also demonstrates that acutely administered blood-borne leptin excites downstream brain stem presympathetic RVLM neurons and RSNA without inducing significant changes in blood pressure and HR and that these effects are mimicked by injecting the MC3/4-R agonist MTII into the cerebral ventricle. These results provide direct electrophysiological evidence in an in vivo model to support the hypothesis that blood-borne leptin activates the sympathetic nervous system by stimulating the release of α-MSH in the vicinity of PVN neurons that also respond to the suppressive effects of the orexogenic peptide NPY on sympathetic activity.
It is now generally agreed that leptin acts on the ARC to regulate the release of α-MSH and NPY into the PVN, the major hypothalamic effector site (3, 6) for leptin's actions. Leptin receptors have been identified on both NPY- and proopiomelanocortin (POMC)-expressing neurons in ARC (13, 28). Functional neuoranatomy studies using c-fos protein expression techniques have demonstrated that the PVN is the major hypothalamic region consistently activated by leptin regardless of dose, delivery route (intravenous or intracerebroventricular), and species (10-12). A recent electrophysiological study demonstrated that leptin increased the frequency of action potentials in POMC-expressing neurons through two mechanisms (7). Leptin causes a direct depolarization of POMC neurons through the activation of a nonspecific cation channel and simultaneously reduces the frequency of inhibitory postsynaptic current mediated by local NPY and GABAergic neurons. Thus leptin appears to have both pre- and postsynaptic actions that increase the neuronal activity of hypothalamic POMC neurons, which then increase endogenous melanocortin agonist release (e.g. α-MSH) within the PVN area. The critical role for the melanocortin system in leptin signaling emerged with the cloning of MC-4 receptor gene and the recognition that it is expressed primarily in the brain regions regulating autonomic functions, including the hypothalamic PVN (20, 25). In mice, intracerebroventricular administration of the synthetic MC3/4 receptor agonist MTII inhibits feeding, and this effect can be completely blocked by the melanocortin antagonist SHU-9119 (16). Furthermore, administration of SHU-9119 alone significantly enhanced feeding, indicating that central melanocortinergic neurons exert a tonic inhibition of feeding behavior (16).
The known connections of parvocellular PVN neurons with autonomic regions of the brain stem and spinal cord suggest that excitation of PVN neurons by leptin or melanocortin might result in increases in sympathetic nerve activity, AP, and HR. However, our data show that the intracerebroventricular melanocortin agonist MTII activated PVN or RVLM neurons and increased RSNA, but without significant changes in MAP and HR. This finding is consistent with that of Haynes et al. (18), who demonstrated that the acute administration of intracerebroventricular MTII increased sympathetic nerve traffic to brown adipose tissue and renal and lumbar beds without changing arterial pressure. In their study, SHU-9119 completely blocked leptin-induced renal but not brown adipose tissue sympathoexcitation, suggesting differential central pathways regulating leptin-induced sympathetic responses to different peripheral tissues. Microinjection of leptin into ventromedial hypothalamus (30) produced a similar result, with an increase in RSNA but no change in MAP. The explanation for this now well-recognized dissociation between MAP and RSNA in the response to acute administration of leptin is not known, but a possible explanation might be that the renal nerve subserves several physiological functions (8), all of which may not be linked to the immediate regulation of arterial pressure. In contrast, chronic intravenous or intracerebroventricular infusion of leptin can induce increases in blood pressure and heart rate in conscious rats (4, 5, 29).
Few prior studies have been done to test the central mechanism by which leptin excites SNA and the cardiovascular system. In our study, increased PVN neuronal activity induced by blood-borne murine leptin could be blocked by melanocortin receptor antagonist (SHU-9119), and RSNA could be stimulated by intracerebroventricular injection of MTII, strongly supporting the concept that the leptin signal driving renal sympathetic nerve activity is mediated by melanocortin receptors at the hypothalamic level. In this study, we did not measure sympathetic drive to brown adipose tissue, which has a primarily thermogenic/metabolic function. Leptin activation of that sympathetic pathway may utilize an alternate central pathway via raphe pallidus (23, 24).
The mechanism by which released NPY inhibits PVN neurons is not well known. In the present study, regional application of NPY within PVN significantly reduced the PVN neuronal activity, and more specifically inhibited the activity of PVN neurons that also responded to ICA leptin. A recent study in hypothalamus suggested that NPY induces the release of GABA, with GABA-mediated presynaptic inhibition (6). Inhibition might also be mediated postsynaptically via activation of local inhibitory interneurons or via a direct influence of NPY on the recorded neurons. These possibilities were not tested in this study. In leptin-deficient mice, the NPY levels in the hypothalamus are dramatically increased (32), while increasing leptin inhibits NPY gene expression (15). This reciprocal relationship between leptin and NPY further supports the role of NPY as an inhibitory mediator of leptin-related functions. Leptin and NPY have opposite effects on many other autonomic functions (2, 27). Interactions between leptin and NPY have been found in the control of RSNA and blood pressure (21), but in the present study this relationship was not directly examined.
In summary, we have demonstrated that the sympathoexcitatory response to blood-borne leptin, delivered acutely via the intracarotid route, is mediated by melanocortin receptors in the hypothalamic paraventricular region. We have also demonstrated that systemically administered murine leptin can excite PVN neurons that are inhibited by NPY. These results provide strong electrophysiological support for the general hypothesis that α-MSH and NPY interact in a reciprocal fashion within the PVN to modulate the sympathetic response to leptin.
This work was supported by NIH Program Project Grant PO1 HL-014388 (Principal Investigator, F. M. Abboud; Project Director, R. B. Felder).
Murine leptin was supplied through an agreement with Amgen.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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