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Ghrelin conditions an avoidance in rodents

S.L Dickson, C. Cook, S.M.Luckman, E Schele

Presented at Neuroscience 2017 Washington DC, USA

Feelings of hunger carry a negative valence (emotion) signal that appears to be conveyed through agouti-related peptide (AgRP) neurons in the hypothalamic arcuate nucleus1. The circulating hunger hormone, ghrelin, activates these neurons although it remains unclear whether it also carries a negative-valence signal. Given that ghrelin also activates pathways in the midbrain that are important for reward, it remains possible that ghrelin could act as a positive reinforcer and hence, carry a positive-valence signal. Here we used condition preference/avoidance tests to explore the reinforcing/aversive properties of ghrelin, delivered by intracerebroventricular (ICV) injection (2 µg/injection once a day for 4 days). We found that ICV ghrelin conditioned avoidance, both in a conditioned place preference/avoidance test (CPP/CPA, in which the animals avoid a chamber previously paired to ghrelin injection) and in a conditioned flavor preference/avoidance test (CFP/CFA, in which the animals consume/avoid a taste previously paired to ghrelin injection). These effects of ghrelin to induce a CPA were observed when conditioning to ghrelin occurred in the absence of food (77% reduction in time spent in ghrelin-paired compartment, P<0.001) and presence of food (in this case an 82% reduction; P<0.001). We did not find evidence, however, that brain ghrelin delivery to rats induces malaise (in the pica test). After conditioning, the preference for the flavor that had been conditioned to ICV ghrelin injection (31 ± 7 %) was dramatically reduced by over half (P < 0.001) compared with the initial preference (71 ± 3 %), which was determined prior to conditioning. Our data indicate that ICV ghrelin carries a negative-valence signal consistent with its role as a circulating hunger hormone and with its effects to activate AgRP neurones

1 Betley JN et al., Nature. 2015 521:180-5.

Computational model of a single oxytocin neuron; spiking, secretion and plasma oxytocin dynamics.

J Maicas Royo, Gareth Leng, Duncan J Macgregor

presented at Intl Union of Physio Sciences World Congress and Word congress on Neurohyp. Hormones , Brazil

The approximately 9000 oxytocin magnocellular neurones of the rat are located in the hypothalamus. From there, they project their axons to the posterior pituitary where they secrete their products into the blood stream. The pattern and quantity of that release depends on the neuronal inputs, their spiking dynamics and on non-linear interaction between spiking activity and exocytosis: the same number of spikes will trigger more secretion when they arrive close together. Here we present a computational model for single oxytocin neurones that can replicate the spiking, secretion and plasma oxytocin dynamics from a wide variety of previously published experimental data. The spiking model mimics electrophysiological data of oxytocin cells responding to cholecystokinin (CCK), a peptide produced in the gut after food intake. The secretion model, which is a modification o an existent vasopressin secretion model [1], matches two independent in vitro experiments. We mimic the plasma clearance of oxytocin with a two-compartment model, replicating the dynamics found after infusion and injection of different oxytocin concentrations. Combining the spiking, secretion and diffusion models, we can infer the spiking activity that produces a given oxytocin concentration in plasma. These inferences match spiking data recorded during constant infusions of NaCl that linearly increase plasma osmotic pressure, or intravenous injection of CCK. In addition, we show how the presence of an after hyperpolarization potential (AHP) in oxytocin neurons dramatically reduces the variability of their spiking activity [2], but also that of oxytocin secretion and plasma levels. The AHP thus acts as a filter, protecting the final product of oxytocin cells from noisy fluctuations.  

Oxytocin single neurones. A spiking and secretion mathematical model and the role of the AHP

Jorge Maicas Royo, Duncan MacGregor, Gareth  Leng

Presented at the joint meeting of the American Physiological Society and  Physiological Society , Dublin, Ireland

 

Magnocellular oxytocin (OT) neurons in the supraoptic nucleus of the hypothalamus project to the posterior pituitary, where they secrete the hormone into the blood stream. OT is essential for breastfeeding and important for parturition. In these cases spike activity is organised into intense bursts[1]. OT has also anorexigenic effects and is involved in osmotic regulation, showing then a continuous increase in spike activity rather than a bursting pattern[1]. In OT cells, spikes are generated in response to afferent inputs that produce fluctuations in membrane potential. That spontaneous spiking activity can be closely matched by models which assume that this afferent input arrives randomly[2]. When spikes propagate to the axon terminals in the posterior pituitary, they trigger exocytosis of hormone containing vesicles. However, OT secretion is a non-linear function of firing rate: the same number of spikes provokes much more secretion if they are close together[3]. It is this feature that makes bursts such an important feature of the milk-ejection reflex. Because of this non-linearity, variance in firing rate that results from the randomly fluctuating synaptic inputs would be expected to be amplified to produce a more variable secretion. Thus there seems to be a conflict: the non-linearity of stimulus-secretion coupling that makes bursts so effective at releasing large pulses of OT during reflex milk ejection will also make secretion very variable in response to a “steady” input. Here we present a mathematical model that replicates the behaviour of spike production and axonal secretion in single OT neurones. The secretion model, based on an existing vasopressin secretion model[4], was matched to in vitro [3][5] experimental data, and accurately reproduces the non-linearity of stimulus-secretion coupling in the OT terminals. The spiking model accurately reproduces the spiking behaviour of OT cells in a wide range of experimental circumstances. Combining the spiking and secretion models allows us to study the responses of the OT system to a fixed transient challenge, mimicking the excitatory response to systemic injection of the gut peptide cholecystokinin (CCK), and in particular, it allows us to study how the response magnitude is affected by factors that affect the basal firing rate of OT neurones. We show that a key feature of the electrophysiological phenotype of OT neurones – their expression of a slow afterhyperpolarisation (AHP) - is a critically important determinant of the variability of the plasma OT concentration that results from secretion. The AHP moderates the variability of spike activity in OT neurones, with a resulting substantial impact on the variability of secretion. The AHP “smooths” the mean firing rate of OT cells over a time scale of a few seconds, avoiding extreme excursions that would result in large fluctuations in secretion.

Reference 1: Caquineau, Céline, and Gareth Leng (2011). ‘Oxytocin and appetite’. In handbook of behavior, food and nutrition , edited by Victor R. Preedy, Ronald Ross Watson, and Colin R. Martin, 289–302. New York, NY: Springer New York. http://link.springer.com/10.1007/978-0-387-92271-3_20.

Reference 2: Maícas Royo, J, C H. Brown, G Leng, and D J MacGregor (2015). ‘Oxytocin neurones: intrinsic mechanisms governing the regularity of spiking activity’. J Neuroendocrinology , doi:10.1111/jne.12358.

Reference 3: Bicknell RJ (1988). Optimizing release from peptide hormone secretory nerve terminals. J Experimental Biology 139: 51–65.

Reference 4: MacGregor, D J., and G Leng (2013). ‘Spike triggered hormone secretion in vasopressin cells; a model investigation of mechanism and heterogeneous population function’. PLoS Comput Biol 9, no.: e1003187.

Reference 5: Bicknell, R J, D Brown, C Chapman, P D Hancock, and G Leng (1984). ‘Reversible fatigue of stimulussecretion coupling in the rat neurohypophysis.’ J Physiology 348: 601–13.

Ghrelin delivery to the lateral parabrachial nucleus increases food intake and changes food choice, without affecting food motivation or food reward behaviour in rats

Tina Bake, Christian Edvardsson, Karolina P Skibicka, Suzanne L Dickson

Presented at Neuronal Control of Appetite, Metabolism and Weight (Z5), Keystone Symposium. Copenhagen, Denmark

 

The lateral parabrachial nucleus (lPBN) in the brainstem has emerged as a key area involved in feeding control. Circulating appetite-regulating hormones, such as GLP-1 and leptin act at the level of the lPBN to alter feeding behaviour. Ghrelin, a stomach-derived orexigenic hormone, promotes both appetitive and consummatory behaviours, engaging homeostatic and reward pathways. Given that ghrelin receptors are present in the lPBN, we investigated whether ghrelin action at this site alters consummatory and appetitive behaviours. Male Sprague-Dawley rats were injected acutely with ghrelin, a ghrelin receptor antagonist (JMV2959) or vehicle directly into the lPBN and then subjected to tests: i) consumption of different foods (chow, chocolate or saccharin solution), ii) a food choice paradigm (chow, lard and sucrose pellets), iii) a progressive ratio (PR) lever-pressing for sucrose task and iv) a conditioned place preference (CPP) test with chocolate as the reinforcer. Intra-lPBN injection of ghrelin increased the intake of chow, chocolate or saccharin solution, while JMV2959 decreased chocolate and saccharin solution intake. In the food choice test, intra-lPBN ghrelin increased the intake of chow but not lard or sucrose pellets. Intra-lPBN ghrelin did not alter behaviour in the PR or CPP tests. Thus, we identify the lPBN as a novel substrate from which ghrelin can alter consummatory behaviours (food intake and choice) but not appetitive behaviours linked to food reward and motivation.

Ghrelin and Reward-Linked Behaviour

Suzanne L Dickson

presented at International Symposium on Ghrelin and Energy Metabolism Homeostasis, Kyoto, Japan

In recent years, we have seen the emergence of a gut-brain reward axis, through which circulating appetite-regulating hormones from the gastro-intestinal tract, such as ghrelin, interact with pathways that confer reward from natural and artificial rewards. In rodents, ghrelin directly activates the mesoaccumbal dopamine pathway at the level of the ventral tegmental area (VTA) causing dopamine release in the nucleus accumbens. Indeed, ghrelin delivery to the brain ventricles or to the VTA can induce reward-linked behaviours for food and for substances of abuse (eg alcohol). Both appetitive and consummatory behaviours are affected by ventricular or VTA delivery of ghrelin, including motivated behavior for sugar (lever-pressing), anticipatory locomotor activity for a sweet treat and food reward behavior (assessed in the condition place preference test). Recently we found that ghrelin impacts on food choice, unexpectedly promoting the intake of normal chow in (i) rats with free access to high fat and sucrose and (ii) rats trained to binge on high fat diet (in a scheduled feeding paradigm).

 

Brain Ghrelin Signaling; Food Reward and Food Choice Behavior

Suzanne Dickson

presented at the 90th Annual Meeting of the Japan Endocrine Society, Kyoto, Japan

 

In recent years, we have seen the emergence of a gut-brain reward axis, through which circulating appetite-regulating hormones from the gastro-intestinal tract, such as ghrelin, interact with pathways that confer reward from natural and artificial rewards. In rodents, ghrelin directly activates the mesoaccumbal dopamine pathway that confers reward from artificial and natural rewards. Delivery of ghrelin into the brain ventricles or into discrete reward areas alters both appetitive (eg food reward and food motivation) and consummatory behaviours (intake, dietary choice, meal patterns). We are currently detangling the neurocircuits underpinning ghrelin’s effects on reward, motivation and food choice.  I will discuss this in the perspective that ghrelin could convey a valence signal, which is dependent on the physiological context and on its target site of action in the brain.

Lamina terminalis, oxytocin neurons and the rat osmotic homeostasis. A computational model.

Jorge Maícas-Royo. Gareth Leng and Duncan J MacGregor

presented at the International Congress of Neuroendocrinology, Toronto, Canada 2018

 

Magnocellular vasopressin and oxytocin neurons in the rat supraoptic nucleus project their axons to the posterior pituitary, where they secrete their product into the bloodstream. Once in plasma, vasopressin and oxytocin contribute to the osmotic homeostasis of the body fluids by promoting natriuresis, increasing blood pressure and inhibiting diuresis. Both vasopressin and oxytocin magnocellular neurons are osmoresponsive. Their membrane dynamics change depending of the osmolality of their extracellular fluid. They integrate that proximal information with inhibitory (IPSPs) and excitatory input afferents (EPSPs) from the organum vasculosum of the lamina terminalis (OVLT), the subfornical organ (SFO) and the median preoptic nucleus (MnPO). The SFO and OVLT contain osmoreceptive neurons that project to magnocellular neurons directly and also indirectly, via the MnPO. In this work we used a previously published computational model (1) that mimics the spiking and secretion activity of oxytocin neurons to simulate the oxytocin plasma response to different osmotic and volumetric challenges. Integrating this oxytocin model with a model of the inputs that oxytocin neurons receive from the circumventricular organs and the MnPO, our results suggest that inhibitory inputs and excitatory inputs are co-activated by osmotic stimuli. We have studied how the gain of osmotically stimulated oxytocin release changes in the presence of a hypovolemic stimulus, showing that this is best explained by an inhibition of an osmotically-regulated inhibitory drive to the magnocellular neurons.

1.  Maícas-Royo J, Leng G, MacGregor DJ. A predictive, quantitative model of spiking activity and stimulus-secretion coupling in oxytocin neurons. Endocrinology. January 2018.

Ghrelin acts in the supramammillary nucleus to regulate food intake in rats

Marie Le May, Nancy Sabatier, Heike Vogel, John Menzies, Gareth Leng, Suzanne L Dickson

Presented at the European College of Neuropharmacology workshop, March 2017, Nice, France

 

Ghrelin provides a gut-brain signal that is important for consummatory-, reward- and novelty-seeking behaviours [1]. One completely unexplored brain target for ghrelin is the supramammillary nucleus (SuM), a posterior hypothalamic area that binds centrally administered fluoro-ghrelin in mice [2]. Given that this brain area is involved in behaviours associated with food intake, food-reward and novelty [3,4], we sought to determine whether ghrelin activates SuM cells and whether ghrelin action at this site can drive food intake and/or alter exploratory behavior for a novel object.

First, we used electrophysiology to record and test with i.v. ghrelin (10 µg) 53 individual SuM cells from 29 rats in vivo. Out of 53 SuM cells, 17 cells (32 %) responded to ghrelin by a significant activation, 11 cells (21 %) responded by a significant inhibition, and 25 cells (47 %) showed a smaller or non-significant response. We analysed the mean change in firing rate over a 20 min period (between 5 min and 25 min after ghrelin injection). For any cell, a change in firing rate that exceeded a threshold (0.5 spikes/s) and was significant at p<0.01 was considered a significant response. In the activated cells, the mean basal firing rate was 1.7 ± 0.5 spikes/s and was increased by a mean change of 1.5 ± 0.3 spikes/s (n = 17). In the inhibited cells, the mean basal firing rate was 1.4 ± 0.3 spikes/s and was decreased by 0.6 ± 0.1 spikes/s (n = 11). The 25 cells that did not show a clear response to ghrelin had a mean basal firing rate of 0.84 ± 0.2 spikes/s and this was unchanged in the 20 min period that followed ghrelin injection (mean change 0.003 ± 0.04 spikes/s).

For feeding studies, male rats received an intra-SuM injection of ghrelin (0.5µg and 1µg) and vehicle on different days in a cross-over design. After a rest day and habituation to the apparatus used (open-field box), the effect of intra-SuM ghrelin (1µg vs vehicle) on novel object recognition (assessing recognition memory and novelty exploration tendency) was evaluated. Ghrelin (1µg) increased chow intake to 4.6 ± 0.5 g, compared to 1.6 ± 0.4 g with vehicle, during the 3 hr after injection (p=0.0358, N=13, two-way ANOVA with Dunnett’s multiple comparison) and tended to increase intake by 6 hr (p=0.0509). The time spent exploring the novel object (compared to the familiar one) did not differ between treatments (p=0.8322, N=7, Mann Whitney test).

In conclusion, we show that systemic ghrelin administration alters the activity of discrete cell groups in the SUM and that ghrelin action at this site contributes to its orexigenic effects but not to its previously reported effects on recognition memory or for exploration of a novel object. Research supported by Nudge-it.

References

[1] Hansson C., Shirazi R.H., Näslund J., Vogel H., Neuber C., Holm G., Anckarsäter H., Dickson S.L., Eriksson E., Skibicka K.P., 2012. Ghrelin influences novelty seeking behaviour in rodents and men. PLoS ONE 7(12), e50409. doi:10.1371/journal.pone.0050409.

[2] Cabral A., Fernandez G., Perello M., 2013. Analysis of brain nuclei accessible to Ghrelin present in the cerebrospinal fluid. Neuroscience 253, 406-415.

[3] Vogel H., Wolf S., Rabasa C., Rodriguez-Pacheco F., Babaei C.S., Stöber F., Goldschmidt, DiMarchi R.D., Finan B.,Tschöp M.H., Dickson S.L., Schürmann A., Skibicka K.P., 2016. GLP-1 and estrogen conjugate acts in the supramammillary nucleus to reduce food-reward and body weight. Neuropharmacology 110, 396-406.

[4] Ito M., Shirao T., Doya K., Sekino Y., 2009. Three-dimensional distribution of Fos-positive neurons in the supramammillary nucleus of the rat exposed to novel environment. Neuro Research 64, 397-402.

Computational model of a single oxytocin neuron; spiking, secretion and plasma oxytocin dynamics.

Jorge Maicas Royo, Gareth Leng, Duncan J MacGregor .

Presented at meeting of British Society for Neuroendocrinology, Nottingham, UK

 

The approximately 9000 oxytocin magnocellular neurones of the rat are located in the hypothalamus. From there, they project their axons to the posterior pituitary where they secrete their products into the blood stream. The pattern and quantity of that release depends on the neuronal inputs, their spiking dynamics and on non-linear interaction between spiking activity and exocytosis: the same number of spikes will trigger more secretion when they arrive close together. Here we present a computational model for single oxytocin neurones that can replicate the spiking, secretion and plasma oxytocin dynamics from a wide variety of previously published experimental data. The spiking model mimics electrophysiological data of oxytocin cells responding to cholecystokinin (CCK), a peptide produced in the gut after food intake. The secretion model, which is a modification o an existent vasopressin secretion model [1], matches two independent in vitro experiments. We mimic the plasma clearance of oxytocin with a two-compartment model, replicating the dynamics found after infusion and injection of different oxytocin concentrations. Combining the spiking, secretion and diffusion models, we can infer the spiking activity that produces a given oxytocin concentration in plasma. These inferences match spiking data recorded during constant infusions of NaCl that linearly increase plasma osmotic pressure, or intravenous injection of CCK. In addition, we show how the presence of an after hyperpolarization potential (AHP) in oxytocin neurons dramatically reduces the variability of their spiking activity [2], but also that of oxytocin secretion and plasma levels. The AHP thus acts as a filter, protecting the final product of oxytocin cells from noisy fluctuations.  


 [1] MacGregor, DJ and Leng G. ‘Spike triggered hormone secretion in vasopressin cells; a model investigation of mechanism and heterogeneous population function’ (2013). PLoS Comput Biol 9, no. 8: e1003187. doi:10.1371/journal.pcbi.1003187.

[2] Maícas Royo, J, CH Brown, G Leng, and DJ MacGregor (2016). ‘Oxytocin neurones: intrinsic mechanisms governing the regularity of spiking activity’. Journal of Neuroendocrinology 28, no. 4: n/a-n/a. doi:10.1111/jne.12358.

Using a single cell model to explain oxytocin neurons ability to reliably report absolute long term levels of gut peptides involved in satiety.

Jorge Maicas Royo, Duncan MacGregor and Gareth Leng

Meeting of British Society for Neuroendocrinology, Lille, France

In analysing the firing patterns of oxytocin cells in the supraoptic nucleus (SON) we noticed an unexpected feature: the mean firing rate at large binwidths is much less variable than expected from the variability at small binwidths, implying a structure in their activity that “smooths out” perturbations in activity.  We have been using computational modelling in an attempt to determine whether this feature can be explained by the after-hyperpolarising potential (AHP) and if the AHP role in oxytocin cells is thus to help produce a relatively stable firing rate. However, a model of oxytocin neurons with an AHP and hyperpolarising after-potential (HAP) was not able to give a good match to both this behaviour and the interspike interval distribution. Our new model solves this by adding equations for a fast depolarising after potential (DAP). To test the role of the AHP with this new model, we matched recordings of 5 oxytocin cells exposed to apamin, a blocker of the AHP, at two concentrations. With the new model we are able to obtain good matches for the 5 cells, and in particular of every interval –baseline, apamin 1 and apamin 2- by varying only the AHP amplitude and the synaptic input rate from interval to interval. Apart from having now a very accurate model for oxytocin cells, we have identified the membrane properties that enable these cells to be very sensitive to small changes in inputs while still having a stable firing rate.

 

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