The Neural Code for Taste in the Nucleus of the Solitary Tract of Rats with Obesity Following Roux-En-Y Gastric Bypass Surgery.

Previous work has shown that taste responses in the nucleus tractus solitarius (NTS; the first central relay for gustation) are blunted in rats with diet-induced obesity (DIO). Here, we studied whether these effects could be reversed by Roux-en-Y gastric bypass (RYGB) surgery, an effective treatment for obesity. Rats were fed a high energy diet (60% kcal fat; HED) both before and after undergoing RYGB. Electrophysiological responses from NTS cells in unrestrained rats were recorded as they licked tastants from a lick spout. Sweet, salty, and umami tastes, as well as their naturalistic counterparts, were presented. Results were compared with those of lean rats from a previous study. As with DIO rats, NTS cells in RYGB rats were more narrowly tuned, showed weaker responses, and less lick coherence than those in lean rats. Both DIO and RYGB rats licked at a slower rate than lean rats and paused more often during a lick bout. However, unlike DIO rats, the proportion of taste cells in RYGB rats was similar to that in lean rats. Our data show that, despite being maintained on a HED after surgery, RYGB can induce a partial recovery of the deficits seen in the NTS of DIO rats.

Development of a 3D-Printed High Temperature Resin Cecal Fistula Implant for Long-Term and Minimally Invasive Access to the Gut Microbiome.

Microbiota dysbiosis has been associated with chronic diseases ranging from gastrointestinal inflammatory and metabolic conditions to neurological changes affecting the gut-brain neural axis, mental health, and general well-being. However, current animal studies using oral gavage and gnotobiotic animals do not allow for non-invasive long-term access to gut microbiome. The purpose of the present study was to evaluate the feasibility of 3D-printed fistula implants through the body wall and into the cecum of rats to obtain long-term access to gut microbiome. Cecal fistulas were designed and 3D-printed using a high temperature resin (Formlabs; acrylic and methacrylic mixture). Nine male Sprague-Dawley rats underwent the fistula implantation. Food intake, body weight, and body fat were measured to determine the impact of fistula manipulation. Gut microbiome, vagal afferents in the hindbrain, and microglia activation were analyzed to determine if fistula implantation disrupted the gut-brain neural axis. We found that the procedure induced a transient decrease in microbial diversity in the gut that resolved within a few weeks. Fistula implantation had no impact on food intake, body weight, fat mass, or microglia activation. Our study shows that 3D-printed cecal fistula implantation is an effective procedure that allows long-term and minimally invasive access to gut microbiome.

Roux­en­Y gastric bypass surgery triggers rapid DNA fragmentation in vagal afferent neurons in rats.

Previous studies have shown that Roux­en­Y gastric bypass (RYGB), one of the most effective weight loss treatments for obesity, results in neurodegenerative responses in vagal afferent gut­brain connection reflected by microglia activation and reduced sensory input to the nucleus tractus solitarius (NTS). However, it is not known whether RYGB­induced microglia activation is the cause or an effect of the reported neuronal damage. Therefore, the aim of this study was to establish the order of neurodegenerative responses in vagal afferents after RYGB in the nodose ganglia (NG) and NTS in male and female rats. Sprague­Dawley rats were fed regular chow or an energy­dense diet for two weeks followed by RYGB or sham surgery. Twenty­four hours later, animals were sacrificed and NG and NTS were collected. Neuronal cell damage was determined by TUNEL assay. Microglia activation was determined by quantifying the fluorescent staining against the ionizing calcium adapter­binding molecule 1. Reorganization of vagal afferents was evaluated by fluorescent staining against isolectin 4. Results of the study revealed significantly increased DNA fragmentation in vagal neurons in the NG when observed at 24 h after RYGB. The surgery did not produce rapid changes in the density of vagal afferents and microglia activation in the NTS. These data indicate that decreased density of vagal afferents and increased microglia activation in the NTS likely ensue as a res ult of RYGB­induced neuronal damage.

Neuroanatomical Localization of Galanin in Zebrafish Telencephalon and Anticonvulsant Effect of Galanin Overexpression.

Galanin is a neuropeptide widely expressed in the nervous system, but it is also present in non-neuronal locations. In the brain, galanin may function as an inhibitory neurotransmitter. Several studies have shown that galanin is involved in seizure regulation and can modulate epileptic activity in the brain. The overall goal of the study was to establish zebrafish as a model to study the antiepileptic effect of galanin. The goal of this study was achieved by (1) determining neuroanatomical localization of galanin in zebrafish lateral pallium, which is considered to be the zebrafish homologue of the mammalian hippocampus, the brain region essential for initiation of seizures, and (2) testing the anticonvulsant effect of galanin overexpression. Whole mount immunofluorescence staining and pentylenotetrazole (PTZ)-seizure model in larval zebrafish using automated analysis of motor function and qPCR were used in the study. Immunohistochemical staining of zebrafish larvae revealed numerous galanin-IR fibers innervating the subpallium, but only scarce fibers reaching the dorsal parts of telencephalon, including lateral pallium. In three-month old zebrafish, galanin-IR innervation of the telencephalon was similar; however, many more galanin-IR fibers reached the dorsal telencephalon, but in the lateral pallium only scarce galanin-IR fibers were visible. qRT-PCR revealed, as expected, a strong increase in the expression of galanin in the Tg(hsp70l:galn) line after heat shock; however, also without heat shock, the galanin expression was several-fold higher than in the control animals. Galanin overexpression resulted in downregulation of c-fos after PTZ treatment. Behavioral analysis showed that galanin overexpression inhibited locomotor activity in PTZ-treated and control larvae. The obtained results show that galanin overexpression reduced the incidence of seizure-like behavior episodes and their intensity but had no significant effect on their duration. The findings indicate that in addition to antiepileptic action, galanin modulates arousal behavior and demonstrates a sedative effect. The current study showed that galanin overexpression correlated with a potent anticonvulsant effect in the zebrafish PTZ-seizure model.

Taste and odor preferences following Roux-en-Y surgery in humans.

It is well established that bariatric surgery, the most effective method to achieve long-term weight loss in obese subjects, reverses enhanced preference and intake of sweet/fatty foods. Although taste and odor preference changes following bariatric surgery have been previously described, their time course and relationship to weight loss remains an issue. The aim of this study was to determine the relationship between taste and odor preference changes and successful weight loss following bariatric surgery. A cross-sectional study was performed on 195 human subjects with body mass index (BMI) above 30 (at least class I obesity), who were scheduled to receive (n = 54) or had previously received (n = 141) Roux-en-Y gastric bypass (RYGB). A Self-Assessment Manikin test was used to measure each participant's affective reaction (ranging from pleasure to displeasure) to a variety of food-related and odor-related pictures. Results confirmed earlier reports about changes in sweet/fatty foods preference after surgery and revealed a shift in preference toward less calorie-dense foods. Relatedly, endorsements of "favorite" foods were mostly sweet/fatty foods in subjects awaiting surgery but were shifted toward more healthy choices, particularly vegetables, in subjects post-RYGB surgery. However, food preference ratings trended toward pre-surgical levels as the time since surgery increased. Answers to open-ended questions about why their diet changed post-surgery revealed that changes in cravings, rather than changes in taste per se, were the major factor. Surprisingly, patients rating a coffee taste as more pleasing after surgery had a lower post-surgical BMI. No associations of odors with change in BMI were apparent. Results showed that following bariatric surgery taste preferences are significantly altered and that these changes correlate with lowered BMI. However, these changes fade as time since surgery lengthens. These results may suggest diagnostic criteria to identify people at risk for less than optimal changes in BMI following bariatric surgery.

Relationship of neuropeptide FF receptors with pubertal maturation of gilts.

Mechanisms governing the timing of puberty in pigs are poorly understood. A genome-wide association study for age at first estrus in pigs identified candidate genes including neuropeptide FF receptor 2 (NPFFR2), which is a putative receptor for RFamide-related peptides (RFRP). RFRP has been shown to negatively regulate secretion of reproductive hormones from hypothalamic and pituitary tissue of pigs in culture. Here, the porcine NPFFR2 gene was further screened and four potentially functional variants were identified to be associated with age at first estrus in pigs (1,288 gilts). The RFRP neurons in the porcine hypothalamus were localized in the paraventricular and dorsomedial nuclei with RFRP fibers in the lateral hypothalamic area. There were marked changes in expression of NPFF receptors in the anterior pituitary gland and hypothalamus of gilts beginning with the peripubertal period. The hypothesis that NPFF receptor function is related to secretion of luteinizing hormone (LH) in gilts was tested with various NPFF receptor ligands. The NPFF receptor antagonist RF9 stimulated a pulse-like release of LH in prepubertal gilts. The putative NPFF receptor agonist RFRP3 modestly suppressed LH pulses in ovariectomized (OVX) prepubertal gilts. A porcine-specific RFRP2 failed to have an effect on LH secretion in OVX prepubertal gilts despite its high degree of homology to avian gonadotropin-inhibitory hormone. Results indicate that an RFRP system is present in the pig and that NPFFR2 is important for pubertal onset in gilts. It is not clear if this regulation involves major control of LH secretion or another unknown mechanism.

Neural proliferation in the dorsal root ganglia of the adult rat following capsaicin-induced neuronal death.

Glial proliferation is a major component of the nervous system's response to injury. In addition to glial proliferation, injury may induce neuronal proliferation in areas of the adult nervous system not considered neurogenic. We have previously reported increased neural proliferation within adult nodose ganglia following capsaicin-induced neuronal death. However, proliferation within the dorsal root ganglia (DRG) remains to be characterized. We hypothesized that capsaicin-induced neuronal death would increase proliferation of satellite glial cells (SGCs) within the DRG. To test this hypothesis, 6-week-old Sprague-Dawley rats received a neurotoxic dose of capsaicin, and proliferation was quantified and characterized at multiple time points thereafter. Proliferation of satellite glial cells expressing the progenitor cell marker nestin was increased at 1 and 3 days following capsaicin administration as shown by BrdU incorporation. In addition to SGCs was a large population of proliferating resident macrophages, as shown by retrovirally mediated expression of GFP. SGC proliferation at these early time points was followed by recovery of neuronal numbers after a loss of 40% of the neuronal population in the DRG. This recovery in neuronal number correlated with recovery of function as shown by paw withdrawal from a noxious heat source. Further understanding of the role that glial proliferation plays in the recovery of neuronal numbers and function may lead to the development of therapeutic treatments for neurodegenerative conditions.

Withdrawal and restoration of central vagal afferents within the dorsal vagal complex following subdiaphragmatic vagotomy.

Vagotomy, a severing of the peripheral axons of the vagus nerve, has been extensively utilized to determine the role of vagal afferents in viscerosensory signaling. Vagotomy is also an unavoidable component of some bariatric surgeries. Although it is known that peripheral axons of the vagus nerve degenerate and then regenerate to a limited extent following vagotomy, very little is known about the response of central vagal afferents in the dorsal vagal complex to this type of damage. We tested the hypothesis that vagotomy results in the transient withdrawal of central vagal afferent terminals from their primary central target, the nucleus of the solitary tract (NTS). Sprague-Dawley rats underwent bilateral subdiaphragmatic vagotomy and were sacrificed 10, 30, or 60 days later. Plastic changes in vagal afferent fibers and synapses were investigated at the morphological and functional levels by using a combination of an anterograde tracer, synapse-specific markers, and patch-clamp electrophysiology in horizontal brain sections. Morphological data revealed that numbers of vagal afferent fibers and synapses in the NTS were significantly reduced 10 days following vagotomy and were restored to control levels by 30 days and 60 days, respectively. Electrophysiology revealed transient decreases in spontaneous glutamate release, glutamate release probability, and the number of primary afferent inputs. Our results demonstrate that subdiaphragmatic vagotomy triggers transient withdrawal and remodeling of central vagal afferent terminals in the NTS. The observed vagotomy-induced plasticity within this key feeding center of the brain may be partially responsible for the response of bariatric patients following gastric bypass surgery.

Reduction of food intake by cholecystokinin requires activation of hindbrain NMDA-type glutamate receptors.

Intraperitoneal injection of CCK reduces food intake and triggers a behavioral pattern similar to natural satiation. Reduction of food intake by CCK is mediated by vagal afferents that innervate the stomach and small intestine. These afferents synapse in the hindbrain nucleus of the solitary tract (NTS) where gastrointestinal satiation signals are processed. Previously, we demonstrated that intraperitoneal (IP) administration of either competitive or noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonists attenuates reduction of food intake by CCK. However, because vagal afferents themselves express NMDA receptors at both central and peripheral endings, our results did not speak to the question of whether NMDA receptors in the brain play an essential role in reduction of feeding by CCK. We hypothesized that activation of NMDA receptors in the NTS is necessary for reduction of food intake by CCK. To test this hypothesis, we measured food intake following IP CCK, subsequent to NMDA receptor antagonist injections into the fourth ventricle, directly into the NTS or subcutaneously. We found that either fourth-ventricle or NTS injection of the noncompetitive NMDA receptor antagonist MK-801 was sufficient to inhibit CCK-induced reduction of feeding, while the same antagonist doses injected subcutaneously did not. Similarly fourth ventricle injection of d-3-(2-carboxypiperazin-4-yl)-1-propenyl-1-phosphoric acid (d-CPPene), a competitive NMDA receptor antagonist, also blocked reduction of food intake following IP CCK. Finally, d-CPPene injected into the fourth ventricle attenuated CCK-induced expression of nuclear c-Fos immunoreactivity in the dorsal vagal complex. We conclude that activation of NMDA receptors in the hindbrain is necessary for the reduction of food intake by CCK. Hindbrain NMDA receptors could comprise a critical avenue for control and modulation of satiation signals to influence food intake and energy balance.

Adrenergic, nitrergic and peptidergic innervation of the urethral muscle in the boar.

In this study, the innervation of the urethral muscle in adult male pigs was investigated using combined NADPH-diaphorase (NADPH-d) histochemistry and immunocytochemistry. Nerve fibres supplying the urethral muscle were found to show NADPH-d activity and they also expressed immunoreactivity to catecholamine synthesising enzymes including tyrosine hydoxylase (TH) and dopamine-beta-hydroxylase (DbetaH) as well as to: vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY). Different subpopulations of the nerve fibres (NADPH-d positive, TH-, DbetaH-, VIP- and NPY-immunoreactive (IR), but also NADPH-d/VIP- and NADPH-d/NPY-IR) were disclosed. These nerve fibres were observed not only to run among muscle fibres of the urethral muscle, but also within extrinsic nerve trunks. Moreover, in the organ studied, numerous ganglia were found. The intramural ganglia, composed of a few to 30 neurons were located in the proximal, middle and distal regions of the pelvic urethra. In the vicinity of the urethral muscle, there were mainly small ganglia containing two to several neurons, but also larger ganglia consisting of up to tens neurons were encountered in the connective tissue surrounding the pelvic urethra. In the ganglia observed in the neighbourhood of the urethral muscle, different subpopulations of nerve cells were found, namely: catecholaminergic, nitrergic, VIP-IR, NPY-IR and also NADPH-d/DbetaH-, NADPH-d/VIP- and NADPH-d/NPY-positive. Possible sources of the innervation for this muscle were also discussed.

Differences in the chemical coding of nerve fibres supplying major populations of neurons between the caudal mesenteric ganglion and anterior pelvic ganglion in the male pig.

The present study was designed to investigate and to compare the chemical coding of nerve fibres supplying major populations of neurons in the caudal mesenteric (CaMG) and anterior pelvic (APG) ganglion in juvenile male pigs (n=5) using double-labelling immunofluorescence. The co-existence patterns of some biologically active substances including tyrosine hydroxylase (TH) and vesicular acetylcholine transporter (VAChT) as well as vasoactive intestinal polypeptide (VIP), substance P (SP), calcitonin gene-related peptide (CGRP), Leu5-enkephalin (LENK) and serotonin (5-HT) were analysed under a confocal laser scanning microscope. Profound differences in the neurochemical features of the nerve terminals between the ganglia were observed. Moreover, there were also distinct differences in the chemical coding of nerve fibres associated with the particular populations and subpopulations of neurons within the ganglia. In both ganglia, nearly all adrenergic and cholinergic neurons were supplied with VAChT-positive nerve fibres (putative preganglionic fibres). However, in the CaMG, they were more numerous and, in contrast to the APG, many of them also stained for VIP. In the APG, a great number of nerve terminals expressed immunoreactivity to SP and CGRP (putative collaterals of sensory neurons). Interestingly, they densely supplied almost exclusively adrenergic neurons. SP-positive nerve fibres were moderate in number in the CaMG, but, in addition to VAChT-IR nerve terminals, the most numerous populations of nerve fibres in this ganglion were those expressing highly colocalized immunoreactivities to CGRP and LENK, and those which stained for 5-HT (putative processes of enteric neurons). However, these fibres supplied almost exclusively larger, intensely stained for TH and clustered adrenergic neurons. This diversity of the nerve terminals reflects the complexity of nerve circuits involved in the innervation of structures supplied by neurons in the porcine CaMG and APG. It also demonstrates the importance of nerve inputs for the proper function of autonomic neurons and thus their target tissues.

The influence of inflammation on the expression of neuropeptides in the ileum-projecting primary sensory neurones in the pig.

In the present study the ELISA test was used to investigate the influence of chemically-induced ileitis on the dorsal root ganglia (DRG) neurons in the pig. The preliminary retrograde fluorescent tracing study revealed that ileum-projecting sensory neurones (IPN) are located in the thoracic ganglia (Th; Th8-Th13). The ileum wall in experimental (E) pigs was subjected to multiple injection with 4% paraformaldehyde to induce inflammation, while in the control (C) animals the organ was injected with 0.1 M phosphate buffer. Three days later the DRGs (Th8-Th13) collected from all the animals were evaluated for VIP, SP, CGRP, NPY, GAL and SOM content with an ELISA test. It was found that the inflammation increased clearly the tissue level of SP, GAL and SOM.

Immunohistochemical characterisation of cholinergic neurons in the anterior pelvic ganglion of the male pig.

This study investigated immunohistochemical properties of cholinergic neurons in the anterior pelvic ganglion (APG) of juvenile male pigs (n=7). Cholinergic neurons were identified using antibodies against choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT). Immunoblotting was applied to verify the specificity of ChAT-immunostaining. Western blotting performed on APG tissue homogenates detected single immunoreactive protein with a molecular weight matching that of ChAT (71.6 kDa). It was found that many APG neurons expressed immunoreactivity to ChAT or VAChT (40% and 39% of the neurons, respectively). The analysis of adjacent sections from the ganglion revealed complete colocalization of ChAT and VAChT in these nerve cells. Furthermore, virtually all the ChAT-positive neurons were tyrosine hydroxylase (TH)-negative (non-adrenergic) but many of them displayed immunoreactivity to nitric oxide synthase (NOS), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) or somatostatin (SOM). There were also single nerve cell bodies that stained for neither ChAT nor TH. The comparison of the adjacent sections revealed that NOS, VIP, NPY and SOM were simultaneously co-expressed in the majority of the cholinergic somata. ChAT- or VAChT-positive varicose nerve terminals supplied nearly all neuronal profiles within the ganglion often forming loose basket-like formations surrounding the particular nerve cell bodies. The present study for the first time has revealed that nearly all non-adrenergic neurons in the porcine APG are cholinergic in nature, i.e. express immunoreactivity for ChAT and VAChT. Considering a high coincidence between the chemical coding of non-adrenergic (cholinergic) nerve fibres supplying some porcine male reproductive organs described in earlier papers and that of cholinergic pelvic neurons found in this study it is further concluded that pelvic ganglia are probably the major source of cholinergic innervation for the porcine urogenital system.

Uterus-innervating neurones of paracervical ganglion in the pig: immunohistochemical characteristics.

Immunohistochemical characteristics of neurones innervating the porcine uterus located in paracervical ganglia were studied with a combination of retrograde fluorescent tracing and immunofluorescence. Retrograde fluorescent tracer Fast Blue (FB) was injected into the uterine horn and uterine cervix. The presence of biologically active substances, tyrosine hydroxylase (TH), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), galanin (GAL), Met-enkephalin-Arg-Gly-Leu (MEAGL) and calcitonin gene-related peptide (CGRP) was studied in FB-positive neurones localised in paracervical ganglia. FB-positive neurones containing TH, NPY, VIP and MEAGL were numerous, while those containing CGRP were scarce. The results pointed to some species-related differences in immunohistochemical coding of neurones of paracervical ganglion responsible for uterus innervation.