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

Kirthikaa Balapattabi, George E. Farmer, Blayne A. Knapp, Joel T. Little, Martha Bachelor, Joseph P. Yuan, Joseph Thomas Cunningham

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Salt-loading (SL) impairs GABAA inhibition of arginine vasopressin (AVP) neurones in the supraoptic nucleus (SON) of the hypothalamus. Based on previous studies, we hypothesised that SL activates tyrosine receptor kinase B (TrkB), down-regulating the activity of K+/Cl co-transporter2 (KCC2) and up-regulating Na+/K+/Cl co-transporter1 (NKCC1). These changes in chloride transport would result in increased [Cl]i in SON AVP neurones. The study combined virally-mediated chloride imaging with ClopHensorN with a single-cell western blot analysis. An adeno-associated virus with ClopHensorN and a vasopressin promoter (AAV2-0VP1-ClopHensorN) was bilaterally injected in the SON of adult male Sprague-Dawley rats that were either euhydrated (Eu) or salt-loaded (SL) for 7 days. Acutely dissociated SON neurones expressing ClopHensorN were tested for decreases or increases in [Cl]i in response to focal application of the GABAA agonist muscimol (100 μmol L-1). SON AVP neurones from Eu rats showed muscimol-induced chloride influx (P < 0.05;23/35). SON AVP neurones from SL rats either significantly increased chloride efflux (P < 0.05;27/39) or did not change chloride flux (12/39). The SON AVP neurones that responded to muscimol appeared to be viable and expressed KCC2 and β-actin. Neurones that did not respond during chloride imaging did not show KCC2 and β-actin protein expression. The KCC2 antagonist (VU0240551,10 μmol L-1) significantly blocked the chloride influx in cells from Eu rats but did not affect cells from SL rats. A NKCC1 antagonist (bumetanide,10 μmol L-1) significantly blocked the chloride efflux in cells from SL rats but had no effect on cells from Eu rats. Blocking NKCC1 using bumetanide had less of an effect on the muscimol-induced Cl influx in Eu rat neurones compared to the KCC2 antagonist. The TrkB antagonist (AnA-12) (50 μmol L-1) and protein kinase inhibitor (K252a) (100 nmol L-1) each significantly blocked chloride efflux in SON AVP neurones from SL rats. Salt-loading increases [Cl]i in SON AVP neurones via a TrKB-KCC2-NKCC1-dependent mechanism in rats.

Original languageEnglish
Article numbere12752
JournalJournal of Neuroendocrinology
Volume31
Issue number8
DOIs
StatePublished - 1 Jan 2019

Fingerprint

Supraoptic Nucleus
Vasopressins
Chlorides
Arginine Vasopressin
Salts
Neurons
Muscimol
Bumetanide
Receptor Protein-Tyrosine Kinases
Actins
GABA-A Receptor Agonists
Dependovirus
Protein Kinase Inhibitors
Sprague Dawley Rats
Western Blotting

Keywords

  • SON and salt-loading
  • chloride imaging
  • vasopressin

Cite this

Balapattabi, Kirthikaa ; Farmer, George E. ; Knapp, Blayne A. ; Little, Joel T. ; Bachelor, Martha ; Yuan, Joseph P. ; Cunningham, Joseph Thomas. / Effects of salt-loading on supraoptic vasopressin neurones assessed by ClopHensorN chloride imaging. In: Journal of Neuroendocrinology. 2019 ; Vol. 31, No. 8.
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abstract = "Salt-loading (SL) impairs GABAA inhibition of arginine vasopressin (AVP) neurones in the supraoptic nucleus (SON) of the hypothalamus. Based on previous studies, we hypothesised that SL activates tyrosine receptor kinase B (TrkB), down-regulating the activity of K+/Cl− co-transporter2 (KCC2) and up-regulating Na+/K+/Cl− co-transporter1 (NKCC1). These changes in chloride transport would result in increased [Cl−]i in SON AVP neurones. The study combined virally-mediated chloride imaging with ClopHensorN with a single-cell western blot analysis. An adeno-associated virus with ClopHensorN and a vasopressin promoter (AAV2-0VP1-ClopHensorN) was bilaterally injected in the SON of adult male Sprague-Dawley rats that were either euhydrated (Eu) or salt-loaded (SL) for 7 days. Acutely dissociated SON neurones expressing ClopHensorN were tested for decreases or increases in [Cl−]i in response to focal application of the GABAA agonist muscimol (100 μmol L-1). SON AVP neurones from Eu rats showed muscimol-induced chloride influx (P < 0.05;23/35). SON AVP neurones from SL rats either significantly increased chloride efflux (P < 0.05;27/39) or did not change chloride flux (12/39). The SON AVP neurones that responded to muscimol appeared to be viable and expressed KCC2 and β-actin. Neurones that did not respond during chloride imaging did not show KCC2 and β-actin protein expression. The KCC2 antagonist (VU0240551,10 μmol L-1) significantly blocked the chloride influx in cells from Eu rats but did not affect cells from SL rats. A NKCC1 antagonist (bumetanide,10 μmol L-1) significantly blocked the chloride efflux in cells from SL rats but had no effect on cells from Eu rats. Blocking NKCC1 using bumetanide had less of an effect on the muscimol-induced Cl− influx in Eu rat neurones compared to the KCC2 antagonist. The TrkB antagonist (AnA-12) (50 μmol L-1) and protein kinase inhibitor (K252a) (100 nmol L-1) each significantly blocked chloride efflux in SON AVP neurones from SL rats. Salt-loading increases [Cl−]i in SON AVP neurones via a TrKB-KCC2-NKCC1-dependent mechanism in rats.",
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Effects of salt-loading on supraoptic vasopressin neurones assessed by ClopHensorN chloride imaging. / Balapattabi, Kirthikaa; Farmer, George E.; Knapp, Blayne A.; Little, Joel T.; Bachelor, Martha; Yuan, Joseph P.; Cunningham, Joseph Thomas.

In: Journal of Neuroendocrinology, Vol. 31, No. 8, e12752, 01.01.2019.

Research output: Contribution to journalArticleResearchpeer-review

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T1 - Effects of salt-loading on supraoptic vasopressin neurones assessed by ClopHensorN chloride imaging

AU - Balapattabi, Kirthikaa

AU - Farmer, George E.

AU - Knapp, Blayne A.

AU - Little, Joel T.

AU - Bachelor, Martha

AU - Yuan, Joseph P.

AU - Cunningham, Joseph Thomas

PY - 2019/1/1

Y1 - 2019/1/1

N2 - Salt-loading (SL) impairs GABAA inhibition of arginine vasopressin (AVP) neurones in the supraoptic nucleus (SON) of the hypothalamus. Based on previous studies, we hypothesised that SL activates tyrosine receptor kinase B (TrkB), down-regulating the activity of K+/Cl− co-transporter2 (KCC2) and up-regulating Na+/K+/Cl− co-transporter1 (NKCC1). These changes in chloride transport would result in increased [Cl−]i in SON AVP neurones. The study combined virally-mediated chloride imaging with ClopHensorN with a single-cell western blot analysis. An adeno-associated virus with ClopHensorN and a vasopressin promoter (AAV2-0VP1-ClopHensorN) was bilaterally injected in the SON of adult male Sprague-Dawley rats that were either euhydrated (Eu) or salt-loaded (SL) for 7 days. Acutely dissociated SON neurones expressing ClopHensorN were tested for decreases or increases in [Cl−]i in response to focal application of the GABAA agonist muscimol (100 μmol L-1). SON AVP neurones from Eu rats showed muscimol-induced chloride influx (P < 0.05;23/35). SON AVP neurones from SL rats either significantly increased chloride efflux (P < 0.05;27/39) or did not change chloride flux (12/39). The SON AVP neurones that responded to muscimol appeared to be viable and expressed KCC2 and β-actin. Neurones that did not respond during chloride imaging did not show KCC2 and β-actin protein expression. The KCC2 antagonist (VU0240551,10 μmol L-1) significantly blocked the chloride influx in cells from Eu rats but did not affect cells from SL rats. A NKCC1 antagonist (bumetanide,10 μmol L-1) significantly blocked the chloride efflux in cells from SL rats but had no effect on cells from Eu rats. Blocking NKCC1 using bumetanide had less of an effect on the muscimol-induced Cl− influx in Eu rat neurones compared to the KCC2 antagonist. The TrkB antagonist (AnA-12) (50 μmol L-1) and protein kinase inhibitor (K252a) (100 nmol L-1) each significantly blocked chloride efflux in SON AVP neurones from SL rats. Salt-loading increases [Cl−]i in SON AVP neurones via a TrKB-KCC2-NKCC1-dependent mechanism in rats.

AB - Salt-loading (SL) impairs GABAA inhibition of arginine vasopressin (AVP) neurones in the supraoptic nucleus (SON) of the hypothalamus. Based on previous studies, we hypothesised that SL activates tyrosine receptor kinase B (TrkB), down-regulating the activity of K+/Cl− co-transporter2 (KCC2) and up-regulating Na+/K+/Cl− co-transporter1 (NKCC1). These changes in chloride transport would result in increased [Cl−]i in SON AVP neurones. The study combined virally-mediated chloride imaging with ClopHensorN with a single-cell western blot analysis. An adeno-associated virus with ClopHensorN and a vasopressin promoter (AAV2-0VP1-ClopHensorN) was bilaterally injected in the SON of adult male Sprague-Dawley rats that were either euhydrated (Eu) or salt-loaded (SL) for 7 days. Acutely dissociated SON neurones expressing ClopHensorN were tested for decreases or increases in [Cl−]i in response to focal application of the GABAA agonist muscimol (100 μmol L-1). SON AVP neurones from Eu rats showed muscimol-induced chloride influx (P < 0.05;23/35). SON AVP neurones from SL rats either significantly increased chloride efflux (P < 0.05;27/39) or did not change chloride flux (12/39). The SON AVP neurones that responded to muscimol appeared to be viable and expressed KCC2 and β-actin. Neurones that did not respond during chloride imaging did not show KCC2 and β-actin protein expression. The KCC2 antagonist (VU0240551,10 μmol L-1) significantly blocked the chloride influx in cells from Eu rats but did not affect cells from SL rats. A NKCC1 antagonist (bumetanide,10 μmol L-1) significantly blocked the chloride efflux in cells from SL rats but had no effect on cells from Eu rats. Blocking NKCC1 using bumetanide had less of an effect on the muscimol-induced Cl− influx in Eu rat neurones compared to the KCC2 antagonist. The TrkB antagonist (AnA-12) (50 μmol L-1) and protein kinase inhibitor (K252a) (100 nmol L-1) each significantly blocked chloride efflux in SON AVP neurones from SL rats. Salt-loading increases [Cl−]i in SON AVP neurones via a TrKB-KCC2-NKCC1-dependent mechanism in rats.

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