Labile Calcium-Permeable AMPA Receptors Constitute New Glutamate Synapses Formed in Hypothalamic Neuroendocrine Cells during Salt Loading

Effect of salt loading on VP mRNA expression in SON. A, Image of a Nissl-stained coronal section of rat brain at the level of the hypothalamus showing the approximate locations of the 1-mm SON punches (dashed circles). B, The relative VP mRNA expression measured by qPCR in SON punches from control (control) and salt-loaded (NaCl) rats, normalized to control; *p < 0.05.

Effects of chronic salt loading on AMPA receptor subunit expression in the SON. A, qPCR of AMPA receptor subunit mRNA expression in SON punches from control and salt-loaded (NaCl) rats revealed no significant change in any of the AMPA receptor subunit mRNAs, GluA1-4, with salt loading. B1, Representative Western blots of GluA1 and GluA2 total protein expression in SON punches from four control rats (C1–C4) and five (GluA1: Na1–Na5) or four (GluA2: Na1–Na4) salt-loaded rats. B2, Mean optical density measurements of GluA1 and GluA2 protein bands from SON tissue from control (N = 8) and salt-loaded (NaCl, N = 7) rats revealed an increase in total GluA1 protein with salt loading. C, Surface and intracellular GluA1 and GluA2 protein levels in SON punches from control (N = 7, 8, respectively) and salt-loaded (NaCl, N = 5, 7, respectively) rats determined by Western blot following BS³ surface protein crosslinking. Inset, Representative Western blots of SON tissue from a control rat treated with the membrane impermeant crosslinking agent BS³ and probed with antisera to GluA2 and β-actin. Two GluA2 bands were detected: a high molecular weight band representing the crosslinked surface protein aggregate (S) and a band at the predicted molecular weight for GluA2 (∼100 kDa) that represents the unmodified intracellular pool (I). β-Actin served as an intracellular control and was found at its predicted molecular weight (∼40 kDa), confirming the lack of crosslinking of intracellular proteins. D, Surface-to-intracellular (S/I) protein ratios determined following BS³ surface protein crosslinking for GluA1-GluA4 AMPA receptor subunits in SON punches from control and salt-loaded (NaCl) rats. Despite the increase in total GluA1 protein, there was no change in the subunit S/I ratios between SON tissues from control and salt-loaded rats for any of the AMPA receptor subunits; *p < 0.05.

Increase in inwardly rectifying AMPA receptor signaling following salt loading. A, Scatter plots of individual neuronal means and group means of sEPSC frequencies and amplitudes. Identified VP neurons recorded in slices from salt-loaded VP-eGFP rats (NaCl) showed a significant increase in the mean sEPSC frequency and amplitude compared to the control group. B, The paired-pulse ratio of eEPSCs (inset) was unchanged following salt loading, suggesting that the increased glutamatergic synaptic activity was not due to a change in the probability of glutamate release. C, The eEPSC current−voltage relationship showed inward rectification at Vm > 0 mV in SON neurons from salt-loaded rats (red curve, n = 6) but not controls (black curve, n = 4). The mean I-V curves are group averages of normalized individual I-V curves, expressed as percent of eEPSC recorded at −60 mV. D, Representative AMPA receptor-mediated eEPSC responses recorded at holding potentials of –60 mV and +40 mV in SON neurons from control and salt-loaded rats. E, The rectification index (eEPSC amplitude at –60 mV/eEPSC amplitude at +40 mV) increased in SON neurons from salt-loaded rats, suggesting the emergence of calcium-permeable AMPA receptor-containing glutamate synapses with salt loading. VP and OT neurons were pooled together for panels B–E. Numerals in bars represent recorded cell numbers; *p < 0.05, **p < 0.01. Scale bars = 100 pA, 20 ms.

Change in EPSC sensitivity to the calcium-permeable AMPA receptor antagonist NAS following salt loading. A, NAS reduced the mean frequency (left) and amplitude (right) of sEPSCs over time in SON neurons from salt-loaded (NaCl) rats, but not in SON neurons from control rats. B, The effect of NAS on mean sEPSC frequency (Freq), amplitude (Amp), and decay time (Decay) in SON neurons from salt-loaded rats (NaCl) and control rats, expressed as a percentage of baseline. NAS decreased the sEPSC frequency and amplitude in SON neurons from salt-loaded rats but not control rats. C, NAS caused a decrease in the mean amplitude of eEPSCs in SON neurons from salt-loaded rats, but not control rats. NAS had no effect on the paired-pulse ratio of eEPSCs (PPR) in SON neurons from either control or salt-loaded rats. D, NAS had no effect on the eEPSC rectification index in SON neurons from control rats, but reversed the increase in the rectification index in SON neurons from salt-loaded rats. Numerals in bars represent recorded cell numbers; *p < 0.05, **p < 0.01 compared to control or baseline.

Correlation of the membrane surface area to NAS sensitivity. There was a significant correlation between the increase in magnocellular neuron surface area, as measured by membrane capacitance (C_m), and the change in eEPSC amplitude in NAS.