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 GABA(A) 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.

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 and MI rats and their horizontal sections (right). Asterisk indicates the site of ligation in the heart of a MI rat, and arrowheads inside the left ventricle (LV) indicate papillary muscles. Note that a large portion of the LV wall was thinned by infarction in the MI rats. The infarction size in C was 37.8%. B and D: 100-fold magnified view of the boxed parts in A and C, right. Note the severe myocardial necrosis, fibrosis, and calcification in the heart from ligated rat (D) compared with the Sham control (B). RV, right ventricle; LA, left atrium. Scale bar: 5 mm.

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 ventrolateral medulla (RVLM; A) and PVN-intermediolateral horn (IML; D) neurons in Sham (left) and MI (right) rats in cell-attached voltage clamp mode (bin size = 5 s). B and E: summary bar graphs showing mean firing rate (left) and its coefficient of variation (CV) (right) of PVN-RVLM (B) and PVN-IML (E) neurons in Sham (n = 28 for PVN-RVLM; n = 30 for PVN-IML neurons) and MI (n = 29 for both neuron groups) rats. Mean firing rate and CV were obtained from data recorded for 3 min. C and F: fluorescence images of the hypothalamic PVN regions showing distribution of retrogradely labeled PVN-RVLM (C) and PVN-IML (F) neurons at −2.12 mm from bregma. Fluorescence images were superimposed onto the images of the same slice taken under bright light. Insets in C and F represent the injection sites of fluorescence dye located at the RVLM of the medulla and the IML of the spinal cord, respectively. Values are means ± SE. *P < 0.05 by Student's t-test. 3V, 3rd ventricle. Scale bars: 100 μm (C and F) and 1 mm (insets in C and F).

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 receptor [50 μM dl-2-amino-5-phosphonopentanoic acid (AP5) and 20 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX); b] and upward current by antagonist of GABA_A receptor [20 μM bicuculline (Bic); c]. Bottom: sEPSCs (downward) and sIPSCs (upward) on expended timescale before (a) and during application of CNQX + AP5 (b) and CNQX + AP5 and Bic (c) at holding potential of −64 mV. B: representative traces of spontaneous synaptic currents recorded in the PVN-RVLM neurons from Sham (left) and MI (right) rats. The synaptic currents were recorded in normal artificial cerebrospinal fluid (aCSF) and at resting membrane potential (Sham −61 mV and MI −60 mV). All traces are continuous within each set of records. C–F: summary bar graphs showing the mean frequency (C), amplitude (D), and decay time constants (E and F) of sIPSCs and sEPSCs in PVN-RVLM neurons from Sham (n = 7) and MI (n = 7) rats. Values are means ± SE. *P < 0.05 by Student's t-test. τ_fast and τ_slow, fast and slow decay time constants, respectively.

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 presence of 50 μM AP5 and 20 μM CNQX (top), and mIPSCs were recorded under the same recording conditions with addition of 1 μM tetrodotoxin (TTX; bottom). B–E: summary bar graphs showing the mean frequency (B), amplitude (C), and decay time constants (D and E) of sIPSCs and mIPSCs in PVN-RVLM neurons from Sham (n = 24 for sIPSCs; n = 12 for mIPSCs) and MI (n = 23 for sIPSCs, n = 13 for mIPSCs) rats. Values are means ± SE. **P < 0.01 by Student's t-test.

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 stimulation from the same neurons shown in A (left). Amplitude of eIPSCs was normalized to the peak of the 1st eIPSC derived from Sham rats (right). Traces are averages of 40 consecutive sweeps evoked every 100 ms. Stimulation artifacts were removed for clarity. C: summary bar graph showing the PPR in PVN-RVLM neurons from Sham (n = 4) and MI (n = 5) rats. Values are means ± SE. *P < 0.05 by Student's t-test.

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 of 20 μM Bic (bin size = 10 s). C and D: summary bar graphs showing the effect of Bic on the firing rate (C) and its CV (D) in PVN-RVLM neurons from Sham (n = 10) and MI (n = 8) rats. Note that Bic altered the firing rate and its CV in PVN-RVLM neurons from MI rats. Values are means ± SE. *P < 0.05 by Student's t-test for paired sample.

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 were recorded in normal aCSF and at resting membrane potential (Sham −62 mV and MI −61 mV; see Fig. 3 for other details). B: summary bar graphs showing the mean frequency, amplitude, and decay time constants of sIPSCs and sEPSCs in PVN neurons from Sham (n = 7) and MI (n = 6) rats. C: representative traces of mIPSCs recorded with KCl-rich pipettes in PVN-IML neurons from Sham (left) and MI (right) rats. The mIPSCs were recorded in the presence of 50 μM AP5 and 20 μM CNQX and 1 μM TTX at a holding potential of −70 mV. D: summary bar graphs showing the mean frequency, amplitude, and decay time constants of mIPSCs in PVN-IML neurons from Sham (n = 10) and MI (n = 9) rats. Values are means ± SE.

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 and right: representative examples showing time course histograms of firing rate from nonresponsive and responsive neurons to Bic, respectively. C and D: summary bar graphs showing the effect of Bic on the firing rate (C) and its CV (D) in PVN-IML neurons from Sham (n = 10) and MI (n = 8) rats. E: comparison of the proportions of neurons nonresponsive and responsive to Bic in 2 presympathetic neuron groups from Sham and MI rats. Values are means ± SE. ***P < 0.001 by Fisher's exact test.