Intracellular Chloride Regulation in AVP+ and VIP+ Neurons of the Suprachiasmatic Nucleus

Abstract

Several reports have described excitatory GABA transmission in the suprachiasmatic nucleus (SCN), the master pacemaker of circadian physiology. However, there is disagreement regarding the prevalence, timing, and neuronal location of excitatory GABA transmission in the SCN. Whether GABA is inhibitory or excitatory depends, in part, on the intracellular concentration of chloride ([Cl−]i). Here, using ratiometric Cl− imaging, we have investigated intracellular chloride regulation in AVP and VIP-expressing SCN neurons and found evidence suggesting that [Cl−]i is higher during the day than during the night in both AVP+ and VIP+ neurons. We then investigated the contribution of the cation chloride cotransporters to setting [Cl−]i in these SCN neurons and found that the chloride uptake transporter NKCC1 contributes to [Cl−]i regulation in SCN neurons, but that the KCCs are the primary regulators of [Cl−]i in SCN neurons. Interestingly, we observed that [Cl−]i is differentially regulated between AVP+ and VIP+ neurons-a low concentration of the loop diuretic bumetanide had differential effects on AVP+ and VIP+ neurons, while blocking the KCCs with VU0240551 had a larger effect on VIP+ neurons compared to AVP+ neurons.

Compensatory ion transport buffers daily protein rhythms to regulate osmotic balance and cellular physiology

“…However, the same timekeeping mechanisms, rhythms in mTORC activity, and daily variation in ion transport have been observed in other cellular contexts, both ex vivo and in vivo [8][9][10][11]26,64 . Based on our similar recent findings in human, algal and fungal cells 9,65 , we predict that rhythms in ion transport will be observed in any eukaryotic cell with oscillations in mTORC activity, circadian or otherwise.…”

“…During neuronal development, for example, progressive expression of KCC2 increases intracellular Cland mediates the transition between excitatory and inhibitory GABA signalling 23,24 . Furthermore, differential KCC/NKCC activity is thought to mediate GABAergic synaptic plasticity of circadian regulation in the mammalian suprachiasmatic nucleus 25,26 .…”

Effects of salt‐loading on supraoptic vasopressin neurones assessed by ClopHensorN chloride imaging

“…Because our data indicate Cl − efflux in SON AVP neurones from SL rats, we tested the hypothesis that the reduced activity of KCC2 and the decreased chloride efflux contribute to this change in the polarity of the muscimol response . To test for KCC2 in setting [Cl − ] i , we used VU0240551, an antagonist that selectively targets the KCCs . The muscimol‐induced increase in [Cl − ] i was inhibited by VU0240551 in neurones from Eu rats (Figure A,C).…”

“…Increases in [Cl − ] i can change GABA‐mediated inhibition to excitation by reversing the direction of chloride movement across the cell membrane. The balance between neural excitation and inhibition is crucial for maintaining the concentration of circulating AVP within normal limits . In most neurones, [Cl − ] i is regulated by cation chloride cotransporters such as K + /Cl − co‐transporter 2 (KCC2), which causes the efflux of Cl − ions, and Na + /K + /Cl − cotransporter 1 (NKCC1), which causes the influx of Cl − ions .…”

The circadian clock: a tale of genetic–electrical interplay and synaptic integration

“…Excitatory GABA neurotransmission is more prevalent in the dorsal SCN compared to the ventral SCN, and the overall consensus is that there is more excitatory GABA transmission during the night than during the day [29–33]. The regional differences in GABA activity may reflect different intracellular Cl − regulation in arginine vasopressin (AVP)- and vasoactive intestinal polypeptide (VIP)-expressing SCN neurons [34]. Computer simulations and physiological recordings suggest that this inhibitoryexcitatory switch in GABA action is an essential component of the SCN network activity, regulating period length and photoperiod encoding [27,28,35].…”