Nicotinic α4 Receptor-Mediated Cholinergic Influences on Food Intake and Activity Patterns in Hypothalamic Circuits.

Nicotinic acetylcholine receptors (nAChRs) play an important role in regulating appetite and have been shown to do so by influencing neural activity in the hypothalamus. To shed light on the hypothalamic circuits governing acetylcholine's (ACh) regulation of appetite this study investigated the influence of hypothalamic nAChRs expressing the α4 subunit. We found that antagonizing the α4β2 nAChR locally in the lateral hypothalamus with di-hydro-ß-erythroidine (DHβE), an α4 nAChR antagonist with moderate affinity, caused an increase in food intake following free access to food after a 12 hour fast, compared to saline-infused animals. Immunocytochemical analysis revealed that orexin/hypocretin (HO), oxytocin, and tyrosine hydroxylase (TH)-containing neurons in the A13 and A12 of the hypothalamus expressed the nAChR α4 subunit in varying amounts (34%, 42%, 50%, and 51%, respectively) whereas melanin concentrating hormone (MCH) neurons did not, suggesting that DHβE-mediated increases in food intake may be due to a direct activation of specific hypothalamic circuits. Systemic DHβE (2 mg/kg) administration similarly increased food intake following a 12 hour fast. In these animals a subpopulation of orexin/hypocretin neurons showed elevated activity compared to control animals and MCH neuronal activity was overall lower as measured by expression of the immediate early gene marker for neuronal activity cFos. However, oxytocin neurons in the paraventricular hypothalamus and TH-containing neurons in the A13 and A12 did not show differential activity patterns. These results indicate that various neurochemically distinct hypothalamic populations are under the influence of α4β2 nAChRs and that cholinergic inputs to the lateral hypothalamus can affect satiety signals through activation of local α4β2 nAChR-mediated transmission.This work was supported by the Royal Society and the European Union (Latin America/European Liason, LAEL).This is the final version of the article. It first appeared from PLOS via http://dx.doi.org/1371/journal.pone.013332

Nicotinic α4 receptor-mediated cholinergic influences on food intake and activity patterns in hypothalamic circuits

Abstract: Nicotinic acetylcholine receptors (nAChRs) play an important role in regulating appetite and have been shown to do so by influencing neural activity in the hypothalamus. To shed light on the hypothalamic circuits governing acetylcholine's (ACh) regulation of appetite this study investigated the influence of hypothalamic nAChRs expressing the α4 subunit. We found that antagonizing the α4β2 nAChR locally in the lateral hypothalamus with di-hydro-ß-erythroidine (DHβE), an α4 nAChR antagonist with moderate affinity, caused an increase in food intake following free access to food after a 12 hour fast, compared to saline-infused animals. Immunocytochemical analysis revealed that orexin/hypocretin (HO), oxytocin, and tyrosine hydroxylase (TH)-containing neurons in the A13 and A12 of the hypothalamus expressed the nAChR α4 subunit in varying amounts (34%, 42%, 50%, and 51%, respectively) whereas melanin concentrating hormone (MCH) neurons did not, suggesting that DHβE-mediated increases in food intake may be due to a direct activation of specific hypothalamic circuits. Systemic DHβE (2 mg/kg) administration similarly increased food intake following a 12 hour fast. In these animals a subpopulation of orexin/hypocretin neurons showed elevated activity compared to control animals and MCH neuronal activity was overall lower as measured by expression of the immediate early gene marker for neuronal activity cFos. However, oxytocin neurons in the paraventricular hypothalamus and TH-containing neurons in the A13 and A12 did not show differential activity patterns. These results indicate that various neurochemically distinct hypothalamic populations are under the influence of α4β2 nAChRs and that cholinergic inputs to the lateral hypothalamus can affect satiety signals through activation of local α4β2 nAChR-mediated transmission

The effects of systemic DHβE injections on HO, MCH, A13-TH and A12-TH activity.

<p>In animals that showed greater food intake compared to controls, DHβE injections at 2 mg/kg increased cFos expression in laterally located HO neurons (A1-2) (Saline and DHβE, n = 6) and decreased cFos expression in total MCH neurons (A3) (Saline, n = 4; DHβE, n = 5). There was no difference between 2 mg/kg DHβE and Saline groups in TH A13 (Saline and DHβE, n = 6) and A12 cFos expression (Saline and DHβE, n = 4) (B1-2). Abbreviations: HO, hypocretin/orexin; MCH, melanin concentrating hormone; TH, tyrosine hydroxylase; f, fornix; DMH, dorsomedial hypothalamus; LH, lateral hypothalamus; PeF, perifornical region; PeFLH, perifornical region of the hypothalamus; Arc, arcuate nucleus. Scale bar: A, 300 μm; B, 100 μm.</p

The effects of systemic DHβE injection on food intake and PVH activity.

<p>A1. Schematic demonstrating the experimental setup. A2. Animals injected with 2 mg/kg DHβE i.p. consumed more food than saline injected animals (saline, n = 7; 1 mg/kg, n = 7; 2 mg/kg, n = 6). Total cFos (B1) (Saline and DHβE, n = 5) and percent of PVH oxytocin-expressing neurons (B2) (Saline and DHβE, n = 4) were not significantly different between DHβE and Control conditions (B3). Abbreviations: PVH, paraventricular nucleus of the hypothalamus; OXY, oxytocin 3V, third ventricle. Scale bar: 100 μm.</p

Hypothalamic TH and oxytocin neurons expressing the α4 nicotinic subunit.

<p>A. 50.1% of TH-containing neurons in the A13 were colocalized with α4 (A1,2) while 50.7% of A12 TH-containing neurons in the arcuate nucleus showed α4 nAChR co-expression (A1,A2) (α4-TH, n = 3). There was no difference in colocalization across the different arcuate subdivisions analyzed (A2). B. Within the PVH, 41.3% of oxytocin neurons were also immunolabeled for α4 (α4-oxy, n = 4). Abbreviations: TH, tyrosine hydroxylase; Oxy, oxytocin; PVH, paraventricular nucleus of the hypothalamus; Arc, Arcuate nucleus; 3V, third ventricle. Scale bar: 200 μm.</p

The effects of DHβE infusions into the lateral hypothalamus on food intake.

<p>A. Scheme demonstrating the experimental setup. Intra-cerebral cannulae were confined to the lateral hypothalamic region (B2) an example of which is shown in B1. Cumulative measurements of food intake showed that DHβE-infused animals consumed more food 2 hours post ad libitum food access following a 12 hour fast (C) (saline, n = 8; 20 μg DHβE, n = 7). Abbreviations: mt, mammillothalamic tract; f, fornix.</p

Example immunolabeling for α4 (A), oxytocin in the PVH (B), tyrosine-hydroxylase in the A13 (C1) and arcuate nucleus (C2), and HO (D1) and MCH (D2) immunoreactivity throughout the hypothalamus.

<p>Abbreviations: OXY, oxytocin; PVH, paraventricular nucleus of the hypothalamus; TH, tyrosine hydroxylase; HO, Hypocretin/Orexin; MCH, Melanin Concentrating Hormone; 3V, third ventricle; mt, mammillothalamic tract; f, fornix. Scale bar: A, B 100 μm; C-E 300 μm.</p