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Am J Physiol Regul Integr Comp Physiol. Jul 2010; 299(1): R129–R139.
Published online Feb 17, 2010. doi:  10.1152/ajpregu.00391.2009
PMCID: PMC2904143

Reduction in synaptic GABA release contributes to target-selective elevation of PVN neuronal activity in rats with myocardial infarction

Abstract

Neuronal activity in the paraventricular nucleus (PVN) is known to be elevated in rats with heart failure. However, the type of neurons involved and the underlying synaptic mechanisms remain unknown. Here we examined spontaneous firing activity and synaptic currents in presympathetic PVN neurons in rats with myocardial infarction (MI), using slice patch clamp combined with the retrograde labeling technique. In PVN neurons projecting to the rostral ventrolateral medulla (PVN-RVLM), MI induced a significant increase in basal firing rate (1.79 to 3.02 Hz, P < 0.05) and a reduction in the frequency of spontaneous (P < 0.05) and miniature (P < 0.01) inhibitory postsynaptic currents (IPSCs). In addition, MI induced an increase in the paired-pulse ratio of evoked IPSCs (P < 0.05). Bicuculline, a GABAA receptor antagonist, increased the firing rate of PVN-RVLM neurons in sham-operated (1.21 to 2.74 Hz, P < 0.05) but not MI (P > 0.05) rats. In contrast, in PVN neurons projecting to the intermediolateral horn of the spinal cord (PVN-IML), MI did not induce any significant changes in the basal firing rate and the properties of spontaneous and miniature IPSCs. The properties of spontaneous excitatory postsynaptic currents (EPSCs) were not altered in either neuron group. In conclusion, our results indicate that MI induces an elevation of firing activity in PVN-RVLM but not in PVN-IML neurons and that the elevated firing rate is largely due to a decrease in GABA release. These results provide evidence for a novel target-selective synaptic plasticity in the PVN that is associated with the sympathetic hyperactivity commonly seen in heart failure.

Keywords: heart failure, sympathetic hyperactivity, inhibitory postsynaptic current, rostral ventrolateral medulla, intermediolateral horn of spinal cord

elevated sympathetic outflow is commonly observed in congestive heart failure [HF (21)] and other diseases such as hypertension (2), metabolic syndrome (38), and chronic renal failure (8). In patients with HF, sympathetic nerve activity to muscle as well as plasma norepinephrine levels are increased (25). Moreover, the sensitivity to baroreflex and cardiopulmonary reflex inhibition of sympathetic tone is reduced in these patients (37). In a rat model of HF, renal sympathetic nerve activity is elevated, whereas sympathoinhibition in response to acute volume expansion and baroreflex activation is blunted, compared with control rats (see Ref. 33 for review).

The paraventricular nucleus of the hypothalamus (PVN) plays a key role in mediating sympathetic overactivity in HF (44). Presympathetic PVN neurons located in the dorsal and ventral divisions project to the rostral ventrolateral medulla (RVLM; PVN-RVLM neurons) or to the sympathetic preganglionic cells in the intermediolateral cell column of the spinal cord (IML; PVN-IML neurons) (48). Previous reports showed that PVN-IML neurons are involved in regulation of blood pressure induced by baroreceptor stimulation (13, 35, 36). Excitatory and inhibitory inputs to PVN-RVLM neurons increase and decrease blood pressure, respectively, by directly acting on RVLM vasomotor neurons that project to the spinal cord (60).

Previous reports showed that the sympathoexcitation observed in HF rats is associated with changes of neuronal activity in specific areas of the brain, including presympathetic areas of the PVN (45). For example, neuronal activity based on hexokinase activity (46) and expression of Fos family proteins such as Fos-B, Fra-1, and Fra-2 (53) is elevated in both parvocellular and magnocellular divisions of the PVN. The firing rate of spontaneous action potentials is significantly elevated in the PVN neurons of HF rats (66).

The elevation of neuronal activity in the PVN may result from facilitation of excitatory synaptic inputs and/or suppression of inhibitory synaptic inputs to presympathetic PVN neurons in HF rats. Li et al. (32) reported that the expression of a glutamate receptor subtype [N-methyl-d-aspartate (NMDA) NR1] is significantly elevated in the PVN of HF rats. In addition, it is known that excitatory influences mediated by angiotensin are also enhanced in HF (54, 67). On the other hand, depression of inhibitory mechanisms mediated by γ-aminobutyric acid (GABA) and nitric oxide (NO) within the PVN (34, 67) is also found to contribute to increased sympathetic activity in HF. Despite this evidence, little is known about the type of PVN neurons and the synaptic mechanisms underlying such alterations in neuronal transmissions within the PVN of HF rats.

In this study, we investigated the changes in basal firing rate, and the underlying synaptic mechanisms, in presympathetic PVN neurons projecting to the RVLM and IML in rats with myocardial infarction (MI). To this end, we used patch-clamp electrophysiology in brain slices in combination with a retrograde labeling technique. A part of this study has been communicated previously (24).

MATERIALS AND METHODS

Animals.

These studies were performed in accordance with the American Physiological Society's “Guiding Principles for Research Involving Animals and Human Beings” and the guidelines of the Laboratory Animal Care Advisory Committee of Seoul National University. Male Sprague-Dawley rats weighing 180–200 g (6–7 wk old; Orient Bio, Kyunggi-do, Korea) were housed in a temperature-controlled (24–26°C) and light-controlled (12:12-h light-dark cycle) room. Water and commercial rat chow were provided ad libitum. The rats were allowed to acclimatize for 1 wk before cardiac surgery.

General experimental protocol.

All rats were subjected to two surgical operations for coronary artery ligation and retrograde labeling of PVN neurons projecting to the RVLM or IML. The cardiac surgery was performed immediately after the acclimatization period. In the seventh or eighth week after the cardiac surgery, retrograde dye was injected into the RVLM or IML to label the PVN neurons projecting to the RVLM or IML. In the eighth to tenth weeks after the cardiac surgery (1–2 wk after the dye injection surgery), brain slices were prepared to record the electrophysiological activity of the labeled neurons. Previous studies have shown that the neurohumoral changes seen in heart failure are fully developed by this period (Refs. 6, 19, 20; Fig. 1). At the time of brain slice preparation, a heart sample was taken from each rat for measurement of the infarct size.

Fig. 1.
Gross and histomorphology of hearts from myocardial infarction (MI) and sham-operated (Sham) rats. Histological examination of the heart samples from Sham (A and B) and MI (C and D) rats stained with hematoxylin-eosin is shown. A and C: hearts of Sham ...

Induction of myocardial infarction and measurement of infarct size.

Rats were randomly assigned to either the sham-operated control (Sham) group or the MI group. MI was induced by ligation of the left coronary artery according to methods previously reported (1, 46, 53). Under general anesthesia induced with an anesthetic cocktail (75 mg/kg ketamine and 10 mg/kg xylazine), a 16-gauge catheter was inserted into the trachea through the oral cavity and connected to a mechanical ventilator (Harvard Apparatus, Holliston, MA). After left thoracotomy at the third intercostal space, the heart was exposed and the pericardium was carefully incised. The anterior descending coronary artery was then ligated with 6-0 sterile silk suture at the level of tip of the auricle. The heart was returned to its original position, and the chest incision was closed. Before the final stitch, the thoracic cavity was gently pressed to evacuate the air inside through an opening made by placing forceps, and the forceps were removed. The Sham rats underwent the same surgery except for the coronary artery ligation. When the rats were able to restart spontaneous breathing, the tracheal tube was removed and the rats were returned to their cages. About one-third of the rats (28.4%) died within 24 h after the coronary ligation.

For the measurement of infarct size, the heart from each MI rat was isolated, stored in 10% neutral buffered formalin solution, and subjected to measurement of the infarct size according to Ahn et al. (1). The left ventricular part of the heart was cut transversely into three or four sections (thickness = 2 mm). The infarct size of each section was measured as a ratio of the infarcted area to the mean left ventricular circumference with Image J software (developed at the National Institutes of Health). Rats with infarct sizes smaller than 30% (which accounted for ~12% of the surviving MI rats) were excluded from these studies.

Retrograde tracing.

The retrograde dye FluoSphere-Red (F-8793, red fluorescent, Molecular Probes, Eugene, OR) was injected into the RVLM or the IML as reported previously (51). Under anesthesia, the rats’ skulls were fixed in stereotaxic frames (SF-7, Narishige, Tokyo, Japan). Injection points were determined with a rat stereotaxic atlas (47). After exposure of the surface of the skull by incision of the skin of the head, a small hole was made with a dental drill. The injection point was 2.1 mm lateral to the midline, −13.0 mm from the bregma, and 8.0 mm below the dorsal surface. FluoSphere-Red solution (Molecular Probes) was injected into the area of the RVLM. For injection into the IML, the second thoracic vertebra was exposed by incision of the skin and the muscle. After removal of the upper segment of the spinal column with a bone cutter, the dura matter was incised and then the tip of the glass capillary that was filled with FluoSphere-Red solution was lowered into the region of IML from the surface of the spinal cord. The injection point was 0.5 mm lateral to the midline and 0.75 mm below the dorsal surface. In both tracings, 100 nl of dye solution was injected unilaterally with a pneumatic picopump (PV820-G, World Precision Instruments, Sarasota, FL). Typical examples of injections in the RVLM and IML are shown in Fig. 2, C and F, respectively. Results from the rats showing a misplaced injection site (2 of 18 rats with IML injections and 16 of 59 rats with RVLM injections) were excluded from the analysis.

Fig. 2.
Comparison of the firing activity of 2 groups of presympathetic paraventricular nucleus (PVN) neurons from Sham and MI rats. A and D: representative traces (top) and time course histograms (bottom) of the spontaneous action potentials from PVN-rostral ...

Hypothalamic slice preparation.

Hypothalamic brain slices were prepared according to methods previously described (22). To obtain healthy neurons in brain slices from adult rats (400–450 g), the brain was isolated after being transcardially perfused with ice-cold modified artificial cerebrospinal fluid (aCSF) with a composition of (in mM) 210 sucrose, 26 NaHCO3, 5 KCl, 1.2 NaH2PO4, 1.2 CaCl2, 2.4 MgCl2, and 10 glucose for 1.5 min at a rate of 30 ml/min. The temperature of this aCSF was kept at −2 to +2°C throughout the sectioning period. The isolated brain was immersed in oxygenated (95% O2-5% CO2) ice-cold aCSF. Two or three coronal hypothalamic slices (300 μm) were cut just caudally to the optic chiasm with a vibrating tissue slicer (Vibratome 1000 plus, Vibratome, St. Louis, MO). These slices were incubated in oxygenated aCSF for at least 1 h at 32°C until recordings were made at 30–33°C. The composition of the aCSF was (in mM) 126 NaCl, 26 NaHCO3, 5 KCl, 1.2 NaH2PO4, 2.4 CaCl2, 1.2 MgCl2, and 10 glucose. The osmolality and pH of both the normal and modified aCSF were 311 mosmol/kgH2O and 7.4, respectively.

Electrophysiological recording.

A slice was transferred to a recording chamber (0.7 ml) and fixed with a grid of nylon stocking threads supported by an O-shaped silver wire weight while being perfused (4 ml/min) with oxygenated aCSF at 30–33°C. Labeled PVN neurons were selected for recording under an upright fluorescence microscope with a “green” filter cube (WG, Olympus) and visualized by differential interference contrast video microscopy. The patch electrode was located on a target neuron under bright light with the aid of a three-dimensional hydraulic micromanipulator (Narishige, Japan). Pipettes were pulled from borosilicate glass capillaries of 1.7-mm diameter and 0.5-mm wall thickness. The open resistance ranged from 2 to 5 MΩ and the seal resistance from 1 to 5 GΩ. Patch pipettes were filled with one of two solutions, K-gluconate-rich or KCl-rich solution. The K-gluconate-rich solution contained (in mM) 135 K-gluconate, 5 KCl, 20 HEPES, 0.5 CaCl2, 5 EGTA, and 5 ATP-Mg, whereas the KCl-rich solution contained (in mM) 140 KCl, 20 HEPES, 0.5 CaCl2, 5 EGTA, and 5 ATP-Mg. pH and osmolality were adjusted with KOH to 7.2 and 312–313 mosmol/kgH2O, respectively. Electrical signals were recorded with an Axopatch 200B (Axon Instruments, Foster City, CA). The signals were filtered at 1 kHz and digitized at 10 kHz by using an analog-digital converter (Digidata 1200B) and pCLAMP software (version 8.0, Axon Instruments). Resting membrane potentials were corrected for the liquid junction potential (~14.4 mV for K-gluconate-rich solution or ~4.8 mV for KCl-rich solution). The membrane input resistance was calculated by dividing the potential changes (mV) evoked by applied hyperpolarizing current pulses (−60 to −180 pA). Neurons showing a decrease in input resistance (>15%) during recordings (~28% of the total neurons studied) were excluded from the analysis.

We recorded the spontaneous firing activity and the effect of drugs with the use of pipettes filled with K-gluconate-rich solution in the cell-attached voltage-clamp mode. The frequency and coefficient of variation (CV) of firing activity were measured from recording periods lasting 2–3 min, and the effects of bath-applied drugs were studied in 2- to 3-min recording periods before and after drug application. For simultaneous recording of spontaneous inhibitory postsynaptic currents (sIPSCs) and excitatory postsynaptic currents (sEPSCs), synaptic currents were recorded in the whole cell mode at holding potentials near resting level with the use of pipettes filled with a K-gluconate-rich solution (Fig. 3A; see also Fig. 7A). For better analysis of IPSCs, recordings were also obtained with pipettes filled with KCl-rich solution, in the presence of antagonists of glutamate receptors 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 20 μM) plus dl-2-amino-5-phosphonopentanoic acid (dl-AP5; 50 μM) and/or blocker of voltage-dependent Na+ channels tetrodotoxin (TTX; 1 μM) at a holding potential of −70 mV (Figs. 4 and and5;5; see also Fig. 7C). Evoked IPSCs (eIPSCs) were also recorded from labeled PVN neurons by electrical stimulation with a bipolar tungsten electrode (World Precision Instruments) that was placed on the dorsal regions 200–600 μm away from the PVN. Paired stimuli (0.2 ms, 1.0–4.0 mA, 0.2 Hz) were applied with an isolated pulse stimulator (model 2100, A-M Systems, Carlsborg, WA) at 100-ms interstimulus intervals. In each experiment, synaptic currents were evoked at a strength capable of inducing submaximal responses. The amplitude of the eIPSC was determined as an average from 20–40 steady consecutive sweeps. The paired-pulse ratio (PPR) was expressed as the amplitude ratio of the second synaptic response to the first synaptic response.

Fig. 3.
Spontaneous excitatory postsynaptic currents (sEPSCs) and inhibitory postsynaptic currents (sIPSCs) recorded from PVN-RVLM neurons in Sham and MI rats with K-gluconate-rich pipettes. A, top: blockade of downward current by antagonists of ionotropic glutamate ...
Fig. 4.
Comparison of sIPSCs and miniature IPSCs (mIPSCs) of PVN-RVLM neurons recorded with KCl-rich pipettes in Sham and MI rats. A: representative traces of IPSCs recorded in PVN-RVLM neurons from Sham (left) and MI (right) rats. sIPSCs were recorded in the ...
Fig. 5.
Paired-pulse ratio (PPR) of evoked IPSCs (eIPSCs) in PVN-RVLM neurons from Sham and MI rats. A: representative plot of peak amplitude of eIPSCs recorded in the PVN neurons from Sham (left) and MI (right) rats. B: typical current traces of eIPSCs by paired-pulse ...
Fig. 7.
Comparison of synaptic currents in PVN-IML neurons from Sham and MI rats. A: representative traces of spontaneous synaptic currents recorded with K-gluconate-rich pipettes in PVN-IML neurons from Sham (left) and MI (right) rats. The synaptic currents ...

Drugs were added to the perfusing aCSF solution at known concentrations, and the solution was completely washed out in <2 min. CNQX, dl-AP5, bicuculline (Bic), and TTX were purchased from Tocris Cookson (Bristol, UK). All drugs were dissolved directly in the aCSF except for CNQX, which was dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO was ≤0.05%.

Analysis of firing activity and IPSCs.

The time course histograms and average of firing activity were analyzed with the Mini Analysis Program (version 6.0, Synaptosoft, Leonia, NJ). Silent neurons, as well as those displaying a frequency of <0.1 Hz, were not included in the analysis of firing frequency (16 PVN-RVLM and 15 PVN-IML neurons). Neurons with a firing frequency that had changed >20% after application of Bic were considered to be responsive to Bic (29). To compare the effect of Bic on the presympathetic PVN neurons in Sham and MI rats, all the neurons tested were used in analysis including nonresponding neurons against Bic. CV of firing rate was calculated by dividing the standard deviation (SD) of the interspike intervals (ISIs) by the mean ISIs. The frequency, amplitude, and decay time constant of spontaneous and miniature synaptic currents were determined from 3- to 5-min segments of current records according to methods previously described with a Mini Analysis Program (22). The threshold for detection of synaptic current was normally set at ~10 pA. Decay time constants (10–90%) were obtained from the best-fit parameters with a double-exponential equation. For eIPSCs, the amplitude and decay time constant were measured from each synaptic current with pCLAMP (version 8.0, Axon Instruments).

Experimental data are expressed as means ± SE, and the number of neurons tested and analyzed is represented by n. Statistical significance of the data was determined by using independent or paired Student's t-test and ANOVA. Fisher's exact test was used for detection of a significant difference in the pattern of neuronal activity between Sham and MI rats. The level of significance was set at P < 0.05.

RESULTS

This study is based on a total of 235 presympathetic PVN neurons projecting either to the RVLM (PVN-RVLM, 131 neurons from 25 Sham and 18 MI rats) or to the intermediolateral horn of the spinal cord (PVN-IML, 104 neurons from 9 Sham and 7 MI rats) with the brain slice patch-clamp technique. In MI rats, the mean infarction size was 46.61 ± 1.95% (n = 25). These values are similar to, or larger than, those reported in previous studies in which functional heart failure was assessed by hemodynamic or echocardiographic measurements (46, 53, 63). No apparent abnormalities were observed in the hearts of Sham rats (Fig. 1). Table 1 summarizes the basic membrane properties of the two groups of presympathetic PVN neurons recorded with a K-gluconate-rich internal solution. None of these properties was significantly different between the Sham and MI rats, indicating that the basic membrane properties are not altered by MI (P > 0.05).

Table 1.
Membrane properties of presympathetic PVN neurons in Sham and MI rats

MI induced increase in firing rate in PVN-RVLM but not PVN-IML neurons.

Previously, Zhang et al. (66) reported that the firing rate of PVN neurons was higher in MI rats than in sham-treated rats. To further determine whether MI can induce such an increase in firing rate in the identified presympathetic PVN neurons, we analyzed the spontaneous firing activity of two groups of presympathetic PVN neurons (PVN-RVLM and PVN-IML neurons) labeled by retrograde tracing dye in the cell-attached voltage-clamp mode (Fig. 2, A and D). In the experiments on PVN-RVLM neurons (n = 73 from Sham and MI rats), 57 neurons (78.1%) showed spontaneous firing as shown in Fig. 2A while the remaining 16 (21.9%) were silent. The proportions of spontaneously active (75.7 vs. 80.6%, Sham vs. MI) and silent (24.3 vs. 19.4%, Sham vs. MI) neurons were not significantly changed by MI (P > 0.05, Fisher's exact test). Analysis of spontaneous firing activity revealed that the mean firing rate was significantly higher in MI than Sham rats [Sham 1.79 ± 0.30 Hz (n = 28) vs. MI 3.02 ± 0.44 Hz (n = 29); P < 0.05; Fig. 2B, left]. In addition, the CV of the firing rate was significantly lower in MI than Sham rats [Sham 0.76 ± 0.15 (n = 28) vs. MI 0.39 ± 0.07 (n = 29); P < 0.05; Fig. 2B, right]. As illustrated in Fig. 2C, the majority of labeled PVN-RVLM neurons were found in the dorsomedial cap and ventral regions of the PVN (at bregma − 1.8 mm level) and in posterior regions of the PVN (at bregma − 2.12 mm level). A typical example of the injection site in the area of RVLM is illustrated in the inset in Fig. 2C.

In the experiments on PVN-IML neurons (n = 74 from Sham and MI rats), 59 (79.7%) showed spontaneous firing whereas the remaining 15 (20.3%) were silent (Fig. 2D). MI did not alter the proportions of spontaneously active (79.0% vs. 80.6%, Sham vs. MI) and silent (21.0% vs. 19.4%, Sham vs. MI; P > 0.05 by Fisher's exact test) neurons. In contrast to PVN-RVLM neurons, MI did not induce any significant change in the firing rate [Sham 3.26 ± 0.28 Hz (n = 30) vs. MI 3.29 ± 0.38 Hz (n = 29); P > 0.05; Fig. 2E, left] and its CV [Sham 0.28 ± 0.04 (n = 30) vs. MI 0.37 ± 0.05 (n = 29); P > 0.05; Fig. 2E, right]. Surprisingly, the basal firing rate of PVN-IML neurons in Sham rats was higher than that of PVN-RVLM neurons (3.26 ± 0.28 vs. 1.79 ± 0.30 Hz; P < 0.001). Figure 2F shows a representative example of the distribution of labeled PVN-IML neurons, which was similar to that of PVN-RVLM. The inset of Fig. 2F illustrates a typical example of the injection site in the area of the IML of the spinal cord. Collectively, these results show that MI induced an increase in firing rate with a reduction in CV of firing rate in PVN-RVLM neurons but not in PVN-IML neurons.

MI induced reduction in frequency of spontaneous IPSCs in PVN-RVLM neurons.

The results shown in Fig. 2 indicate that MI induced an increase in the frequency of spontaneous firing activity in PVN-RVLM neurons. To determine whether altered synaptic mechanisms contribute to the elevated firing activity of PVN-RVLM neurons in MI rats, we examined spontaneous synaptic currents at the membrane potential measured immediately after establishing the whole cell mode (−55 to −65 mV), using recording pipettes filled with K-gluconate-rich solution. This allowed us to record both inward and outward synaptic currents simultaneously (Fig. 3Aa). The inward synaptic currents were blocked by antagonists of ionotropic glutamate receptors (50 μM AP5 + 20 μM CNQX; Fig. 3Ab), whereas the outward synaptic currents were blocked by an antagonist of GABAA receptor (20 μM Bic; Fig. 3Ac), indicating that the spontaneous inward and outward synaptic currents were EPSCs (sEPSCs) and IPSCs (sIPSCs), respectively. The current records shown in Fig. 3B illustrate that sIPSCs (upward events) occurred less frequently in the PVN-RVLM neurons from MI rats (n = 7) than in those from Sham rats (n = 7). The mean frequency of sIPSCs was significantly less in MI rats than in Sham rats (Sham 2.79 ± 0.66 Hz vs. MI 0.92 ± 0.24 Hz; P < 0.05; Fig. 3C). On the other hand, no significant differences in the mean amplitude (Sham 15.22 ± 1.51 pA vs. MI 15.50 ± 1.20 pA; P > 0.05; Fig. 3D) or in the two decay time constants of sIPSCs (τfast: Sham vs. MI, 5.69 ± 0.81 vs. 7.93 ± 0.91 ms; τslow: Sham vs. MI, 13.01 ± 3.23 vs. 13.84 ± 3.24 ms; P > 0.05, respectively; Fig. 3, E and F) were observed between the two groups. Moreover, in contrast to the changes in sIPSCs, there were no significant differences between Sham and MI groups in the frequency (Sham 3.01 ± 0.79 Hz vs. MI 2.82 ± 0.92 Hz; P > 0.05; Fig. 3C), amplitude (Sham 16.52 ± 1.23 pA vs. MI 15.66 ± 0.98 pA; P > 0.05; Fig. 3D), and decay time constants (τfast: Sham 2.60 ± 0.65 ms vs. MI 3.81 ± 0.40 ms; τslow: Sham 5.77 ± 1.11 ms vs. MI 7.45 ± 1.35 ms; P > 0.05, respectively; Fig. 3, E and F) of sEPSCs (downward events) recorded from seven neurons in each group.

To further confirm the reduction in sIPSC frequency observed in PVN-RVLM neurons in MI rats, we recorded sIPSCs with the use of pipettes filled with KCl-rich solution in the presence of blockers of sEPSCs (CNQX and AP5). This recording condition allows better analysis of sIPSCs because the amplitudes of IPSCs become larger (from ~15 to ~62 pA; Figs. 3 and and4)4) while blocking sEPSCs. Figure 4A, top, illustrates typical examples of such current records of sIPSCs from the neurons in Sham (n = 24) and MI (n = 23) rats. As summarized in Fig. 4, there was a significant reduction in the mean sIPSC frequency in MI rats (Sham vs. MI, 4.05 ± 0.47 vs. 2.25 ± 0.22 Hz; P < 0.01; Fig. 4B) but not in the amplitude (Sham 62.73 ± 5.58 pA vs. MI 60.4 ± 6.98 pA; P > 0.05; Fig. 4C) or in the two decay time constants of sIPSCs (τfast: Sham 5.57 ± 0.67 ms vs. MI 5.28 ± 0.62 ms; τslow,: Sham 19.22 ± 2.72 ms vs. MI 19.33 ± 3.33 ms; P > 0.05, respectively; Fig. 4, D and E).

MI-induced reduction in IPSC frequency in PVN-RVLM neurons is independent of action potential.

The reduction in IPSC frequency could be due to a reduction in the firing activity of presynaptic GABAergic neurons. To determine whether the reduction in IPSC frequency is dependent on the firing activity of GABAergic neurons, we compared the properties of miniature IPSCs (mIPSCs) in Sham (n = 12) and MI (n = 13) rats in the presence of TTX, a blocker of voltage-dependent Na+ channel (Fig. 4A, bottom). The MI-induced changes in mIPSCs were in general not different from those observed in sIPSCs. A significant reduction was seen in the mean frequency (Sham 4.69 ± 0.73 Hz vs. MI 2.28 ± 0.46 Hz; P < 0.01; Fig. 4B) but not in the amplitude (Sham 62.40 ± 10.72 pA vs. MI 62.05 ± 9.05 pA; P > 0.05; Fig. 4C) or in the decay time constants of mIPSCs (τfast: Sham 5.37 ± 0.58 ms vs. MI 5.35 ± 0.54 ms; τslow: Sham 20.45 ± 1.84 ms vs. MI 19.43 ± 1.70 ms; P > 0.05, respectively; Fig. 4, D and E). The results also indicate that the firing activity of presynaptic GABAergic neurons is negligible under our experimental conditions, because the frequencies of sIPSCs and mIPSCs were not significantly different in the Sham or MI groups (P > 0.05 in both Sham and MI rats).

MI induced increase in paired-pulse ratio of evoked IPSCs in PVN-RVLM neurons.

A decrease in the frequency of IPSCs could be due to a reduction in GABA release probability or, alternatively, a reduction in the number of GABAergic synaptic contacts (56, 57). To further elucidate the possible mechanisms contributing to the reduction in IPSC frequency observed in MI rats, we analyzed the IPSCs evoked by paired-pulse stimulation. The PPR in PVN-RVLM neurons from Sham rats was slightly larger than 1, indicating a weak paired-pulse facilitation (Fig. 5, A, left, and B). As shown in Fig. 5A, right, and B, the PPR was significantly increased in neurons from MI [Sham 1.06 ± 0.07 (n = 4), MI 1.72 ± 0.21 (n = 5); P < 0.05; Fig. 5C]. The amplitude of the first eIPSC in MI rats appeared smaller than that in Sham rats, but the difference was not statistically significant [Sham 212.22 ± 51.34 pA (n = 4) vs. MI 142.07 ± 38.85 pA (n = 5); P > 0.05]. In addition, the failure rate (0.09 ± 0.09 vs. 0.16 ± 0.07, Sham and MI, respectively; P > 0.05) and the decay time constants of eIPSCs (data not shown) were not significantly different in the PVN-RVLM neurons from Sham and MI rats.

Effect of bicuculline on firing activity was altered by MI in PVN-RVLM neurons.

Our results (Figs. 335) suggest that GABA release was reduced in PVN-RVLM neurons of MI rats. To further test whether the reduced GABA release contributed to the elevated neuronal activity observed in PVN-RVLM neurons in MI rats (Fig. 2), we examined the influence of endogenous GABA on PVN-RVLM neuronal activity in Sham and MI rats. As shown in Fig. 6, the GABAA receptor antagonist (20 μM Bic) increased the firing rate in 8 of 10 PVN-RVLM neurons tested in Sham rats (Fig. 6A), and the summarized data show that Bic significantly increased the firing rate [from 1.22 ± 0.47 to 2.74 ± 0.52 Hz (n = 10); P < 0.05; Fig. 6C]. However, in MI rats, Bic did not induce a remarkable increase in the firing rate in any of the neurons tested (n = 8; Fig. 6B), and the Bic-induced changes were not significant [from 3.29 ± 0.72 to 3.44 ± 0.74 Hz (n = 8); P > 0.05; Fig. 6C]. In addition, Bic significantly reduced the CV of firing rate in Sham rats (from 0.83 ± 0.14 to 0.40 ± 0.05; P < 0.05) but not in MI rats (from 0.28 ± 0.01 to 0.27 ± 0.02; P > 0.05; Fig. 6D).

Fig. 6.
Effect of Bic on firing activity of PVN-RVLM neurons from Sham and MI rats. A and B: time course histograms (left) and representative traces (right) of firing activity of PVN-RVLM neurons from Sham (A) and MI rats (B) before (a) and after (b) application ...

MI did not induce reduction in frequency of sIPSCs and mIPSCs in PVN-IML neurons.

In contrast to the PVN-RVLM neurons, there was no significant alteration in the firing activity of PVN-IML neurons as shown in Fig. 2E. If the reduction in IPSC frequency were a major cause of the elevated firing rate observed in PVN-RVLM neurons, then a simple expectation would be that MI does not induce a significant change in IPSC frequency in PVN-IML neurons. To determine whether the synaptic currents of PVN-IML neurons were altered by MI, we examined sEPSCs and sIPSCs (see Fig. 3A for details). As illustrated in Fig. 7, there was no remarkable difference in the frequency of sIPSCs between PVN-IML neurons from Sham (n = 7) and MI (n = 6) rats. The summarized data indicate that MI did not alter the mean frequency of sIPSCs in PVN-IML neurons (Sham 0.92 ± 0.28 Hz vs. MI 0.78 ± 0.15 Hz; P > 0.05; Fig. 7B). In addition, we did not observe any significant differences in the mean amplitude (Sham 15.35 ± 1.64 pA vs. MI 16.21 ± 1.49 pA; P > 0.05; Fig. 7B) and in the two decay time constants of sIPSCs between the two groups (τfast: Sham vs. MI, 5.16 ± 0.67 vs. 4.87 ± 0.40 ms; τslow: Sham vs. MI, 12.74 ± 1.24 vs. 11.26 ± 1.98 ms; P > 0.05, respectively; Fig. 7B). Furthermore, the properties of sEPSCs were not significantly different between Sham and MI rats for frequency (Sham 1.80 ± 0.36 Hz vs. MI 1.88 ± 0.29 Hz; P > 0.05, respectively; Fig. 7B), amplitude (Sham 17.01 ± 1.68 pA vs. MI 19.27 ± 2.08 pA; P > 0.05, respectively; Fig. 7B), and decay time constants (τfast: Sham 3.17 ± 0.47 ms vs. MI 2.85 ± 0.23 ms; τslow: Sham 5.90 ± 1.07 ms vs. MI 6.05 ± 0.62 ms; P > 0.05, respectively; Fig. 7B). We also compared the properties of mIPSCs of PVN-IML neurons from Sham (n = 10) and MI (n = 9) rats. Similar to those observed in sIPSCs, there were no significant differences between Sham and MI rats in the mean frequency (Sham 1.92 ± 0.31 Hz vs. MI 1.99 ± 0.53 Hz; P > 0.05; Fig. 7D), amplitude (Sham 53.89 ± 8.46 pA vs. MI 56.23 ± 10.62 pA; P > 0.05; Fig. 7D), and decay time constants of mIPSCs (τfast: Sham 4.74 ± 0.46 ms vs. MI 4.25 ± 0.39 ms; τslow: Sham 16.91 ± 1.3 ms vs. MI 17.72 ± 2.69 ms; P > 0.05, respectively; Fig. 7D). We also found that the basal mIPSC frequency of PVN-IML neurons was lower than that of PVN-RVLM neurons (1.92 ± 0.31 vs. 4.69 ± 0.73 Hz in Sham rats; P < 0.01). Collectively, the results indicate that MI did not alter the IPSC frequency in PVN-IML neurons, which is consistent with the results shown in Fig. 3.

Effect of bicuculline on firing rate was not altered by MI in PVN-IML neurons.

Our results (Fig. 7) indicate that MI does not alter the frequency of IPSCs in PVN-IML neurons. In this situation, one can expect that the effect of GABAA receptor blockade should not be different in PVN-IML neurons between Sham and MI rats. In the majority of neurons tested, Bic did not change the frequency of spontaneous firing rate [8 of 10 neurons in Sham rats (Fig. 8A, left); 6 of 8 neurons in MI rats (Fig. 8B, left)]. However, Bic increased the firing rate in two cells from each of the Sham and MI rats (Fig. 8, A and B, right). As summarized in Fig. 8C, the overall effects of Bic (20 μM) on the firing rate were not significantly different between the two groups [from 3.37 ± 0.45 to 3.48 ± 0.41 Hz (n = 10) (P > 0.05) in Sham rats; from 3.87 ± 0.34 to 3.75 ± 0.47 Hz (n = 8) (P > 0.05) in MI rats], and there were no significant differences in the effects of Bic on CV of the firing rate [from 0.23 ± 0.06 to 0.25 ± 0.07 in Sham rats (P > 0.05); from 0.21 ± 0.05 to 0.25 ± 0.05 in MI rats (P > 0.05); Fig. 8D]. In addition, we further compared the proportions of neurons whose firing rate was increased or not increased by Bic in two groups of presympathetic PVN neurons from Sham and MI rats (Fig. 8E). The proportions of Bic-responsive and nonresponsive neurons differed significantly in PVN-RVLM neurons between Sham and MI rats (P < 0.001 by Fisher's exact test) but did not differ in PVN-IML neurons between Sham and MI rats (P > 0.05 by Fisher's exact test; Fig. 8E). Together, the results of this experiment show that GABAA receptor blockade did not increase the firing activity in PVN-IML neurons in either Sham or MI rats, which supports earlier observations that MI does not alter synaptic GABA release in PVN-IML neurons.

Fig. 8.
Effect of Bic on firing rate of PVN-IML neurons from Sham and MI rats. A and B: time course histograms of firing activity of PVN-IML neurons from Sham (A) and MI (B) rats before, during, and after application of 20 μM Bic (bin size = 10 s). Left ...

DISCUSSION

In the present study, we demonstrate that MI induces an increase in the frequency of spontaneous action potentials in PVN-RVLM presympathetic neurons, and that the increase is largely due to a reduction in basal GABAergic synaptic activity. However, such an increase did not occur in PVN-IML presympathetic neurons. This study provides evidence for a novel pathway-specific synaptic mechanism underlying the elevated firing activity previously reported in the PVN neurons of rats with HF.

Our present in vitro findings, by identifying the type of presympathetic neurons activated and the synaptic mechanism, further extend previous reports that have shown an elevated neuronal activity in unspecified PVN neurons by using long-term activity markers such as hexokinase (46) and Fra-like immunoreactivity (53) as well as by recording firing activity of unspecified PVN neurons in vivo from rats with MI (66). It is known that stimulation of PVN-RVLM neurons induces direct excitatory effects on cardiovascular RVLM neurons projecting to the spinal cord (59, 60). The excitation of these RVLM neurons, mediated by glutamate and vasopressin (52, 59), can increase the activity of the sympathetic preganglionic spinal neurons via receptors such as NMDA (7) and adrenergic receptors (11). Recently Chen and Toney (14) further demonstrated that the discharge of identified PVN-RVLM neurons is tightly associated with changes in cardiac rhythm, blood pressure, and renal sympathetic nerve activity in vivo. Collectively, our findings suggest that it is PVN-RVLM neurons that drive the sympathetic tone responsible for the sustained elevated level of renal sympathetic nerve activity in rats with MI (34).

In view of the findings that PVN-IML presympathetic neurons are also involved in regulating vasomotor components of sympathetic outflow (13, 35, 36) and that the activity of PVN-IML neurons is elevated in spontaneously hypertensive rats (30), it is rather unexpected that the excitability of PVN-IML presympathetic neurons is not altered in MI rats. However, our finding is in good agreement with other reports showing that spinally projecting PVN neurons are largely unaffected by baroreceptor signals (group II PVN-IML neurons; Refs. 13, 17). Some of the renal sympathetic nerve activity (~40%) does not contribute to blood pressure maintenance and is driven by a supraspinal autonomic center other than the RVLM (5). In line with this idea, it has been reported that PVN-IML neurons can be preferentially activated by inputs other than from baroreceptors, such as cold exposure (10) and fear (12). Together, our results suggest that PVN-IML neurons play a minor role in mediating the sympathetic overactivity seen in HF, and further study is needed to better understand the contribution of PVN-IML neurons in mediating vasomotor and nonvasomotor sympathetic activities in various normal and disease states.

In the present study, we found that two presympathetic PVN neurons are different at the basal level of GABAergic inputs, although the two groups are not different in their passive membrane properties. PVN-IML neurons receive two- to threefold less inhibitory synaptic inputs than PVN-RVLM neurons (0.92 vs. 2.79 Hz for sIPSCs; 1.92 vs. 4.69 Hz for mIPSCs), and the frequency of spontaneous firing activity of PVN-IML neurons was about twofold higher than that of PVN-RVLM neurons (3.26 vs. 1.79 Hz). These results indicate that the level of tonic GABAergic inhibition is much stronger in PVN-RVLM than PVN-IML neurons in Sham rats. The GABAergic projections into the PVN are known to originate from the subparaventricular zone, the anterior hypothalamic area, the dorsomedial hypothalamic nucleus, the medial preoptic area, the lateral hypothalamic area, and a series of subnuclei of the bed nucleus of the stria terminalis (see Ref. 16 for review). In contrast, virtually no GABAergic neurons were located in the PVN (4, 16, 49). Therefore, one could expect that the two presympathetic neuron groups are likely to receive GABAergic afferents originating from different areas, and hence the two descending sympathoexcitatory pathways convey different information dependent on the information mediated by respective GABAergic projection neurons.

Elevation of the firing activity of PVN-RVLM could be due to changes in excitatory or inhibitory synaptic transmission or, alternatively, a consequence of changes in intrinsic membrane excitability, as previously shown in hypertensive rats (29). The results indicate that the elevated firing activity in PVN-RVLM neurons in MI rats is largely due to a reduction in GABAergic inhibitory synaptic transmission. This is supported by several lines of evidence: 1) the frequencies of sIPSCs and mIPSCs in MI rats are reduced compared with Sham rats (Fig. 4); 2) blockade of GABAA receptors with Bic in Sham rats increases the firing rate of PVN neurons (Fig. 6); and 3) the frequency of EPSCs was not altered in MI rats (Fig. 3). Considering that GABA is the major inhibitory neurotransmitter that tonically regulates presympathetic neurons (31, 39, 42) and sympathetic tone in rats (29, 39, 63), our results are generally consistent with previous findings showing blunted PVN GABA function in the regulation of sympathetic renal nerve discharge in rats with MI (34, 63). In addition, other GABA-related mechanisms are also possible for the elevated firing rate observed in PVN-RVLM neurons. Park et al. (42, 43) recently showed that the firing activity of PVN neurons, including those projecting to the RVLM, can be increased by blockade of tonic GABA receptors, which are located at extrasynaptic sites (see Ref. 18 for review). The physiological conditions that inhibit or facilitate GABA release can increase (28) or decrease (31) the firing activity of PVN-RVLM neurons, respectively. Furthermore, the downregulation of transmembrane anion transporter KCC2 can also elevate the firing rate of PVN neurons (23). Whether, in addition to affecting synaptic GABA function, MI can also alter the tonic GABAergic inhibitory modality and/or the activity of transmembrane anion transporters in the presympathetic PVN neurons requires further investigation.

Unlike the changes in inhibitory synaptic transmission, the properties of glutamatergic EPSCs were not significantly altered in the PVN-RVLM neurons of MI rats. Given that the expression of NMDA receptors was found to be increased in HF rats with sympathetic hyperactivity (32), one could expect an increase in the decay time constant of EPSC because blockade of NMDA receptors induces more rapid decay of evoked EPSCs with little change in their peak amplitude (58). The lack of such changes in the EPSC kinetics in our study could have arisen from factors that can mask channel activation under our experimental conditions, including Mg2+ in the recording solution (9). Further studies are warranted to better explore the potential role of changes in excitatory synaptic transmission as a mechanism underlying the elevated neuronal activity in presympathetic PVN neurons of MI rats.

What could be the mechanism underlying the reduction of GABA release in PVN-RVLM neurons in MI rats? Two extensively studied neurotransmitter systems known to modulate GABAergic transmission include NO and angiotensin (see Refs. 34 and 67 for review). NO increases the frequency of IPSCs in magnocellular neurons in the PVN (3) and supraoptic nucleus (41) and preautonomic PVN neurons projecting to the spinal cord (27) and medulla oblongata (31). NO-induced increase in GABAergic transmission effectively suppresses the firing activity of presympathetic PVN neurons in rat hypothalamic slices (27, 31). In vivo studies have also shown that the inhibitory effect of NO on sympathetic nerve discharge was blocked by a GABAA receptor antagonist (64) and that the number of neuronal nitric oxide synthase-positive neurons was reduced in the PVN of the HF rats showing sympathoexcitation (34, 65). On the basis of these studies, one may hypothesize that the release of GABA in the PVN is under a tonic regulation of the NO system, as supported both by in vitro (50) and in vivo studies (34, 64). In this scenario, the reduced NO synthesis in the PVN of MI rats could be one of the major mechanisms leading to the decrease in GABA IPSC frequency and increased firing activity of presympathetic PVN neurons. An alternative mechanism to be considered is a role of angiotensin II, a major neurotransmitter within the PVN known to reduce GABAergic inputs and to increase the firing activity of the presympathetic PVN neurons projecting to the spinal cord (26) and RVLM (28). Although angiotensin II levels in the CSF (55) and expression of AT1 receptors (62) were shown to be elevated in HF rats, the AT1 receptor antagonist losartan alone did not alter either IPSC frequency or the firing activity of PVN neurons in a rat brain slice preparation (26, 28) or basal sympathetic nerve activity when microinjected to the PVN (15). Therefore, it is less likely that the reduced IPSC frequency was due to an increased tonic action of angiotensin II in PVN preautonomic neurons in brain slices from MI rats. Collectively, given the well-established roles of NO in modulating GABA function, and its critical role in elevated sympathetic activity in HF (34, 65), a diminished NO tone seems to be the most likely mechanism underlying the reduced GABA release and elevated neuronal activity in the PVN of MI rats.

A few limitations must be considered in interpreting the results of this study. First, it is possible that some of the PVN-RVLM and/or PVN-IML neurons recorded in the present study belong to the third group of presympathetic PVN neurons projecting to both the RVLM and the IML of the spinal cord (48, 51). Under our experimental conditions, the proportions of the third presympathetic neurons in PVN-RVLM and/or PVN-IML neurons are unlikely to be significant because any contamination with the third presympathetic neurons will reduce the significance in differences between PVN-RVLM and PVN-IML neurons. Second, the firing rate of PVN-IML neurons in the slice preparation (3.26 Hz) is largely comparable to those of PVN-IML neurons (1.8–2.7 Hz; Ref. 13) and unidentified PVN neurons in whole animals (2.7 Hz; Ref. 66), whereas the proportion of spontaneously active neurons in PVN-IML neurons (79.0% in this study) is comparable to one report (68–78%; Ref. 13) but not to another report from whole animals (3% in vivo; Ref. 36). Therefore, one should bear in mind that the slice preparation is isolated from ongoing physiological influences as well as the effects of anesthetics present in the whole animal study.

In summary, this study demonstrates that MI induces an elevation of firing rate in PVN-RVLM but not PVN-IML presympathetic neurons and that the elevation of the firing rate is largely due to a disinhibition of GABAergic inhibitory synaptic inputs. The results provide a cellular mechanism for the elevated neuronal activity previously reported in HF rats (46, 53, 66) and also a synaptic mechanism for the blunted GABAergic transmission in the PVN of HF rats (63). Our findings indicate that the PVN-RVLM-IML but not the PVN-IML pathway plays a major role in regulating sympathetic outflow from the upper thoracic level (T2) and can be an effective target in elucidating the pathophysiology of sympathetic overactivity seen in HF.

Perspectives and Significances

The hypothalamic PVN has two groups of presympathetic neurons that project to sympathetic preganglionic neurons in the spinal cord: PVN-RVLM and PVN-IML. Neuronal excitability is elevated only in PVN-RVLM neurons in rats with MI, as shown in this study, but in both PVN-RVLM and PVN-IML neurons in spontaneously hypertensive rats (29, 30). Such target-specific and/or disease-specific plasticity in presympathetic neurons at the level of the PVN provides good experimental evidence to support the differential control of sympathetic outflow (40). In view of the presence of multiple descending pathways converging at the sympathetic preganglionic neurons in the spinal cord, it would be essential to understand the relative contribution of a given pathway underlying the sympathetic overactivity found in diseases such as heart failure, hypertension (2), chronic renal failure (8), and metabolic syndrome (38). This understanding would help to design a safer and more effective modality to counteract the sustained elevation of sympathetic tone in patients.

GRANTS

This work was supported by grants of Ministry of Science & Technology (2004-01466; P. D. Ryu), the Korean Health 21 R&D Project (02-PJ1-PG3-21302-0013; P. D. Ryu), Korea Science and Engineering Foundation (KOSEF) (R01-2008-000-20623-0, J. B. Park), and National Heart, Lung, and Blood Institute Grant R01-HL-090948 (J. E. Stern).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

ACKNOWLEDGMENTS

The authors thank Dr. Allan Herbison for critical reading of the manuscript.

Present address of K. Lee: Centre for Neuroendocrinology and Dept. of Physiology, School of Medical Sciences, University of Otago, Dunedin 9054, New Zealand.

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