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NPY signaling inhibits extended amygdala CRF neurons to suppress binge alcohol drinking.

Pleil KE, Rinker JA, Lowery-Gionta EG, Mazzone CM, McCall NM, Kendra AM, Olson DP, Lowell BB, Grant KA, Thiele TE, Kash TL - Nat. Neurosci. (2015)

Bottom Line: It is thought to do so by hijacking brain systems that regulate stress and reward, including neuropeptide Y (NPY) and corticotropin-releasing factor (CRF).The central actions of NPY and CRF have opposing functions in the regulation of emotional and reward-seeking behaviors; thus, dysfunctional interactions between these peptidergic systems could be involved in the development of these pathologies.Together, these data provide both a cellular locus and signaling framework for the development of new therapeutics for treatment of neuropsychiatric diseases, including alcohol use disorders.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA. [2] Bowles Center for Alcohol Studies, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.

ABSTRACT
Binge alcohol drinking is a tremendous public health problem because it leads to the development of numerous pathologies, including alcohol abuse and anxiety. It is thought to do so by hijacking brain systems that regulate stress and reward, including neuropeptide Y (NPY) and corticotropin-releasing factor (CRF). The central actions of NPY and CRF have opposing functions in the regulation of emotional and reward-seeking behaviors; thus, dysfunctional interactions between these peptidergic systems could be involved in the development of these pathologies. We used converging physiological, pharmacological and chemogenetic approaches to identify a precise neural mechanism in the bed nucleus of the stria terminalis (BNST), a limbic brain region involved in pathological reward and anxiety behaviors, underlying the interactions between NPY and CRF in the regulation of binge alcohol drinking in both mice and monkeys. We found that NPY Y1 receptor (Y1R) activation in the BNST suppressed binge alcohol drinking by enhancing inhibitory synaptic transmission specifically in CRF neurons via a previously unknown Gi-mediated, PKA-dependent postsynaptic mechanism. Furthermore, chronic alcohol drinking led to persistent alterations in Y1R function in the BNST of both mice and monkeys, highlighting the enduring, conserved nature of this effect across mammalian species. Together, these data provide both a cellular locus and signaling framework for the development of new therapeutics for treatment of neuropsychiatric diseases, including alcohol use disorders.

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Y1R-mediated effects on inhibition in the BNST are specific to CRF neurons. (a) Sample image of the dorsal BNST in a naïve CRF–Cre x Ai3 reporter mouse taken from a coronal brain slice depicting CRF–Cre-positive neurons in green (scale bar = 150 μM). Fluorescence expression shown is typical in this mouse line and was confirmed for all mice used in experiments for panels b–h. (b) Representative traces of mIPSCs from CRF–Cre-positive (CRF+) and CRF–Cre-negative (CRF−) neurons in the BNST before and after bath application of LeuPro NPY (300 nM). (c–d) Basal mIPSC frequency did not differ in CRF+ and CRF− BNST neurons (unpaired t-test: p > 0.20; CRF+ n = 11, N = 6, CRF− n = 7, N = 4 for all panels), but basal mIPSC amplitude was greater in CRF+ than CRF− neurons (t(16) = 2.25,*p = 0.039). (e) Bath application of LeuPro NPY increased mIPSC frequency in CRF+ neurons in the BNST by 54.7 ± 19.3% (paired t-test baseline vs. washout: t(10) = 2.83, *p = 0.018) but did not alter mIPSC frequency in CRF− neurons (−1.3 ± 7.8%; p > 0.85; magnitude of average percent change in frequency depicted in inset bar graph). (f) LeuPro NPY slightly but significantly decreased mIPSC amplitude in CRF+ neurons (−12.4 ± 5.2%; t(10) = 2.40, *p = 0.038) but did not alter mIPSC amplitude in CRF− neurons (−0.1 ± 4.2%; p > 0.95; magnitude of effects depicted in inset bar graph). (g) Representative averaged traces (top) and weighted tau values (bottom) of CRF+ and CRF− neurons in the BNST before and after bath application of LeuPro NPY showing that LeuPro NPY increased the decay magnitude of mIPSCs in CRF+ neurons (paired t-test: t(10) = 2.40, *p = 0.037) but did not alter the decay of mIPSC events in CRF− neurons (p > 0.50); rise time of mIPSCs was not altered in either group (p’s > 0.10, data not shown). (h) Cumulative probability distributions of mIPSC amplitude showing that there was a leftward shift in the distribution by the 75th percentile of events in CRF+ neurons (paired t-test: t(10) = 2.34, *p = 0.041), indicating that application of LeuPro NPY led to a greater number of mIPSC with smaller amplitude, while there was no effect on the distribution of mIPSC amplitude in CRF− neurons (p’s > 0.45). All data in c–h are presented as mean ± SEM.
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Figure 4: Y1R-mediated effects on inhibition in the BNST are specific to CRF neurons. (a) Sample image of the dorsal BNST in a naïve CRF–Cre x Ai3 reporter mouse taken from a coronal brain slice depicting CRF–Cre-positive neurons in green (scale bar = 150 μM). Fluorescence expression shown is typical in this mouse line and was confirmed for all mice used in experiments for panels b–h. (b) Representative traces of mIPSCs from CRF–Cre-positive (CRF+) and CRF–Cre-negative (CRF−) neurons in the BNST before and after bath application of LeuPro NPY (300 nM). (c–d) Basal mIPSC frequency did not differ in CRF+ and CRF− BNST neurons (unpaired t-test: p > 0.20; CRF+ n = 11, N = 6, CRF− n = 7, N = 4 for all panels), but basal mIPSC amplitude was greater in CRF+ than CRF− neurons (t(16) = 2.25,*p = 0.039). (e) Bath application of LeuPro NPY increased mIPSC frequency in CRF+ neurons in the BNST by 54.7 ± 19.3% (paired t-test baseline vs. washout: t(10) = 2.83, *p = 0.018) but did not alter mIPSC frequency in CRF− neurons (−1.3 ± 7.8%; p > 0.85; magnitude of average percent change in frequency depicted in inset bar graph). (f) LeuPro NPY slightly but significantly decreased mIPSC amplitude in CRF+ neurons (−12.4 ± 5.2%; t(10) = 2.40, *p = 0.038) but did not alter mIPSC amplitude in CRF− neurons (−0.1 ± 4.2%; p > 0.95; magnitude of effects depicted in inset bar graph). (g) Representative averaged traces (top) and weighted tau values (bottom) of CRF+ and CRF− neurons in the BNST before and after bath application of LeuPro NPY showing that LeuPro NPY increased the decay magnitude of mIPSCs in CRF+ neurons (paired t-test: t(10) = 2.40, *p = 0.037) but did not alter the decay of mIPSC events in CRF− neurons (p > 0.50); rise time of mIPSCs was not altered in either group (p’s > 0.10, data not shown). (h) Cumulative probability distributions of mIPSC amplitude showing that there was a leftward shift in the distribution by the 75th percentile of events in CRF+ neurons (paired t-test: t(10) = 2.34, *p = 0.041), indicating that application of LeuPro NPY led to a greater number of mIPSC with smaller amplitude, while there was no effect on the distribution of mIPSC amplitude in CRF− neurons (p’s > 0.45). All data in c–h are presented as mean ± SEM.

Mentions: When we examined the effect of Y1R activation on GABAergic transmission in the BNST of naïve mice, we noted a considerable amount of variability in the magnitude of the effect across individual neurons, which has also been reported in the basolateral amygdala29. Because CRF has been shown to increase alcohol drinking in mice16, 35, the opposite phenotype that we found with Y1R activation in the BNST, and both peptides are densely expressed in the BNST13, 36, we hypothesized that Y1R activation in the BNST decreases binge alcohol drinking by directly inhibiting CRF neurons. We first evaluated the ability of Y1R activation to alter synaptic function at CRF neuron synapses using slice electrophysiology in the BNST of CRF-reporter mice (Fig. 4a,b). CRF+ neurons had greater mIPSC amplitude, but not frequency, than CRF− neurons (Fig. 4c,d). Y1R activation with bath application of LeuPro NPY greatly increased mIPSC frequency in CRF+ neurons by 54.7 ± 19.3% but did not alter mIPSC frequency in CRF− neurons (−1.3 ± 7.8%; Fig. 4e), suggesting that the effects of Y1R in the BNST are specific to CRF neurons. Further, LeuPro NPY decreased the amplitude of mIPSCs in CRF+, but not CRF−, neurons by a small but significant amount (Fig. 4f) and increased the decay time of mIPSC events in CRF+ neurons but not CRF− neurons (Fig. 4g). Analysis of the cumulative probability distribution of mIPSC events confirmed that Y1R activation led to an increase in smaller-amplitude mIPSCs in CRF+ but not CRF− neurons (Fig. 4h). Together, results suggest that activation of postsynaptic Y1R on CRF neurons in the BNST results in the insertion of GABAARs with longer decay kinetics, such as those with α2/3 subunits, leading to more smaller-amplitude IPSCs with longer decay.


NPY signaling inhibits extended amygdala CRF neurons to suppress binge alcohol drinking.

Pleil KE, Rinker JA, Lowery-Gionta EG, Mazzone CM, McCall NM, Kendra AM, Olson DP, Lowell BB, Grant KA, Thiele TE, Kash TL - Nat. Neurosci. (2015)

Y1R-mediated effects on inhibition in the BNST are specific to CRF neurons. (a) Sample image of the dorsal BNST in a naïve CRF–Cre x Ai3 reporter mouse taken from a coronal brain slice depicting CRF–Cre-positive neurons in green (scale bar = 150 μM). Fluorescence expression shown is typical in this mouse line and was confirmed for all mice used in experiments for panels b–h. (b) Representative traces of mIPSCs from CRF–Cre-positive (CRF+) and CRF–Cre-negative (CRF−) neurons in the BNST before and after bath application of LeuPro NPY (300 nM). (c–d) Basal mIPSC frequency did not differ in CRF+ and CRF− BNST neurons (unpaired t-test: p > 0.20; CRF+ n = 11, N = 6, CRF− n = 7, N = 4 for all panels), but basal mIPSC amplitude was greater in CRF+ than CRF− neurons (t(16) = 2.25,*p = 0.039). (e) Bath application of LeuPro NPY increased mIPSC frequency in CRF+ neurons in the BNST by 54.7 ± 19.3% (paired t-test baseline vs. washout: t(10) = 2.83, *p = 0.018) but did not alter mIPSC frequency in CRF− neurons (−1.3 ± 7.8%; p > 0.85; magnitude of average percent change in frequency depicted in inset bar graph). (f) LeuPro NPY slightly but significantly decreased mIPSC amplitude in CRF+ neurons (−12.4 ± 5.2%; t(10) = 2.40, *p = 0.038) but did not alter mIPSC amplitude in CRF− neurons (−0.1 ± 4.2%; p > 0.95; magnitude of effects depicted in inset bar graph). (g) Representative averaged traces (top) and weighted tau values (bottom) of CRF+ and CRF− neurons in the BNST before and after bath application of LeuPro NPY showing that LeuPro NPY increased the decay magnitude of mIPSCs in CRF+ neurons (paired t-test: t(10) = 2.40, *p = 0.037) but did not alter the decay of mIPSC events in CRF− neurons (p > 0.50); rise time of mIPSCs was not altered in either group (p’s > 0.10, data not shown). (h) Cumulative probability distributions of mIPSC amplitude showing that there was a leftward shift in the distribution by the 75th percentile of events in CRF+ neurons (paired t-test: t(10) = 2.34, *p = 0.041), indicating that application of LeuPro NPY led to a greater number of mIPSC with smaller amplitude, while there was no effect on the distribution of mIPSC amplitude in CRF− neurons (p’s > 0.45). All data in c–h are presented as mean ± SEM.
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Figure 4: Y1R-mediated effects on inhibition in the BNST are specific to CRF neurons. (a) Sample image of the dorsal BNST in a naïve CRF–Cre x Ai3 reporter mouse taken from a coronal brain slice depicting CRF–Cre-positive neurons in green (scale bar = 150 μM). Fluorescence expression shown is typical in this mouse line and was confirmed for all mice used in experiments for panels b–h. (b) Representative traces of mIPSCs from CRF–Cre-positive (CRF+) and CRF–Cre-negative (CRF−) neurons in the BNST before and after bath application of LeuPro NPY (300 nM). (c–d) Basal mIPSC frequency did not differ in CRF+ and CRF− BNST neurons (unpaired t-test: p > 0.20; CRF+ n = 11, N = 6, CRF− n = 7, N = 4 for all panels), but basal mIPSC amplitude was greater in CRF+ than CRF− neurons (t(16) = 2.25,*p = 0.039). (e) Bath application of LeuPro NPY increased mIPSC frequency in CRF+ neurons in the BNST by 54.7 ± 19.3% (paired t-test baseline vs. washout: t(10) = 2.83, *p = 0.018) but did not alter mIPSC frequency in CRF− neurons (−1.3 ± 7.8%; p > 0.85; magnitude of average percent change in frequency depicted in inset bar graph). (f) LeuPro NPY slightly but significantly decreased mIPSC amplitude in CRF+ neurons (−12.4 ± 5.2%; t(10) = 2.40, *p = 0.038) but did not alter mIPSC amplitude in CRF− neurons (−0.1 ± 4.2%; p > 0.95; magnitude of effects depicted in inset bar graph). (g) Representative averaged traces (top) and weighted tau values (bottom) of CRF+ and CRF− neurons in the BNST before and after bath application of LeuPro NPY showing that LeuPro NPY increased the decay magnitude of mIPSCs in CRF+ neurons (paired t-test: t(10) = 2.40, *p = 0.037) but did not alter the decay of mIPSC events in CRF− neurons (p > 0.50); rise time of mIPSCs was not altered in either group (p’s > 0.10, data not shown). (h) Cumulative probability distributions of mIPSC amplitude showing that there was a leftward shift in the distribution by the 75th percentile of events in CRF+ neurons (paired t-test: t(10) = 2.34, *p = 0.041), indicating that application of LeuPro NPY led to a greater number of mIPSC with smaller amplitude, while there was no effect on the distribution of mIPSC amplitude in CRF− neurons (p’s > 0.45). All data in c–h are presented as mean ± SEM.
Mentions: When we examined the effect of Y1R activation on GABAergic transmission in the BNST of naïve mice, we noted a considerable amount of variability in the magnitude of the effect across individual neurons, which has also been reported in the basolateral amygdala29. Because CRF has been shown to increase alcohol drinking in mice16, 35, the opposite phenotype that we found with Y1R activation in the BNST, and both peptides are densely expressed in the BNST13, 36, we hypothesized that Y1R activation in the BNST decreases binge alcohol drinking by directly inhibiting CRF neurons. We first evaluated the ability of Y1R activation to alter synaptic function at CRF neuron synapses using slice electrophysiology in the BNST of CRF-reporter mice (Fig. 4a,b). CRF+ neurons had greater mIPSC amplitude, but not frequency, than CRF− neurons (Fig. 4c,d). Y1R activation with bath application of LeuPro NPY greatly increased mIPSC frequency in CRF+ neurons by 54.7 ± 19.3% but did not alter mIPSC frequency in CRF− neurons (−1.3 ± 7.8%; Fig. 4e), suggesting that the effects of Y1R in the BNST are specific to CRF neurons. Further, LeuPro NPY decreased the amplitude of mIPSCs in CRF+, but not CRF−, neurons by a small but significant amount (Fig. 4f) and increased the decay time of mIPSC events in CRF+ neurons but not CRF− neurons (Fig. 4g). Analysis of the cumulative probability distribution of mIPSC events confirmed that Y1R activation led to an increase in smaller-amplitude mIPSCs in CRF+ but not CRF− neurons (Fig. 4h). Together, results suggest that activation of postsynaptic Y1R on CRF neurons in the BNST results in the insertion of GABAARs with longer decay kinetics, such as those with α2/3 subunits, leading to more smaller-amplitude IPSCs with longer decay.

Bottom Line: It is thought to do so by hijacking brain systems that regulate stress and reward, including neuropeptide Y (NPY) and corticotropin-releasing factor (CRF).The central actions of NPY and CRF have opposing functions in the regulation of emotional and reward-seeking behaviors; thus, dysfunctional interactions between these peptidergic systems could be involved in the development of these pathologies.Together, these data provide both a cellular locus and signaling framework for the development of new therapeutics for treatment of neuropsychiatric diseases, including alcohol use disorders.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA. [2] Bowles Center for Alcohol Studies, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.

ABSTRACT
Binge alcohol drinking is a tremendous public health problem because it leads to the development of numerous pathologies, including alcohol abuse and anxiety. It is thought to do so by hijacking brain systems that regulate stress and reward, including neuropeptide Y (NPY) and corticotropin-releasing factor (CRF). The central actions of NPY and CRF have opposing functions in the regulation of emotional and reward-seeking behaviors; thus, dysfunctional interactions between these peptidergic systems could be involved in the development of these pathologies. We used converging physiological, pharmacological and chemogenetic approaches to identify a precise neural mechanism in the bed nucleus of the stria terminalis (BNST), a limbic brain region involved in pathological reward and anxiety behaviors, underlying the interactions between NPY and CRF in the regulation of binge alcohol drinking in both mice and monkeys. We found that NPY Y1 receptor (Y1R) activation in the BNST suppressed binge alcohol drinking by enhancing inhibitory synaptic transmission specifically in CRF neurons via a previously unknown Gi-mediated, PKA-dependent postsynaptic mechanism. Furthermore, chronic alcohol drinking led to persistent alterations in Y1R function in the BNST of both mice and monkeys, highlighting the enduring, conserved nature of this effect across mammalian species. Together, these data provide both a cellular locus and signaling framework for the development of new therapeutics for treatment of neuropsychiatric diseases, including alcohol use disorders.

Show MeSH
Related in: MedlinePlus