Pheromone-sensing neurons regulate peripheral lipid metabolism in <i>Caenorhabditis elegans</i>

It is now established that the central nervous system plays an important role in regulating whole body metabolism and energy balance. However, the extent to which sensory systems relay environmental information to modulate metabolic events in peripheral tissues has remained poorly understood. In addition, it has been challenging to map the molecular mechanisms underlying discrete sensory modalities with respect to their role in lipid metabolism. In previous work our lab has identified instructive roles for serotonin signaling as a surrogate for food availability, as well as oxygen sensing, in the control of whole body metabolism. In this study, we now identify a role for a pair of pheromone-sensing neurons in regulating fat metabolism in C. elegans, which has emerged as a tractable and highly informative model to study the neurobiology of metabolism. A genetic screen revealed that GPA-3, a member of the Gα family of G proteins, regulates body fat content in the intestine, the major metabolic organ for C. elegans. Genetic and reconstitution studies revealed that the potent body fat phenotype of gpa-3 null mutants is controlled from a pair of neurons called ADL(L/R). We show that cAMP functions as the second messenger in the ADL neurons, and regulates body fat stores via the neurotransmitter acetylcholine, from downstream neurons. We find that the pheromone ascr#3, which is detected by the ADL neurons, regulates body fat stores in a GPA-3-dependent manner. We define here a third sensory modality, pheromone sensing, as a major regulator of body fat metabolism. The pheromone ascr#3 is an indicator of population density, thus we hypothesize that pheromone sensing provides a salient 'denominator' to evaluate the amount of food available within a population and to accordingly adjust metabolic rate and body fat levels

Comparison of longitudinal in vivo measurements of retinal nerve fiber layer thickness and retinal ganglion cell density after optic nerve transection in rat.

To determine the relationship between longitudinal in vivo measurements of retinal nerve fiber layer thickness (RNFLT) and retinal ganglion cell (RGC) density after unilateral optic nerve transection (ONT).Nineteen adult Brown-Norway rats were studied; N = 10 ONT plus RGC label, N = 3 ONT plus vehicle only (sans label), N = 6 sham ONT plus RGC label. RNFLT was measured by spectral domain optical coherence tomography (SD-OCT) at baseline then weekly for 1 month. RGCs were labeled by retrograde transport of fluorescently conjugated cholera toxin B (CTB) from the superior colliculus 48 hours prior to ONT or sham surgery. RGC density measurements were obtained by confocal scanning laser ophthalmoscopy (CSLO) at baseline and weekly for 1 month. RGC density and reactivity of microglia (anti-Iba1) and astrocytes (anti-GFAP) were determined from post mortem fluorescence microscopy of whole-mount retinae.RNFLT decreased after ONT by 17% (p<0.05), 30% (p<0.0001) and 36% (p<0.0001) at weeks 2, 3 and 4. RGC density decreased after ONT by 18%, 69%, 85% and 92% at weeks 1, 2, 3 and 4 (p<0.0001 each). RGC density measured in vivo at week 4 and post mortem by microscopy were strongly correlated (R = 0.91, p<0.0001). In vivo measures of RNFLT and RGC density were strongly correlated (R = 0.81, p<0.0001). In ONT-CTB labeled fellow eyes, RNFLT increased by 18%, 52% and 36% at weeks 2, 3 and 4 (p<0.0001), but did not change in fellow ONT-eyes sans CTB. Microgliosis was evident in the RNFL of the ONT-CTB fellow eyes, exceeding that observed in other fellow eyes.In vivo measurements of RNFLT and RGC density are strongly correlated and can be used to monitor longitudinal changes after optic nerve injury. The strong fellow eye effect observed in eyes contralateral to ONT, only in the presence of CTB label, consisted of a dramatic increase in RNFLT associated with retinal microgliosis

The relationship between RGC density measured <i>in vivo</i> by CSLO and RGC density measured <i>post mortem</i> by epifluorescence microscopy at week-4.

<p>The relationship between RGC density measured <i>in vivo</i> by CSLO and RGC density measured <i>post mortem</i> by epifluorescence microscopy at week-4.</p

Timeline of procedures, overview of experimental design.

<p>Timeline of procedures, overview of experimental design.</p

Longitudinal measurements of RNFLT for all three experimental groups.

<p>Values of RNFLT were normalized to the baseline average of each eye; symbols represent the group average, error bars indicate SEM. RNFLT decreased in Group-1 ONT eyes by 17%, 30% and 36% at weeks 2, 3 and 4. Group 2 operated eyes (“ONT sans CTB”) exhibited a similar pattern of longitudinal RNFLT change. In Group-1 ONT <i>fellow eyes</i>, RNFLT <i>increased</i> by 18%, 52% and 36% at weeks 2, 3 and 4. There were no significant differences from baseline for any group at week-1. There was no significant change in RNFLT at any follow-up time point in Group-2 non-operated fellow control eyes (“ONT sans CTB Fellow”), Group-3 sham operated (“Sham”) or Group-3 fellow control eyes (“Sham Fellow”). *Significant change from baseline assessed by two-way repeated measures ANOVA with Bonferroni post-hoc tests on raw values (see text for details).</p

Representative individual example of longitudinal CSLO imaging <i>in vivo</i> (same animal as shown in Fig. 1) to assess the change in CTB-labeled RGC density over time after ONT in the right eye (OD) and in the non-operated fellow eye (OS).

<p>RGC density in the ONT eye decreased rapidly; RGC density in the fellow eye remained relatively constant, though decreased slightly over time due to fading of CTB fluorescence. Scale bar = 2 mm.</p

Longitudinal measurements of RGC density <i>in vivo</i> by CSLO in Group-1 (ONT versus ONT fellow eyes) and Group-3 (sham ONT versus sham fellow eyes) animals.

<p>The density of CTB-labeled RGCs decreased in ONT eyes by 18%, 69%, 85% and 92% relative to baseline at follow-up weeks 1, 2, 3 and 4, respectively. RGC density significantly decreased at week-3 by 20% and decreased 19% by week-4. RGC density did not change significantly in sham or sham-fellow eyes. *Significant change from baseline assessed by two-way repeated measures ANOVA with Bonferroni post-hoc tests on raw values. Error bars indicate SEM.</p

Representative individual example showing results for longitudinal SD-OCT measurements of RNFLT over time, beginning at baseline (BL) and continuing weekly for 4-weeks (WK1-WK4) after unilateral optic nerve transection (ONT) in the right eye (OD) and the non-operated fellow eye (OS).

<p>In each panel, the infrared (IR) reflectance image of the ocular fundus is shown at the left and the accompanying SDOCT B-scan is shown at the right; the red circle in the IR image shows the position and path of peripapillary SD-OCT scan. The B-scan images also show the segmentations used to derive RNFLT measurements: green, internal limiting membrane (ILM); red, posterior border of RNFL. RNFLT decreased over time in the ONT eye, as expected, but increased substantially over time in fellow eye. Scale bars = 1 mm.</p

Density of Iba-1 positive (Iba-1+) retinal microglial cells in each of the experimental groups as well as in a group of naïve control eyes.

<p>Microglial cell density was elevated 4 weeks after ONT, whether CTB was present or not, but also substantially elevated in ONT-fellow eyes only if CTB was present. *One-way ANOVA with Bonferroni post-hoc tests (p<0.05). Error bars indicate SEM.</p

The relationship between RGC density measured <i>in vivo</i> by CSLO and RNFLT measured <i>in vivo</i> by SD-OCT.

<p>Longitudinal data for both groups that had RGCs labeled by CTB (Groups 1 and 3) are plotted together in panel A; error bars indicate SEM. In panel B, only the longitudinal data for the individual ONT eyes are plotted.</p