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Effect of modulating leptin receptor expressing neurons in lateral hypothalamus and ventral tegmental area on motivation in mice
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Behavioural and systems
aversive vs neutral faces) were entered into second-level design matrices to determine associations with neuroticism and 5-HTTLPR. Statistically significant clusters were determined using 3dClustSim and a voxel-level significance threshold of P < 0.01. Results: The personality measure neuroticism did not differ between 5-HTTLPR genotype groups (P = 0.86). Regarding amygdala psycho-physiological-interaction (PPI), we observed a statistically significant cluster within an mPFC ROI, wherein s’ carriers showed statistically significantly greater left amygdala functional connectivity compared to LALA individuals ([−8, 40, 0], z = 3.23, k = 284 voxels). Whole-brain analyses revealed a statistically significant negative association between neuroticism and left and right amygdala functional connectivity within a cluster including left lateral orbitofrontal cortex (lOFC), ventrolateral prefrontal cortex (vlPFC) and temporal pole (left amygdala: [−28, 24, −12], z = 4.37, k = 1061 voxels; right amygdala: [−46, 20, −18], z = 4.19, k = 1674 voxels). We observed a significant moderation effect of 5-HTTLPR and neuroticism on functional connectivity between both amygdalae and left lOFC/vlPFC/temporal pole (interaction effect on extracted cluster connectivity estimate: left amygdala [95% CI]: −0.064 [−0.11, −0.018], puncorr = 0.0067, qFDR = 0.027; right amygdala: −0.057 [−0.097, −0.017], puncorr = 0.006, qFDR = 0.027). Specifically, 5HTTLPR moderated the association between neuroticism and functional connectivity between both amygdalae and left lOFC/vlPFC, such that s’ carriers exhibited a more negative association relative to LALA individuals. Conclusion: In summary, we observed independent and interactive effects of 5-HTTLPR genotype and neuroticism on threat-related amygdala functional connectivity within regions critically involved in threat processing, emotion regulation and visual processing. These findings benefit our understanding of genetic sources of variability in threat-related neural pathways and highlight novel aspects of brain function, which may mediate 5-HTTLPR and neuroticism-related risk for mood and affective disorders. Reference(s) [1] LeDoux, J.E., 2000. Emotion circuits in the brain. Annual Review of Neuroscience, 23, 155–84. [2] McGuffin, P., Alsabban, S., Uher, R., 2011. The truth about genetic variation in the serotonin transporter gene and response to stress and medication. The British Journal of Psychiatry, 198(6), 424–7. [3] Klein, D.N., Kotov, R., Bufferd, S.J., 2011. Personality and depression: explanatory models and review of the evidence. Annual Review of Clinical Psychology, 7, 269–95.
[4] McLaren, D.G., Ries, M.L., Xu, G., Johnson, S.C., 2012. A generalized form of context-dependent psychophysiological interactions (gPPI): a comparison to standard approaches. Neuroimage, 61(4), 1277–86. P.2.014 Effect of modulating leptin receptor expressing neurons in lateral hypothalamus and ventral tegmental area on motivation in mice V. De Vrind1*, R. Van Zessen1, A. Omrani1, R. Adan1. 1Brain Center Rudolph Magnus, Translational Neuroscience, Utrecht, The Netherlands Background: Leptin is an adipose tissue derived hormone which circulates in levels proportional to the amount of adipose tissue. It is involved in body weight homeostasis and is well-known for reducing food intake. Of importance here, leptin has been shown to reduce the increase in motivation for food reward caused by food restriction in rodents ([1] and unpublished data in rats from our lab). Similarly, leptin repletion in weight-reduced humans leads to increased feelings of satiation [2] and to decreased functional connectivity in pathways involved in reward valuation [3]. Studying the role that leptin plays in motivation for food may help in further understanding the underlying mechanisms in body weight regulation and how decreases in body weight alter the incentive motivation towards food. Ventral tegmental area (VTA) dopamine (DA) neurons play a critical role in motivation. Therefore, in this set of experiments we will examine the extent to which the VTA contributes to the behavioral effects seen upon leptin repletion in weight-reduced subjects. Methods: For anatomical studies, immunohistochemistry was performed on leptin receptor-cre (LepRcre) mice crossed with a Rosa26-YFP reporter line. For behavioral studies adult male LepRcre mice were injected with AAV-DIO-hM3Dq (DREADDq) which induces neuronal activation upon clozapine-N-oxide (CNO) injection. Animals were tested on multiple behavioral tasks, such as the progressive ratio (PR), open field and free feeding task, to assess effects on, motivation, locomotion and feeding, respectively. Tests were done during both food restriction (low leptin levels) and ad libitum (high leptin levels) to compare the effect of different baseline leptin levels on behavioral aspects. Results: Immunohistochemistry done on LepRcre/ Rosa26-YFP mice showed that the LepR is indeed expressed in the VTA. However, only ∼5% of the total
Behavioural and systems amount of VTA DA neurons expressed the LepR and this represented ∼25% of all LepRs in the VTA. Consistently, activating LepR neurons using DREADDq in the VTA did not affect locomotion, feeding or motivation for food. This suggested that leptin’s effects on motivation originate from leptin responsive areas that provide input to the VTA. By injecting a fluorescently labeled retrograde herpes simplex virus into the VTA of a LepRcre mouse, we showed that the lateral hypothalamus (LH) contained the largest population of LepR neurons projecting to the VTA. Therefore, further behavioral studies were focused on the LH. Preliminary results show that activating LH LepR neurons increases performance on both the open field (111.9% ± 3.5 total distance covered; CNO as percentage of saline) and the PR (active lever presses 140.8% ± 16.8; CNO as percentage of saline) but not on the free feeding task. Conclusion: Our anatomical and behavioral data support the idea that not leptin sensitive neurons in the VTA, but instead in the LH, mediate the increased motivation for food under conditions of negative energy balance. Reference(s) [1] Sharma, S., 2012. Progressive-ratio responding for palatable high-fat and high-sugar food in mice. J. Vis. Exp., http://dx.doi.org/10.3791/3754 [2] Kissileff, H., 2012. Leptin reverses declines in satiation in weight-reduced obese humans. Am. J. Clin. Nutr., https://dx.doi.org/10.3945%2Fajcn.111. 012385 [3] Hinkle, W., 2013. Effects of reduced weight maintenance and leptin repletion on functional connectivity of the hypothalamus in obese humans. Plos One, https:// dx.doi.org/10.1371/journal.pone.0059114
P.2.015 Enduring neurobehavioural effects in mice induced by early-life microbiotagut-brain axis disruption: reversal by oxytocin L. Morais1,2*, P.J. Kannedy3, G. Moloney1, A.V. Golubeva4, K. Rea4, Y. Borre4, K.A. Scott4, E. Patterson4, R. Stilling4, A. Moya-Pérez4, A.E. Hoban4, S. El Aidy4, S. Beers4, O. O’Sullivan4, R. Moloney4, P. Ross4, P.D. Cotter4, C. Stanton4, T. Dinan3, J.F. Cryan1. 1APC Microbiome Institute- University College Cork, Neuroscience&Anatomy, Cork, Ireland; 2 APC Microbiome Institute- University College Cor, Neurosciece, Cork, Ireland; 3APC Microbiome
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Institute- University College Cork, Psychiatry and Neurobehavioural Science, Cork, Ireland; 4APC Microbiome Institute- University College Cork, APC Microbiome Institute, Cork, Ireland Mounting evidence points to a role for the microbiotagut-brain axis in regulating many aspects of behaviour and brain health. The bidirectional communication between the microbiota and the brain involves neural, endocrine, metabolic and immunological pathways [1]. Early-life is a critical developmental window for the microbiome and the brain as the microbial colonization in the infant coincides with key neurodevelopmental period [2]. The initial seeding of the gut microbiome occurs during birth as the infant emerges through the mother’s birth canal. However, birth by Caesarean-section (C-section) affects early colonization of the gut microbiota rewiring the entire microbiota-gut-brain axis [3]. The neuropeptide oxytocin (OXT) is released during birth and it has been stongly implicated in social behaviour and anxiety and may contribute to a wide range of psycopathologies. Recently, oxytocin brain levels was shown to be regulated by the presence of commensal bacteria and thereby improve social behavioural deficits [4]. The aim of the present study was to assess the consequences of the early-life microbiota disruption on mouse behaviour and on the microbiota gut-brain axis. Following birth by C-Section or per vaginum pups were given to foster dams. Additional pregnant females were allowed to deliver spontaneously and the litters were used as full-term vaginal delivery control group. Social and anxiety-like behaviour and subsequent physiological, molecular and microbiota parameters were assessed. In early-life, animals born by C-section exhibited anxietylike behaviour as measured by an increase in the ultrasonic vocalization in the postnatal day (P) 9 ( p < 0.05). In adulthood, C-section offspring exhibited deficits in social novelty preference ( p < 0.001), and anxiety-like behaviour in the marble burying test ( p < 0.001). Analyses of the hypothalamic-pituitary-adrenal axis activity revealed that C-section offspring have an increased corticosterone release in response to stress in adulthood ( p < 0.05). One potential mechanism mediating the behavioural changes and HPA axis dysfunction due to C-section may be increased intestinal permeability. Thus, we have investigated gastrointestinal permeability to lipopolysaccharide (LPS) in early-life (P9), adolescence (P25) and adulthood (P150). At P9 and P25, LPS concentrations in the plasma were increased in mice born by C-section ( p < 0.05). The increased gut permeability to LPS was associated with an exaggerated immune-response in C-section born mice, as measured by an increased production of IL-10 ( p < 0.05) and TNF-α ( p < 0.05).
Behavioural and systems
aversive vs neutral faces) were entered into second-level design matrices to determine associations with neuroticism and 5-HTTLPR. Statistically significant clusters were determined using 3dClustSim and a voxel-level significance threshold of P < 0.01. Results: The personality measure neuroticism did not differ between 5-HTTLPR genotype groups (P = 0.86). Regarding amygdala psycho-physiological-interaction (PPI), we observed a statistically significant cluster within an mPFC ROI, wherein s’ carriers showed statistically significantly greater left amygdala functional connectivity compared to LALA individuals ([−8, 40, 0], z = 3.23, k = 284 voxels). Whole-brain analyses revealed a statistically significant negative association between neuroticism and left and right amygdala functional connectivity within a cluster including left lateral orbitofrontal cortex (lOFC), ventrolateral prefrontal cortex (vlPFC) and temporal pole (left amygdala: [−28, 24, −12], z = 4.37, k = 1061 voxels; right amygdala: [−46, 20, −18], z = 4.19, k = 1674 voxels). We observed a significant moderation effect of 5-HTTLPR and neuroticism on functional connectivity between both amygdalae and left lOFC/vlPFC/temporal pole (interaction effect on extracted cluster connectivity estimate: left amygdala [95% CI]: −0.064 [−0.11, −0.018], puncorr = 0.0067, qFDR = 0.027; right amygdala: −0.057 [−0.097, −0.017], puncorr = 0.006, qFDR = 0.027). Specifically, 5HTTLPR moderated the association between neuroticism and functional connectivity between both amygdalae and left lOFC/vlPFC, such that s’ carriers exhibited a more negative association relative to LALA individuals. Conclusion: In summary, we observed independent and interactive effects of 5-HTTLPR genotype and neuroticism on threat-related amygdala functional connectivity within regions critically involved in threat processing, emotion regulation and visual processing. These findings benefit our understanding of genetic sources of variability in threat-related neural pathways and highlight novel aspects of brain function, which may mediate 5-HTTLPR and neuroticism-related risk for mood and affective disorders. Reference(s) [1] LeDoux, J.E., 2000. Emotion circuits in the brain. Annual Review of Neuroscience, 23, 155–84. [2] McGuffin, P., Alsabban, S., Uher, R., 2011. The truth about genetic variation in the serotonin transporter gene and response to stress and medication. The British Journal of Psychiatry, 198(6), 424–7. [3] Klein, D.N., Kotov, R., Bufferd, S.J., 2011. Personality and depression: explanatory models and review of the evidence. Annual Review of Clinical Psychology, 7, 269–95.
[4] McLaren, D.G., Ries, M.L., Xu, G., Johnson, S.C., 2012. A generalized form of context-dependent psychophysiological interactions (gPPI): a comparison to standard approaches. Neuroimage, 61(4), 1277–86. P.2.014 Effect of modulating leptin receptor expressing neurons in lateral hypothalamus and ventral tegmental area on motivation in mice V. De Vrind1*, R. Van Zessen1, A. Omrani1, R. Adan1. 1Brain Center Rudolph Magnus, Translational Neuroscience, Utrecht, The Netherlands Background: Leptin is an adipose tissue derived hormone which circulates in levels proportional to the amount of adipose tissue. It is involved in body weight homeostasis and is well-known for reducing food intake. Of importance here, leptin has been shown to reduce the increase in motivation for food reward caused by food restriction in rodents ([1] and unpublished data in rats from our lab). Similarly, leptin repletion in weight-reduced humans leads to increased feelings of satiation [2] and to decreased functional connectivity in pathways involved in reward valuation [3]. Studying the role that leptin plays in motivation for food may help in further understanding the underlying mechanisms in body weight regulation and how decreases in body weight alter the incentive motivation towards food. Ventral tegmental area (VTA) dopamine (DA) neurons play a critical role in motivation. Therefore, in this set of experiments we will examine the extent to which the VTA contributes to the behavioral effects seen upon leptin repletion in weight-reduced subjects. Methods: For anatomical studies, immunohistochemistry was performed on leptin receptor-cre (LepRcre) mice crossed with a Rosa26-YFP reporter line. For behavioral studies adult male LepRcre mice were injected with AAV-DIO-hM3Dq (DREADDq) which induces neuronal activation upon clozapine-N-oxide (CNO) injection. Animals were tested on multiple behavioral tasks, such as the progressive ratio (PR), open field and free feeding task, to assess effects on, motivation, locomotion and feeding, respectively. Tests were done during both food restriction (low leptin levels) and ad libitum (high leptin levels) to compare the effect of different baseline leptin levels on behavioral aspects. Results: Immunohistochemistry done on LepRcre/ Rosa26-YFP mice showed that the LepR is indeed expressed in the VTA. However, only ∼5% of the total
Behavioural and systems amount of VTA DA neurons expressed the LepR and this represented ∼25% of all LepRs in the VTA. Consistently, activating LepR neurons using DREADDq in the VTA did not affect locomotion, feeding or motivation for food. This suggested that leptin’s effects on motivation originate from leptin responsive areas that provide input to the VTA. By injecting a fluorescently labeled retrograde herpes simplex virus into the VTA of a LepRcre mouse, we showed that the lateral hypothalamus (LH) contained the largest population of LepR neurons projecting to the VTA. Therefore, further behavioral studies were focused on the LH. Preliminary results show that activating LH LepR neurons increases performance on both the open field (111.9% ± 3.5 total distance covered; CNO as percentage of saline) and the PR (active lever presses 140.8% ± 16.8; CNO as percentage of saline) but not on the free feeding task. Conclusion: Our anatomical and behavioral data support the idea that not leptin sensitive neurons in the VTA, but instead in the LH, mediate the increased motivation for food under conditions of negative energy balance. Reference(s) [1] Sharma, S., 2012. Progressive-ratio responding for palatable high-fat and high-sugar food in mice. J. Vis. Exp., http://dx.doi.org/10.3791/3754 [2] Kissileff, H., 2012. Leptin reverses declines in satiation in weight-reduced obese humans. Am. J. Clin. Nutr., https://dx.doi.org/10.3945%2Fajcn.111. 012385 [3] Hinkle, W., 2013. Effects of reduced weight maintenance and leptin repletion on functional connectivity of the hypothalamus in obese humans. Plos One, https:// dx.doi.org/10.1371/journal.pone.0059114
P.2.015 Enduring neurobehavioural effects in mice induced by early-life microbiotagut-brain axis disruption: reversal by oxytocin L. Morais1,2*, P.J. Kannedy3, G. Moloney1, A.V. Golubeva4, K. Rea4, Y. Borre4, K.A. Scott4, E. Patterson4, R. Stilling4, A. Moya-Pérez4, A.E. Hoban4, S. El Aidy4, S. Beers4, O. O’Sullivan4, R. Moloney4, P. Ross4, P.D. Cotter4, C. Stanton4, T. Dinan3, J.F. Cryan1. 1APC Microbiome Institute- University College Cork, Neuroscience&Anatomy, Cork, Ireland; 2 APC Microbiome Institute- University College Cor, Neurosciece, Cork, Ireland; 3APC Microbiome
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Institute- University College Cork, Psychiatry and Neurobehavioural Science, Cork, Ireland; 4APC Microbiome Institute- University College Cork, APC Microbiome Institute, Cork, Ireland Mounting evidence points to a role for the microbiotagut-brain axis in regulating many aspects of behaviour and brain health. The bidirectional communication between the microbiota and the brain involves neural, endocrine, metabolic and immunological pathways [1]. Early-life is a critical developmental window for the microbiome and the brain as the microbial colonization in the infant coincides with key neurodevelopmental period [2]. The initial seeding of the gut microbiome occurs during birth as the infant emerges through the mother’s birth canal. However, birth by Caesarean-section (C-section) affects early colonization of the gut microbiota rewiring the entire microbiota-gut-brain axis [3]. The neuropeptide oxytocin (OXT) is released during birth and it has been stongly implicated in social behaviour and anxiety and may contribute to a wide range of psycopathologies. Recently, oxytocin brain levels was shown to be regulated by the presence of commensal bacteria and thereby improve social behavioural deficits [4]. The aim of the present study was to assess the consequences of the early-life microbiota disruption on mouse behaviour and on the microbiota gut-brain axis. Following birth by C-Section or per vaginum pups were given to foster dams. Additional pregnant females were allowed to deliver spontaneously and the litters were used as full-term vaginal delivery control group. Social and anxiety-like behaviour and subsequent physiological, molecular and microbiota parameters were assessed. In early-life, animals born by C-section exhibited anxietylike behaviour as measured by an increase in the ultrasonic vocalization in the postnatal day (P) 9 ( p < 0.05). In adulthood, C-section offspring exhibited deficits in social novelty preference ( p < 0.001), and anxiety-like behaviour in the marble burying test ( p < 0.001). Analyses of the hypothalamic-pituitary-adrenal axis activity revealed that C-section offspring have an increased corticosterone release in response to stress in adulthood ( p < 0.05). One potential mechanism mediating the behavioural changes and HPA axis dysfunction due to C-section may be increased intestinal permeability. Thus, we have investigated gastrointestinal permeability to lipopolysaccharide (LPS) in early-life (P9), adolescence (P25) and adulthood (P150). At P9 and P25, LPS concentrations in the plasma were increased in mice born by C-section ( p < 0.05). The increased gut permeability to LPS was associated with an exaggerated immune-response in C-section born mice, as measured by an increased production of IL-10 ( p < 0.05) and TNF-α ( p < 0.05).