GABAergic inhibition through synergistic astrocytic neuronal interaction transiently decreases vasopressin neuronal activity during hypoosmotic challenge

Abstract

The neuropeptide vasopressin is crucial to mammalian osmotic regulation. Local hypoosmotic challenge transiently decreases and then increases vasopressin secretion. To investigate mechanisms underlying this transient response, we examined the effects of hypoosmotic challenge on the electrical activity of rat hypothalamic supraoptic nucleus (SON) vasopressin neurons using patch-clamp recordings. We found that 5 min exposure of hypothalamic slices to hypoosmotic solution transiently increased inhibitory postsynaptic current (IPSC) frequency and reduced the firing rate of vasopressin neurons. Recovery occurred by 10 min of exposure, even though the osmolality remained low. The γ-aminobutyric acid (GABA)A receptor blocker, gabazine, blocked the IPSCs and the hypoosmotic suppression of firing. The gliotoxin l-aminoadipic acid blocked the increase in IPSC frequency at 5 min and the recovery of firing at 10 min, indicating astrocytic involvement in hypoosmotic modulation of vasopressin neuronal activity. Moreover, β-alanine, an osmolyte of astrocytes and GABA transporter (GAT) inhibitor, blocked the increase in IPSC frequency at 5 min of hypoosmotic challenge. Confocal microscopy of immunostained SON sections revealed that astrocytes and magnocellular neurons both showed positive staining of vesicular GATs (VGAT). Hypoosmotic stimulation in vivo reduced the number of VGAT-expressing neurons, and increased co-localisation and molecular association of VGAT with glial fibrillary acidic protein that increased significantly by 10 min. By 30 min, neuronal VGAT labelling was partially restored, and astrocytic VGAT was relocated to the ventral portion while it decreased in the somatic zone of the SON. Thus, synergistic astrocytic and neuronal GABAergic inhibition could ensure that vasopressin neuron firing is only transiently suppressed under hypoosmotic conditions.

Figure 1

Hypoosmotic challenge of rat brain slices decreases then restores firing rate and evokes correlated changes in inhibitory postsynaptic current (IPSC) frequency of vasopressin neurones in the SON. A, Whole cell patch clamp recordings from vasopressin neurones. Aa, Firing activity of a phasically-firing neurone in normal aCSF (305 mOsm, naCSF) before and after superfusing the slice with −20 mOsm hypoosmotic aCSF (haCSF). Number at bottom left indicates initial membrane potential. Ab, Post hoc immunostaining of recorded supraoptic neurones. Confocal microscopic images (63× objective) of the recorded cells, which were filled with Lucifer yellow (LY) and showed immunoreactivity for vasopressin-neurophysin (VP-NP), but not for oxytocin neurophysin (OT-NP). The dashed circle in merged channel points to the overlapping of LY and VP-NP staining. Ac, Mean (± SEM) changes (%) in firing rate (Ac1) and absolute value of the membrane potential (RMP, Ac2) after 5 and 10 min superfusion with haCSF, relative to naCSF (0 min in haCSF). In all figures, *P <0.05 vs. 0 min and †P <0.05 compared to 5 min by ANOVA. B. IPSC (Ba) recorded from a neurone at −10 mV. Regions indicated by boxes are expanded below the traces. Bb1, Mean ± SEM changes (%) in IPSC frequency after 5 and 10 superfusion of haCSF relative to 0 min.

Figure 2

GABA_A receptor-mediated IPSCs are inhibitory in vasopressin neurones. A, current-clamp recordings of firing activity of a putative vasopressin neurone in the presence of gabazine (10 µM) in naCSF and then with gabazine in haCSF. Boxes in Aa indicate regions expanded in Ab. B, Changes in mean ± SEM firing frequency (%, Ba) and absolute value of the membrane potential (%, Bb) after 5 and 10 min superfusion with haCSF containing gabazine, relative to 0 min. C, Representative trace in voltage-clamp recording of IPSCs in the presence of gabazine at −10 mV.

Figure 3

Disruption of astrocyte functions blocks hypoosmotic suppression or later recovery of vasopressin neurone firing. A, Firing activity of a vasopressin neurone in naCSF containing the gliotoxin l-aminoadipic acid (L-AAA, 0.25 mM) before and after superfusion with haCSF containing L-AAA, in full recording (Aa) and expanded episodes (Ab). B, Mean ± SEM changes (%) in firing rate (Ba) and absolute value of the membrane potential (RMP, Bb) of 6 cells after 5 and 10 min superfusion, relative to 0 min. No significant differences were observed.

Figure 4

Blocking astrocytic GABA transport reduces effects of hypoosmotic challenge on IPSC frequency. Aa–c, Effects of superfusion with L-AAA in haCSF on IPSC frequency, relative to 0 min. Ba–c, Effects of superfusion with β-alanine (0.15 mM) in haCSF on IPSC frequency, relative to 0 min.

Figure 5

Hypoosmotic challenge modulates expression of vesicular GABA transporter (VGAT) in both astrocytes and neurones of the SON. A, Confocal microscopic images of the SON in a hypothalamus slice. Aa, Representative images showing (from left to right) nuclei, bright-field (B–F) view of the slice, GFAP, VGAT and the merged view. The open arrowhead indicates a neuronal nucleus and the open arrow points to an astrocytic process. Ab1 and Ab2, Negative controls in the presence of goat antibody against VGAT without secondary antibody (Ab1) and Alexa Fluor 555-labelled secondary antibody without primary antibody (Ab2), respectively. B, Time-dependent effects of in vivo hypoosmotic challenge on GFAP and VGAT expression. Ba, Representative confocal images (from left to the right) showing staining of nuclei, GFAP, VGAT, their merges and the expanded insets (from the squared areas in the merged channel), respectively. From top to the bottom, the panels showing times at 0 min (immediately), 10 min and 30 min of after i.p. application of hypoosmotic solution (20 ml/kg, i.p.). The letters in the nuclei channels show the orientation of the SON in the slices: V, ventral; D, dorsal; M, medial; L, lateral side. White arrows indicate the VGAT staining in the ventral glial lamina (bottom). Bb, Same images as Ba but in higher amplification. Bc, Summary graphs showing the general change in VGAT intensity (left) and the number of VGAT-positive somata of magnocellular neurones (right). C, Effects of in vivo hypoosmotic challenge on GFAP levels. Left panels (Ca) show Western blot bands and the bar graph (Cb) summarises quantitative data expressed as percentage of control GFAP level relative to actin (loading control). D, co-immunoprecipitation of GFAP with VGAT after 0, 10 and 30 minutes of in vivo hypoosmotic challenge. IgG-HC, IgG heavy chain, used as negative control; TL, total lysates, used as positive control.