Journal: bioRxiv

Article Title: Like sisters but not twins – vasopressin and oxytocin excite BNST neurons via cell type-specific expression of oxytocin receptor to reduce anxious arousal

doi: 10.1101/2024.09.06.611656

Figure Lengend Snippet: Steady state firing frequency (SSF) as a function of current (input/output, I/O) is shown before (pre), during, and after AVP application (washout, post). A) In Type I neurons , there was a significant treatment effect, with AVP inducing a leftward shift of the I/O relationship, without affecting its slope ( P =0.0137, n=11, mixed-effect analysis). Post hoc test showed a significant AVP effect on SSF in most currents injected. A’) Representative traces of Type I BNST neuron responses to a depolarizing current pulse injection of 40 pA for 1 sec before (Pre), during (AVP) and after AVP (Post). B) Application of OTR antagonist, OTA, blocked the excitatory effect of AVP in Type I neurons ( P =0.0574, F(1.466, 11.73)=3.999, n=9, mixed-effects model). B’) Representative traces of Type I neuron responses to a depolarizing current pulse injection of 60 pA for 1 sec before, during OTA alone, and during OTA+AVP. C) In the presence of the V1aR antagonist, SR49059, AVP application significantly changed the I/O relationship ( P= 0.0009, n=7, mixed-effect analysis). Multiple comparisons showed an excitatory effect of AVP at all currents injected except 40 pA ( P =0.0792). The SR49059 showed an excitatory effect on its own at (pA) 80 ( P =0.0432) and 100 ( P =0.0496). C’) Representative traces of Type I neuron responses to a depolarizing current pulse injection of 100 pA for 1 sec, before, during SR49059 alone, and during SR49059+AVP). D) In a presence of V1bR antagonist, Nelivaptan, AVP significantly shifted the I/O relationship ( P <0.0001, n=10, mixed-effect analysis). Multiple comparisons showed an excitatory effect of AVP on SSF at all current injected except 170 ( P =0.2845) or 180 pA ( P =0.2847). Nelivaptan alone had no effect on the SSF at any of the currents injected except at 140 pA ( P =0.0474). D’) Representative traces of Type I neuron responses to a depolarizing current pulse injection of 80 pA for 1 sec before, during Nelivaptan alone and Nelivaptan+AVP. E) In Type III neurons , AVP induced a leftward shift of the I/O relationship, without affecting its slope ( P =0.0007, n=11, mixed-effect analysis), with a significant AVP effect in all currents injected except 180 ( P =0.0584). The leftward shift of the I/O relationship in Type III neurons recovered during washout of AVP for all currents injected . E’) Representative traces of Type III neuron responses to a depolarizing current pulse injection of 140 pA before, during (AVP) and after AVP (Post). F) in the presence of OTR antagonist, OTA, there was no treatment effect of AVP on the I/O relationship ( P =0.3949, n=9, mixed-effect analysis). F’) Representative traces of Type III neuron response to a depolarizing current pulse injection of 100 pA for 1 sec, before, during OTA alone, and during OTA+AVP. G) In in the presence of the V1aR antagonist, SR49059, AVP application significantly changed the I/O relationship ( P =0.0068, n=7, mixed-effect analysis). Multiple comparisons showed an excitatory effect of AVP on SSF at most current injected except (in pA) at 70 ( P =0.1359), 80 ( P =0.2954), 100 ( P =0.1874), and 110 ( P =0.1720). Application of SR49059 alone did not have an effect on the SSF at any of the currents injected. G’) Representative traces of Type III neuron response to a depolarizing current pulse injection of 140 pA for 1 sec, before, during SR49059, and during SR49059+AVP). H) When the V1bR antagonist, Nelivaptan, was applied before AVP, AVP significantly shifted the I/O relationship ( P <0.0001, n=10, mixed-effect analysis). Multiple comparisons showed a significant excitatory effect of AVP on SSF at most currents injected except 170 ( P =0.2845) and 180 pA ( P =0.2847). H’) Representative traces of Type III neuron responses to a depolarizing current pulse injection of 110 pA for 1 sec, before, during Nelivaptan alone and during Nelivaptan+AVP. * P <0.05, ** P <0.01, *** P <0.001 vs. pre

Article Snippet: The following antagonists were used: a V1aR/OTR antagonist (d(CH 2 ) 5 1 ,Tyr(Me) 2 ,Arg )-vasopressin, Manning compound ; 1 μM, Tocris, Bio-Techne Corporation; MN, #3377); selective OTR antagonist (OTA; d(CH2)5(1), D-Tyr(2), Thr(4), Orn(8), des-Gly-NH2(9)]-Vasotocin trifluoroacetate salt ; 0.4 μM, Chemical Repository, #V-905 NIMH); selective V1aR antagonist SR49059 (2 S )-1-[[(2 R ,3 S )-5-Chloro-3-(2-chlorophenyl)-1-[(3,4-dimethoxyphenyl)sulfonyl]-2,3-dihydro-3-hydroxy-1 H -indol-2-yl]carbonyl]-2-yrrolidinecarboxamide; 5 μM, Tocris, Bio-Techne Corporation, #2310); and a V1bR antagonist Nelivaptan ((2 S ,4 R )-1-[(3 R )-5-Chloro-1-[(2,4-dimethoxyphenyl)sulfonyl]-2,3-dihydro-3-(2-methoxyphenyl)-2-oxo-1 H -indol-3-yl]-4-hydroxy- N , N -dimethyl-2-pyrrolidinecarboxamide; 1 μM, Tocris, Bio-Techne Corporation, #6195).

Techniques: Injection

Journal: bioRxiv

Article Title: Like sisters but not twins – vasopressin and oxytocin excite BNST neurons via cell type-specific expression of oxytocin receptor to reduce anxious arousal

doi: 10.1101/2024.09.06.611656

Figure Lengend Snippet: A) In Type III neurons from wild-type Sprague-Dawley rats, application of AVP in the presence of the V1aR/OTR antagonist ((d(CH 2 ) 5 1 ,Tyr(Me) 2 ,Arg 8 )-vasopressin) did not change the I/O relationship ( P =0.2742, n=8, mixed-effect analysis). B) In Type III neurons, application of endogenous OTR agonist oxytocin (OT) induced a leftward shift of the I/O relationship ( P =0.0077, n=8), with significant OT effect on SSF in most currents injected except at 80pA ( P =0.3822) and 180pA ( P =0.2500). C) Selective and potent OTR agonist ([Thr 4 ,Gly 7 ]-oxytocin (TGOT) affected the I/O relationship ( P =0.0196, n=8) with significant excitatory effect in the first part of the curve from 70pA to 110pA and trending at 120 ( P =0.0673). These effects were fully reversed with washout. C’) Examples of evoked responses of a Type III neuron to a depolarizing pulse of 90 pA for 1 sec before, during, and after TGOT application. D) Selective V1aR agonist, FE201874, induced an excitatory effect on the I/O relationship ( P =0.0117, n=10), with significant effects on SSF as shown, which were fully reversed at washout. D’) Examples of evoked responses of a Type III neuron to a depolarizing pulse of 90pA for 1 sec before, during, and after FE201874 application. E) Selective V1bR agonist, d[Cha4]-AVP did not induce a significant effect on I/O relationship ( P =0.5334, n=8), * P <0.05, ** P <0.01, *** P <0.001 vs. pre. F) Nissl (left) and anatomical annotations (right) from the Allen Mouse Brain Atlas and Allen Reference Atlas - Mouse Brain . High somatodendritic OTR-mCherry expression in the BNST DL after male OTR-Cre transgenic rats were injected with a Cre-dependent AAV-DREADDs-mCherry in the BNST DL . G) Example of evoked responses of a Type I/OTR mCherry fluorescent neuron and H) Type III/OTR mCherry neuron to a hyperpolarizing and depolarizing current pulses of 1 sec. I) In Type III/OTR fluorescent neurons, mixed-effect analysis revealed a significant incremental effect of AVP on SSF ( P <0.0001, n=10), with a significant excitatory effect of AVP on SSF at most current injected except a trend at 100 pA ( P =0.0678) and 110 pA ( P =0.0763). I’) Representative trace of Type III/OTR fluorescent neuron responses to a depolarizing current pulse injection of 130 pA for 1 sec, before, during and after AVP application. J) Fluorescent OTR-BNST DL -mCherry neurons were characterized as Type I-III neurons based on their electrophysiological properties, with majority 35/45 (77.8%) characterized as Type III, 7/45 (15.6%) as Type I, and 3/45 (6.7%) as Type II. K) The great majority of recorded fluorescent CRF-BNST DL -mCherry neurons (21/26) were characterized as Type III (80.8%), whereas 2/26 as Type II (7.7%), and 3/26 as Type I (11.5%). L) In Type III/CRF neurons from CRF-Cre transgenic rats, there was a significant incremental effect of AVP on SSF ( P =0.0057, n=7), with multiple comparisons showed a significant effect of AVP at all current injected except at 90 pA ( P =0.2288). L’) Representative traces of Type III/CRF fluorescent neuron responses to a depolarizing current pulse injection of 100 pA for 1 sec before, during, and after AVP application. M) Nissl (left) and anatomical annotations (right) from the Allen Mouse Brain Atlas and Allen Reference Atlas – Mouse Brain . High somatodendritic CRF-mCherry expression in the BNST DL after male CRF-Cre transgenic rats were injected with a Cre-dependent AAV-DREADDs-mCherry in the BNST DL . N) Example of evoked responses of a Type III/CRF fluorescent neuron to a hyperpolarizing and depolarizing current pulses of 1 sec. O) In Type III/CRF neurons, there was a significant TGOT effect on action potential frequency ( P =0.0167, n=7). Multiple comparisons showed a significant effect of TGOT at (in pA): 140 and 150 ( P =0.0370), and trends at 130 ( P = 0.0807), 160 ( P =0.0978), and 170 ( P =0.0867). O’) Representative traces of Type III/CRF fluorescent neuron responses to a depolarizing current pulse injection of 150 pA before, during and after TGOT application. * P <0.05, ** P <0.01, *** P <0.001 vs pre.

Article Snippet: The following antagonists were used: a V1aR/OTR antagonist (d(CH 2 ) 5 1 ,Tyr(Me) 2 ,Arg )-vasopressin, Manning compound ; 1 μM, Tocris, Bio-Techne Corporation; MN, #3377); selective OTR antagonist (OTA; d(CH2)5(1), D-Tyr(2), Thr(4), Orn(8), des-Gly-NH2(9)]-Vasotocin trifluoroacetate salt ; 0.4 μM, Chemical Repository, #V-905 NIMH); selective V1aR antagonist SR49059 (2 S )-1-[[(2 R ,3 S )-5-Chloro-3-(2-chlorophenyl)-1-[(3,4-dimethoxyphenyl)sulfonyl]-2,3-dihydro-3-hydroxy-1 H -indol-2-yl]carbonyl]-2-yrrolidinecarboxamide; 5 μM, Tocris, Bio-Techne Corporation, #2310); and a V1bR antagonist Nelivaptan ((2 S ,4 R )-1-[(3 R )-5-Chloro-1-[(2,4-dimethoxyphenyl)sulfonyl]-2,3-dihydro-3-(2-methoxyphenyl)-2-oxo-1 H -indol-3-yl]-4-hydroxy- N , N -dimethyl-2-pyrrolidinecarboxamide; 1 μM, Tocris, Bio-Techne Corporation, #6195).

Techniques: Injection, Expressing, Transgenic Assay

Journal: bioRxiv

Article Title: Like sisters but not twins – vasopressin and oxytocin excite BNST neurons via cell type-specific expression of oxytocin receptor to reduce anxious arousal

doi: 10.1101/2024.09.06.611656

Figure Lengend Snippet:

Article Snippet: The following antagonists were used: a V1aR/OTR antagonist (d(CH 2 ) 5 1 ,Tyr(Me) 2 ,Arg )-vasopressin, Manning compound ; 1 μM, Tocris, Bio-Techne Corporation; MN, #3377); selective OTR antagonist (OTA; d(CH2)5(1), D-Tyr(2), Thr(4), Orn(8), des-Gly-NH2(9)]-Vasotocin trifluoroacetate salt ; 0.4 μM, Chemical Repository, #V-905 NIMH); selective V1aR antagonist SR49059 (2 S )-1-[[(2 R ,3 S )-5-Chloro-3-(2-chlorophenyl)-1-[(3,4-dimethoxyphenyl)sulfonyl]-2,3-dihydro-3-hydroxy-1 H -indol-2-yl]carbonyl]-2-yrrolidinecarboxamide; 5 μM, Tocris, Bio-Techne Corporation, #2310); and a V1bR antagonist Nelivaptan ((2 S ,4 R )-1-[(3 R )-5-Chloro-1-[(2,4-dimethoxyphenyl)sulfonyl]-2,3-dihydro-3-(2-methoxyphenyl)-2-oxo-1 H -indol-3-yl]-4-hydroxy- N , N -dimethyl-2-pyrrolidinecarboxamide; 1 μM, Tocris, Bio-Techne Corporation, #6195).

Techniques: