**3.2 Gut-brain Neural Circuits**

#### *3.2.1 Humans*

Nutrient signaling and sensing are fundamental processes that animals including humans and flies undergo [131]. Proper coordination and communication between gut and brain is necessary to regulate metabolic homeostasis and physiology in all animals. In this regard, many research groups have shown the role of enteric neurons and endocrine signals as important mediators of these processes. The way the enteric nervous system communicates to the brain via neural circuits is a multifaceted question and poorly explored. In mammals such as humans, alterations in neuropeptides and brain – gut hormone levels can derail people otherwise on the path to a healthy life. These changes can also lead to diseases such as neurodegenerative diseases, metabolic syndrome and diabetes [132].

Gluconeogenesis is a biochemical pathway by which animals make sugars from non-carbohydrate precursors and sources [133]. It is used to regulate homeostasis and a stable internal state in post – fed state [134]. Studies in rats showed that stimulation of intestinal gluconeogenesis (IGN) sends a signal from sodium – glucose co- transporters present at the intestinal mucosa to the brain, initiating a neural gut – brain axis [135–137]. Diets rich in protein [138–140] and fiber [141] promote IGN stressing on the importance of nutrient sensing for initiating several gut – brain axis [137]. It has been found that μ – opoid receptors (MOR) regulate IGN. These receptors (present in the nerves in the portal vein wall) react to neuropeptides to stimulate a gut – brain neural circuit that affects IGN, hunger and satiety mechanisms [141]. Further analysis of MOR deficient mice shows the role of MORs in regulating food intake, referred as "reward" system [142, 143]. Analyses of MOR-knockouts (MOR-KO) demonstrate how they play a role in managing satiety effects of alimentary proteins, through a neural gut-brain circuit [140].

Vagus nerve (VN; pneumogastric nerve) is the longest cranial nerve [144] in humans which runs from the medulla oblongata in brain to colon in GI [145]. It innervates other structures as well such as larynx, pharynx, heart and lungs thus affects digestive, cardiovascular and respiratory system – all at one [146]. Vagal efferent send down signals from the brain to gut, which accounts for about 10% – 20% of all the nerve fibers. Remaining 80% is accounted for by the vagal afferents carrying information from the gut to the brain [147]. Vagal sensory neurons in the GI keep an eye on stomach volume and luminal contents through different neural circuits [148]. VN contains and branches into several sensory neurons (~2300 in mouse) that further innervate and render support and supply to other internal organs. A variety of sensory neurons, one side facing the brainstem and the terminal one facing the organ such as GI [149] have been revealed. Free terminals of vagal afferents are rooted within lamina propria of intestinal villi [148]. Some mammalian models like in cat and rat, it has been shown how these sensory neurons detect different nutrients in diets with the help of unambiguous and explicit fibers [150–152]. Vagal afferent endings in the intestine express several mechanosensitive as well as chemical receptors [153]. Glucagon- like peptide 1 (GLP1) is a gut

hormone receptor that intercedes the nutrient sensing mechanism via VN [154]. GLP1R (GLP1 receptor) is present in many cells [155]. Agonists for GLP1R show how it affects brain further proving its presence in both, gut and brain [156]. Another receptor of vagal afferents, GPR65 near the intestinal villi, plays a role in nutrient detection drawing attention to how these sensory neurons are a part of the gut – brain axis [157, 158]. It detects serotonin and impact gut motility [147]. Such receptors detect several hormones present in the gut, like choleocystokinin (CKK), ghrelin and leptin which play a role in the regulation of hunger and satiety [159–161]. Because of its role in gut motility and mobility, VN and its afferent neurons present in the gut play a role in Intestinal Bowel Syndrome (IBS) [162] and new treatment plans around the same are being looked at in rat [162] and mice [158] models.

To take the findings in vagal nerves forward, nerves allowing communication of cNST (caudal nucleus of the solitary tract) with gut were focused on. Information about sugar detection to cNST via gut – brain axis is a topic of research nowadays. In live mice, it is noticed that glucose detection by cNST is robust and VN transaction silences that activation [163]. Nodose ganglion of vagus nerve when silenced prevents the sugar preference of cNST [163] suggesting the presence of a physical gut – brain axis. It has been shown that inactivation of sugar-activated cSNT prevents the mice to choose sugar from water or an artificial sweetener [163]. This study specifically calls attention to how organisms have paths for detecting nutrient signals, sensing them in the diet and also have circuitries to carry forward the signals and communicate with the rest of the body, purposely the brain.

### *3.2.2 Drosophila*

With the help of several markers and reporter genes, it is found that *Drosophila* intestine is innervated by neurons- efferent and sensory [14]. Other studies have stated that fly's gut receives innervations from three regions – stomato-gastric nervous system [164–167]; the *corpora cardiaca*, neurosecretory structures [168]; and neurons located in the CNS extending their axons toward three different portions of the digestive tract [14, 169–172]. The expression of Ret receptor tyrosine kinase in gut innervating neurons in adult fly has recently been shown to contribute to the development of stomato- gastric ganglia in flies [173, 174]. In contrast to mammalian gastrointestinal tracts which are profusely innervated throughout their entire length, the innervation of the fly's digestive tract is restricted to only three different portions. The first is the anterior-most slice comprising the pharynx, esophagus, crop and anterior midgut. The second is midgut/hindgut junction and third is the posterior hindgut [14, 164, 167, 172]. Muscle valves present in all three regions support and regulate peristaltic regulation and intestinal transit functions of gut-innervating neurons. Most neurites terminate on the visceral muscles and some reach the underlying epithelium, particularly in the esophagus, proventriculus, pyloric valve, and rectal ampulla [14, 175] suggesting neuronal regulation of epithelial properties such as secretion or absorption. In flies, not all innervation is efferent. Gustatory neuron afferents from the pharynx send their axons to the suboesophageal zone (SEZ, the primary taste center of the fly brain), where they target a distinct domain adjacent to the projections of other (leg/labellum) gustatory receptor neurons [176–179]. Dendrites of peripheral sensory neurons can be seen in the anterior and posterior-most regions of the digestive tract [14], and appear most abundant in the esophagus and anterior midgut.

In the anterior portion of adult and larval midgut, serotonin positive neurites and various neuropeptides including Akh, Dh44, Myosuppressin, and possibly Allatostatin C and FMRFamide (or an FMRFamide-like peptide such as the NPY-like

#### *Gut Feeding the Brain:* Drosophila *Gut an Animal Model for Medicine to Understand… DOI: http://dx.doi.org/10.5772/intechopen.96503*

neuropeptide short neuropeptide F [sNPF]) [131, 168, 172, 180–182] have been described suggesting chemical diversity of enteric innervation. Four serotonergic neurons are found to innervate the enteric nervous system in fly larva. These neurons project from the antennal nerve (AN) near the SEZ and extend throughout the ENs [14]. These projections end at the anterior region of the midgut and are primarily around the proventriculus region and the foregut. These innervations are considered structurally analogous to the mammalian VN because of similar projections from the brain to the different structures of the foregut. It is yet to be seen if there are functional similarities as well [172].

Pigment- dispersing factor (Pdf), Ion transport peptide, and Proctolin positive neurites have been reported in the larval and adult hindgut [169, 183–186]. All three innervated regions receive insulinergic innervation from the CNS; the *pars intercerebralis* (PI) insulin-producing cells extend axons beyond the ring gland that innervate the anterior midgut and crop in adult flies (**Figure 3**), and the insulinlike peptide 7 (Ilp7)-producing neurons of the abdominal ganglion innervate the midgut/hindgut junction and the rectal ampulla [170, 187]. Interestingly, putative dendritic termini of both kinds of insulin-producing neurons have been found in very close proximity in the CNS. This data suggests the release of different insulins to the different portions of the digestive tract may be co-regulated centrally [14].

Functional studies of insect innervation have primarily focused on the control of peristalsis and peptide hormone secretion so far. Studies of peristaltic regulation in flies have primarily concerned the effects of neuropeptides (Allatostatins, Myosuppressin, or Drosulfakinins) on *ex vivo* intestinal preparations [188–191] ascribing distinct roles for these peptides in the modulation of crop or anterior midgut contractions in adults. Both intestinal and non-intestinal roles of Pdfexpressing neurons in the regulation of muscle peristalsis for a set of hindgutinnervating neurons located in the abdominal ganglion of the CNS have been demonstrated [171, 192]. It is found that this neural source of Pdf (a neuropeptide related to mammalian vasoactive intestinal polypeptides, known for its roles in the central circadian clock) promotes peristalsis of hindgut muscles and sustains the defecation cycle in larvae [192]. Pdf can also promote contractions of the muscles of the ureters, the proximal part of the malpighian tubules [171]. Hence, the digestive tract is used by some enteric neurons as a docking site to exert their functions on other internal organs at some distance.

Recently epithelial roles for gut-innervating neurons e.g. role in the control of fluid balance have been revealed by a semi-automated analysis of defecation behavior in adult flies, providing quantitative readouts for food intake, fluid/ion balance, and intestinal transit [14, 193]. The HGN1 neurons (2–5 CNS neurons located in the posterior segments of the abdominal ganglion) innervate the hindgut and the rectum (**Figure 3**), with a subset of their neurites projecting through the visceral muscles to reach the underlying epithelium [14]. HGN1 neuronal silencing experiments resulted in increased defecation rate. These neurons are shown to be required for the post-mating changes in intestinal fluid retention due to their epithelial innervation. It has been established that HGN1 neurons and the Pdf hindgutinnervating neurons have their direct action on the hindgut and anal sphincter muscles [192]. A role for gut-innervating neurons in the maintenance of epithelial turnover has also been suggested by the finding of anatomical proximity between enteric neurites in the posterior midgut and adult somatic intestinal progenitors, and the reduced ISC to EC differentiation resulting from downregulating Hedgehog (Hh) signaling (albeit pan-neuronally) [194]. The more anterior innervation of the proventriculus may also play a role in maintaining gut permeability. This is inferred from the finding that inactivation of a relatively broad subset of neurons, including a subset of anterior midgut-innervating neurons results in an abnormal

#### **Figure 3.**

*Innervation of adult Drosophila intestine. Enteric innervation (shown in green) along Drosophila gut-brain axis (central nervous system and gastrointestinal system). Neurons from brain and enteric ganglia innervate anterior portion of gut. Neurons from ventral nerve cord send axons in the hindgut which also extend anteriorly along the posterior midgut. Adopted from Miguel-Aliaga et al., 2018.*

proventricular structure, increased permeability of the epithelial barrier, and increased susceptibility to oral bacterial infection: all suggestive of defects in the production of peritrophic matrix [175].

In adult flies, inactivation of insulin-producing neurons results in contrasting effects on the hyperphagic response triggered by nutrient scarcity. Silencing of the insulin-producing cells of the brain PI that innervates the anterior midgut lowered this response, whereas silencing of the hindgut-innervating Ilp7 neurons increased it, and also resulted in higher circulating glucose [14, 195]. Not much is known about the importance of sparse sensory innervation of the intestine. One remarkable exception are the pharyngeal taste neurons. *Pox-neuro* (*poxn*) mutant flies lack gustatory function in the legs and labial palps but retain expression of sweet taste receptors in their pharynx and a preference for sweet compounds, highlighting the pharyngeal contribution to sugar detection [196, 197]. Further understanding of the taste circuit relaying this pharyngeal sensory signal is provided by IN1 neurons (subset of interneurons) receiving input from the pharyngeal sensory neurons.

*Gut Feeding the Brain:* Drosophila *Gut an Animal Model for Medicine to Understand… DOI: http://dx.doi.org/10.5772/intechopen.96503*

The activity of IN1 neurons is exquisitely dependent on the amount and duration of feeding [198]. Posterior to the pharynx, in the gastrointestinal tract, the contribution of sensory innervation to nutritional homeostasis remains to be investigated.

Post-ingestive sensory feedback from the gut has been assumed to inhibit feeding based on work in other insects for example severing the recurrent nerve or the medial abdominal nerve, which transmit information from the gut to the brain, results in overconsumption in blowflies [199]. Work done in flies lends support to this idea; whereas severing the medial abdominal nerve did not disturb food consumption, severing the recurrent nerve elevated consumption of sucrose but not water or bitter tasting solutions [200]. The existence of neuronal stretch receptors on the gut that monitor the volume of ingested food is supported by both neurophysiological and anatomical data in numerous other insects [4, 80, 199, 201]. However, the existence and molecular nature of these receptors in *Drosophila* remains to be established. Interestingly, six peripheral neurons on the proventriculus have been shown to express the gustatory receptor Gr43a (function as fructose receptor), which is also expressed by some pharyngeal neurons [202–204]. These proventricular neurons extend dendritic processes into the foregut lumen, and a subset of their axons innervate the midgut, whereas another subset extends along the esophagus, forming a nerve bundle with axons of gustatory receptor neurons projecting toward SEZ. Hence, they may relay nutritional information back to central/more anterior neurons or act locally on the gut. Establishing their roles will require genetic tools able to target the enteric subset without affecting the central or peripheral Gr43a-expressing neurons.
