Scientists explore brainstem circuits to find nausea intervention strategies

By Neha MathurJun 20 2022Reviewed by Aimee Molineux

In a recent study published in Cell, researchers uncovered the organization of nausea-associated brainstem circuits to show that selectively blocking the excitatory neurons responsive to nausea-promoting growth/differentiation factor 15 (GDF15) suppresses nausea-related behaviors.

Background

Nausea is an unpleasant and discomforting sensation of visceral malaise that remains poorly understood at the molecular and cellular levels. Hence, it is challenging to develop effective drug targets for nausea intervention.

Several visceral poisons induce the sensation of nausea through the area postrema, a minuscule sensory organ in the brainstem that detects blood-borne factors, such as the gut hormone glucose insulinotropic peptide (GIP). Notably, GIP activates inhibitory neurons that project locally from the area postrema eliciting inhibitory currents in nausea-promoting excitatory neurons through g-aminobutyric acid (GABA) receptors. Moreover, GIP blocks behavioral responses to visceral poisons in the area postrema.

Genetic approaches, such as single-cell complementary deoxyribonucleic acid (cDNA) sequencing, have provided a cellular atlas of the area postrema. They revealed that the area postrema has four excitatory and three inhibitory neuron types.  One of its excitatory neuron type expresses multiple receptors for nausea-inducing stimuli, including the GDF15 receptor (GFRAL), the calcium-sensing receptor (CaSR), and the glucagon-like peptide 1 (GLP1) receptor (GLP1R).

While in humans, GFRAL, GLP1R, and CaSR agonists cause nausea or vomiting, in small animals, such as mice, which cannot vomit, GFRAL evokes conditioned flavor avoidance in which simultaneous administration of poison and a novel flavor makes mice avoid that flavor in the future. This makes GFRAL neurons a key node in nausea circuits.

About the study

In the present study, the authors hypothesized that at least some of the area postrema inhibitory neurons suppress the activity and function of nausea-promoting excitatory neurons and poison responses and used channelrhodopsin (ChR2)-assisted circuit mapping (CRACM) to study the connectivity patterns of area postrema inhibitory neurons.

They injected area postrema of Gad2-ires-Cre, Rosa26-lsl-L10GFP mice with an adeno-associated virus (AAV) containing a cyclic recombinase (Cre)-dependent ChR2-mCherry allele. The researchers performed histological analysis of mCherry expression to confirm proper AAV targeting of the area postrema in every animal post hoc. Note that Gad2-ires-Cre mice have Cre recombinase expression directed to glutamate decarboxylase 2 (GAD2) positive neurons.

The team measured post-synaptic responses in area postrema excitatory neurons in mice. They also observed the consequences of activating area postrema inhibitory neurons using chemogenetic approaches deploying the synthetic agonist clozapine-N-oxide (CNO).

Further, the researchers established a behavioral paradigm in mice to study conditioned flavor avoidance. To this end, they gave water-restricted mice some cherry- or grape-flavored saccharin solution on the conditioning day. Then, they injected mice with saline (control), poisons, and CNO. They used a two-choice assay to measure the behavioral preference of the test animals.

Finally, the team obtained gastric inhibitory polypeptide receptor (GIPR)-Cre mice and used two-color expression analysis to validate the efficient targeting of cluster six area postrema neurons which mediate GIP-induced anti-nausea effects.

Study findings

Whole-cell recordings in area postrema tissue slices revealed light-gated currents in mCherry-positive inhibitory neurons. The authors observed a reduction in the frequency of post-synaptic responses in 27% of area postrema inhibitory neurons and 38% of excitatory neurons in the adjacent nucleus of the solitary tract (NTS) regions. Further, 89% of excitatory neurons displayed light-evoked inhibitory post-synaptic currents (IPSCs). Furthermore, they noted optogenetic activation produced large outward chloride currents in area postrema excitatory neurons. Together, these findings indicated that a majority of area postrema excitatory neurons formed functional connections with some NTS excitatory neurons.

CNO-induced proto-oncogene, AP-1 transcription factor subunit (Fos) expression in mCherry-labeled area postrema inhibitory neurons validated that in the absence of induction of nausea, mice displayed a modest preference for the experienced flavor, including cherry and grape. Conversely, they showed behavioral avoidance for the previously experienced flavors when evoked by various poisons and the GFRAL agonist GDF15.

CNO silenced GDF15 responses in mice but had no effect in control mice, in the presence and the absence of poisons. These findings indicated that at least some area postrema inhibitory neurons inhibit the activity and function of nausea-promoting excitatory neurons. The authors also noted some GIPR-negative cells in the NTS region close by the area postrema, which may have resulted from transient GIPR expression, too low to detect via ribonucleic acid (RNA) in situ hybridization.

Conclusions

The study established GIPR neurons and area postrema inhibitory neurons cluster six as the pharmacological target for suppressing behavioral responses to at least some nausea-inducing toxins. The human body releases GIP after eating calorie-rich foods containing small amounts of harmful chemicals.

To summarize, area postrema located brainstem circuits could inform decision regarding diets for those experiencing nausea based on reward, need, and toxin risk.