Sequential appetite suppression by oral and visceral feedback to the brainstem.

The termination of a meal is controlled by dedicated neural circuits in the caudal brainstem. A key challenge is to understand how these circuits transform the sensory signals generated during feeding into dynamic control of behaviour. The caudal nucleus of the solitary tract (cNTS) is the first site in the brain where many meal-related signals are sensed and integrated1-4, but how the cNTS processes ingestive feedback during behaviour is unknown. Here we describe how prolactin-releasing hormone (PRLH) and GCG neurons, two principal cNTS cell types that promote non-aversive satiety, are regulated during ingestion. PRLH neurons showed sustained activation by visceral feedback when nutrients were infused into the stomach, but these sustained responses were substantially reduced during oral consumption. Instead, PRLH neurons shifted to a phasic activity pattern that was time-locked to ingestion and linked to the taste of food. Optogenetic manipulations revealed that PRLH neurons control the duration of seconds-timescale feeding bursts, revealing a mechanism by which orosensory signals feed back to restrain the pace of ingestion. By contrast, GCG neurons were activated by mechanical feedback from the gut, tracked the amount of food consumed and promoted satiety that lasted for tens of minutes. These findings reveal that sequential negative feedback signals from the mouth and gut engage distinct circuits in the caudal brainstem, which in turn control elements of feeding behaviour operating on short and long timescales.

Enteroendocrine cell types that drive food reward and aversion.

Animals must learn through experience which foods are nutritious and should be consumed, and which are toxic and should be avoided. Enteroendocrine cells (EECs) are the principal chemosensors in the GI tract, but investigation of their role in behavior has been limited by the difficulty of selectively targeting these cells in vivo. Here, we describe an intersectional genetic approach for manipulating EEC subtypes in behaving mice. We show that multiple EEC subtypes inhibit food intake but have different effects on learning. Conditioned flavor preference is driven by release of cholecystokinin whereas conditioned taste aversion is mediated by serotonin and substance P. These positive and negative valence signals are transmitted by vagal and spinal afferents, respectively. These findings establish a cellular basis for how chemosensing in the gut drives learning about food.

Breaking down a gut-to-brain circuit that prevents malabsorption.

The ileal brake is an important reflex that ensures proper absorption of nutrients. This involves intestinal GLP-1 release, which recruits an enteric-sympathetic-spinal pathway to inhibit gastric motility and appetite. This visceral alarm system could be targeted to treat obesity and gastrointestinal dysfunction.

Satb2 neurons in the parabrachial nucleus mediate taste perception.

The neural circuitry mediating taste has been mapped out from the periphery to the cortex, but genetic identity of taste-responsive neurons has remained elusive. Here, we describe a population of neurons in the gustatory region of the parabrachial nucleus that express the transcription factor Satb2 and project to taste-associated regions, including the gustatory thalamus and insular cortex. Using calcium imaging in awake, freely licking mice, we show that Satb2 neurons respond to the five basic taste modalities. Optogenetic activation of these neurons enhances taste preferences, whereas chronic inactivation decreases the magnitude of taste preferences in both brief- and long-access taste tests. Simultaneous inactivation of Satb2 and calcitonin gene-related peptide neurons in the PBN abolishes responses to aversive tastes. These data suggest that taste information in the parabrachial nucleus is conveyed by multiple populations of neurons, including both Satb2 and calcitonin gene-related peptide neurons.

Parabrachial CGRP Neurons Establish and Sustain Aversive Taste Memories.

Food aversions develop when the taste of a novel food is associated with sickness, which often occurs after food poisoning or chemotherapy treatment. We identified calcitonin-gene-related peptide (CGRP) neurons in the parabrachial nucleus (PBN) as sufficient and necessary for establishing a conditioned taste aversion (CTA). Photoactivating projections from CGRPPBN neurons to either the central nucleus of the amygdala or the bed nucleus of the stria terminalis can also induce robust CTA. CGRPPBN neurons undergo plasticity following CTA, and inactivation of either Arc or Grin1 (genes involved in memory consolidation) prevents establishment of a strong CTA. Calcium imaging reveals that the novel food re-activates CGRPPBN neurons after conditioning. Inhibition of these neurons or inactivation of the Grin1 gene after conditioning attenuates CTA expression. Our results indicate that CGRPPBN neurons not only play a key role for learning food aversions but also contribute to the maintenance and expression of those memories.

HSD2 neurons in the hindbrain drive sodium appetite.

Sodium-depleted animals develop an appetite for aversive concentrations of sodium. Here we show that chemogenetic activation of aldosterone-sensitive neurons that express 11ß-hydroxysteroid dehydrogenase type 2 (HSD2) in the nucleus of the solitary tract is sufficient to drive consumption of sodium-containing solutions in mice, independently of thirst or hunger. These HSD2-positive neurons are necessary for full expression of sodium appetite and have distinct downstream targets that are activated during sodium depletion.

Caloric restriction selectively reduces the GABAergic phenotype of mouse hypothalamic proopiomelanocortin neurons.

KEY POINTS: Hypothalamic proopiomelanocortin (POMC) neurons release peptide products that potently inhibit food intake and reduce body weight. These neurons also release the amino acid transmitter GABA, which can inhibit downstream neurons. Although the release of peptide transmitters from POMC neurons is regulated by energy state, whether similar regulation of GABA release might occur had not been examined. The present results show that the GABAergic phenotype of POMC neurons is decreased selectively by caloric deficit and not altered by high-fat diet or stress. The fact the GABAergic phenotype of POMC neurons is sensitive to energy state suggests a dynamic physiological role for this transmitter and highlights the importance of determining the functional consequence of GABA released from POMC neurons in terms of the regulation of normal energy balance. ABSTRACT: In addition to peptide transmitters, hypothalamic neurons, including proopiomelanocortin (POMC) and agouti-related peptide (AgRP) neurons, also release amino acid transmitters that can alter energy balance regulation. While recent studies show that the GABAergic nature of AgRP neurons is increased by caloric restriction, whether the GABAergic phenotype of POMC neurons is also regulated in an energy-state-dependent manner has not been previously examined. The present studies used fluorescence in situ hybridization to detect Gad1 and Gad2 mRNA in POMC neurons, as these encode the glutamate decarboxylase enzymes GAD67 and GAD65, respectively. The results show that both short-term fasting and chronic caloric restriction significantly reduce the percentage of POMC neurons expressing Gad1 mRNA in both male and female mice, with less of an effect on Gad2 expression. Neither acute nor chronic intermittent restraint stress altered Gad1 expression in POMC neurons. Maintenance on a high-fat diet also did not affect the portion POMC neurons expressing Gad1, suggesting that the GABAergic phenotype of POMC neurons is particularly sensitive to energy deficit. Because changes in Gad1 expression have been previously shown to correlate with altered terminal GABA release, fasting is likely to cause a decrease in GABA release from POMC neurons. Altogether, the present results show that the GABAergic nature of POMC neurons can be dynamically regulated by energy state in a manner opposite to that in AgRP neurons and suggest the importance of considering the functional role of GABA release in addition to the peptide transmitters from POMC neurons.

Separate GABA afferents to dopamine neurons mediate acute action of opioids, development of tolerance, and expression of withdrawal.

GABA release from interneurons in VTA, projections from the nucleus accumbens (NAc), and rostromedial tegmental nucleus (RMTg) was selectively activated in rat brain slices. The inhibition induced by µ-opioid agonists was pathway dependent. Morphine induced a 46% inhibition of IPSCs evoked from the RMTg, 18% from NAc, and IPSCs evoked from VTA interneurons were almost insensitive (11% inhibition). In vivo morphine treatment resulted in tolerance to the inhibition of RMTg, but not local interneurons or NAc, inputs. One common sign of opioid withdrawal is an increase in adenosine-dependent inhibition. IPSCs evoked from the NAc were potently inhibited by activation of presynaptic adenosine receptors, whereas IPSCs evoked from RMTg were not changed. Blockade of adenosine receptors selectively increased IPSCs evoked from the NAc during morphine withdrawal. Thus, the acute action of opioids, the development of tolerance, and the expression of withdrawal are mediated by separate GABA afferents to dopamine neurons.

Interglomerular lateral inhibition targeted on external tufted cells in the olfactory bulb.

Lateral inhibition between neurons occurs in many different sensory systems, where it can perform such functions as contrast enhancement. In the olfactory bulb, lateral inhibition may occur between odorant receptor-specific glomeruli that are linked anatomically by GABAergic granule cells (GCs) and cells within the glomerular layer, although evidence supporting lateral inhibition at a functional level is modest. Here, we used patch-clamp, imaging, and glutamate uncaging methods in rat olfactory bulb slices to test for the presence of interglomerular lateral inhibition, as well as its underlying mechanisms. We found that a conditioning stimulus applied at one or a small group of glomeruli could suppress stimulus-evoked excitation of output mitral cells (MCs) at another glomerulus for interstimulus intervals of 20-50 ms and glomerular separations of up to 600 µm. The observed lateral inhibition was entirely dependent on circuitry within the glomerular layer, rather than GCs, and it involved GABAergic synaptic inputs that were targeted mainly onto tufted cells, which act as intermediaries in the excitation between olfactory sensory neurons and MCs. The key cell type responsible for mediating lateral interactions between glomeruli were GABAergic short-axon cells. These results suggest a functional segregation of GABAergic cells within the bulb, with one set located in the glomerular layer mediating suppression of MC spiking across glomeruli, and a second set, the GCs, synchronizing different glomeruli.

Expression of GABAergic and glutamatergic phenotypic markers in hypothalamic proopiomelanocortin neurons.

Hypothalamic proopiomelanocortin (POMC) neurons have traditionally been defined by their peptide transmitters, which are important regulators of energy balance and reward. Recent work shows that POMC neurons can also release the amino acid transmitters γ-aminobutyric acid (GABA) and glutamate, although studying GABAergic and glutamatergic populations of POMC neurons has been hindered by the difficulty in reliably identifying amino acid (AA) transmitter phenotypes. In the present study, fluorescent in situ hybridization and immunohistochemistry were used to identify POMC neurons and to detect the presence of mRNA for the transporters responsible for packaging either GABA (vesicular GABA transporter [vGAT]) or glutamate (vesicular glutamate transporter [vGLUT]) into vesicles, as well as the enzymes responsible for GABA synthesis, glutamic acid decarboxylase (GAD)65 and GAD67. Approximately 7% of POMC neurons expressed vGlut2 and the highest percentage of vGlut2-positive POMC cells were located in the rostral arcuate nucleus. Despite the reports of GABA release from POMC neurons, vGat was not detected in POMC neurons, although Gad65 and Gad67 were present in ~40% of POMC neurons. Approximately half of the vGlut2-expressing POMC cells also expressed Gad65. Markers of neurotransmitter phenotype were better detected by using in situ hybridization techniques rather than transgenic expression of fluorophores under the control of the vGat or Gad67 promoters. It is now clear that the expression of markers of AA phenotype provides a useful means to identify distinct subpopulations of POMC neurons. Additionally, the method described will be useful to explore the possibility that plasticity of AA phenotype is an important aspect of POMC neuron function.