Krzysztof Czaja

Chapter 3: Histology of the peripheral nerve and changes occurring during nerve regeneration.

Peripheral nerves are complex organs that can be found throughout the body reaching almost all tissues and organs to provide motor and/or sensory innervation. A parenchyma (the noble component made by the nerve fibers, i.e., axons and Schwann cells) and a stroma (the scaffold made of various connective elements) can be recognized. Although morphological analysis is the most common approach for studying peripheral nerve regeneration, researchers are not always aware of several histological peculiarities of these organs. Therefore, the aim of this review is to describe, at a structural and ultrastructural level, the main features of the parenchyma and the stroma of the normal undamaged nerve as well as the most important morphological changes that occur after nerve damage and during posttraumatic nerve regeneration. The paper is aimed at providing the reader with the basic framework information on nerve morphology. This would enable the correct interpretation of morphological data obtained by many experimental studies on peripheral nerve repair and regeneration such as those outlined in several other papers included in this special issue of the International Review of Neurobiology.

Cannabinoid receptor 1-expressing neurons in the nucleus accumbens

Endocannabinoid signaling critically regulates emotional and motivational states via activation of cannabinoid receptor 1 (CB1) in the brain. The nucleus accumbens (NAc) functions to gate emotional and motivational responses. Although expression of CB1 in the NAc is low, manipulation of CB1 signaling within the NAc triggers robust emotional/motivational alterations related to drug addiction and other psychiatric disorders, and these effects cannot be exclusively attributed to CB1 located at afferents to the NAc. Rather, CB1-expressing neurons in the NAc, although sparse, appear to be critical for emotional and motivational responses. However, the cellular properties of these neurons remain largely unknown. Here, we generated a knock-in mouse line in which CB1-expressing neurons expressed the fluorescent protein td-Tomato (tdT). Using these mice, we demonstrated that tdT-positive neurons within the NAc were exclusively fast-spiking interneurons (FSIs). These FSIs were electrically coupled with each other, and thus may help synchronize populations/ensembles of NAc neurons. CB1-expressing FSIs also form GABAergic synapses on adjacent medium spiny neurons (MSNs), providing feed-forward inhibition of NAc output. Furthermore, the membrane excitability of tdT-positive FSIs in the NAc was up-regulated after withdrawal from cocaine exposure, an effect that might increase FSI-to-MSN inhibition. Taken together with our previous findings that the membrane excitability of NAc MSNs is decreased during cocaine withdrawal, the present findings suggest that the basal functional output of the NAc is inhibited during cocaine withdrawal by multiple mechanisms. As such, CB1-expressing FSIs are targeted by cocaine exposure to influence the overall functional output of the NAc.

Diet-driven microbiota dysbiosis is associated with vagal remodeling and obesity

Obesity is one of the major health issues in the United States. Consumption of diets rich in energy, notably from fats and sugars (high-fat/high-sugar diet: HF/HSD) is linked to the development of obesity and a popular dietary approach for weight loss is to reduce fat intake. Obesity research traditionally uses low and high fat diets and there has been limited investigation of the potential detrimental effects of a low-fat/high-sugar diet (LF/HSD) on body fat accumulation and health. Therefore, in the present study, we investigated the effects of HF/HSD and LF/HSD on microbiota composition, gut inflammation, gut-brain vagal communication and body fat accumulation. Specifically, we tested the hypothesis that LF/HSD changes the gut microbiota, induces gut inflammation and alters vagal gut-brain communication, associated with increased body fat accumulation. Sprague-Dawley rats were fed an HF/HSD, LF/HSD or control low-fat/low-sugar diet (LF/LSD) for 4weeks. Body weight, caloric intake, and body composition were monitored daily and fecal samples were collected at baseline, 1, 6 and 27days after the dietary switch. After four weeks, blood and tissues (gut, brain, liver and nodose ganglia) were sampled. Both HF/HSD and LF/HSD-fed rats displayed significant increases in body weight and body fat compared to LF/LSD-fed rats. 16S rRNA sequencing showed that both HF/HSD and LF/HSD-fed animals exhibited gut microbiota dysbiosis characterized by an overall decrease in bacterial diversity and an increase in Firmicutes/Bacteriodetes ratio. Dysbiosis was typified by a bloom in Clostridia and Bacilli and a marked decrease in Lactobacillus spp. LF/HSD-fed animals showed a specific increase in Sutterella and Bilophila, both Proteobacteria, abundances of which have been associated with liver damage. Expression of pro-inflammatory cytokines, such as IL-6, IL-1β and TNFα, was upregulated in the cecum while levels of tight junction protein occludin were downregulated in both HF/HSD and LF/HSD fed rats. HF/HSD and LF/HSD-fed rats also exhibited an increase in cecum and serum levels of lipopolysaccharide (LPS), a pro-inflammatory bacterial product. Immunofluorescence revealed the withdrawal of vagal afferents from the gut and at their site of termination the nucleus of the solitary tract (NTS) in both the HF/HSD and LF/HSD rats. Moreover, there was significant microglia activation in the nodose ganglia, which contain the vagal afferent neuron cell bodies, of HF/HSD and LF/HSD rats. Taken together, these data indicate that, similar to HF/HSD, consumption of an LF/HSD induces dysbiosis of gut microbiota, increases gut inflammation and alters vagal gut-brain communication. These changes are associated with an increase in body fat accumulation.

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.

N-methyl-D-aspartate receptor subunit phenotypes of vagal afferent neurons in nodose ganglia of the rat.

Most vagal afferent neurons in rat nodose ganglia express mRNA coding for the NR1 subunit of the heteromeric N‐methyl‐D‐aspartate (NMDA) receptor ion channel. NMDA receptor subunit immunoreactivity has been detected on axon terminals of vagal afferents in the dorsal hindbrain, suggesting a role for presynaptic NMDA receptors in viscerosensory function. Although NMDA receptor subunits (NR1, NR2B, NR2C, and NR2D) have been linked to distinct neuronal populations in the brain, the NMDA receptor subunit phenotype of vagal afferent neurons has not been determined. Therefore, we examined NMDA receptor subunit (NR1, NR2B, NR2C, and NR2D) immunoreactivity in vagal afferent neurons. We found that, although the left nodose contained significantly more neurons (7,603), than the right (5,978), the proportions of NMDA subunits expressed in the left and right nodose ganglia were not significantly different. Immunoreactivity for NMDA NR1 subunit was present in 92.3% of all nodose neurons. NR2B immunoreactivity was present in 56.7% of neurons; NR2C‐expressing nodose neurons made up 49.4% of the total population; NR2D subunit immunoreactivity was observed in just 13.5% of all nodose neurons. Double labeling revealed that 30.2% of nodose neurons expressed immunoreactivity to both NR2B and NR2C, whereas NR2B and NR2D immunoreactivities were colocalized in 11.5% of nodose neurons. NR2C immunoreactivity colocalized with NR2D in 13.1% of nodose neurons. Our results indicate that most vagal afferent neurons express NMDA receptor ion channels composed of NR1, NR2B, and NR2C subunits and that a minority phenotype that expresses NR2D also expresses NR1, NR2B, and NR2C. J. Comp. Neurol. 496:877–885, 2006.

CCK-Induced Reduction of Food Intake and Hindbrain MAPK Signaling Are Mediated by NMDA Receptor Activation

The dorsal vagal complex of the hindbrain, including the nucleus of the solitary tract (NTS), receives neural and humoral afferents that contribute to the process of satiation. The gut peptide, cholecystokinin (CCK), promotes satiation by activating gastrointestinal vagal afferents that synapse in the NTS. Previously, we demonstrated that hindbrain administration of N-methyl-D-aspartate (NMDA)-type glutamate receptor antagonists attenuate reduction of food intake after ip CCK-8 injection, indicating that these receptors play a necessary role in control of food intake by CCK. However, the signaling pathways through which hindbrain NMDA receptors contribute to CCK-induced reduction of food intake have not been investigated. Here we report CCK increases phospho-ERK1/2 in NTS neurons and in identified vagal afferent endings in the NTS. CCK-evoked phospho-ERK1/2 in the NTS was attenuated in rats pretreated with capsaicin and was abolished by systemic injection of a CCK1 receptor antagonist, indicating that phosphorylation of ERK1/2 occurs in and is mediated by gastrointestinal vagal afferents. Fourth ventricle injection of a competitive NMDA receptor antagonist, prevented CCK-induced phosphorylation of ERK1/2 in hindbrain neurons and in vagal afferent endings, as did direct inhibition of MAPK kinase. Finally, fourth ventricle administration of either a MAPK kinase inhibitor or NMDA receptor antagonist prevented the reduction of food intake by CCK. We conclude that activation of NMDA receptors in the hindbrain is necessary for CCK-induced ERK1/2 phosphorylation in the NTS and consequent reduction of food intake.

Vagal afferent neurons projecting to the stomach and small intestine exhibit multiple N-methyl-D-aspartate receptor subunit phenotypes

Previous reports suggest that NMDA receptors participate in control of food intake via vagal afferent neurons that innervate the upper gastrointestinal (GI) tract. While messenger RNA coding for the NR1 NMDA receptor subunit is present in a majority of vagal afferent neurons of nodose ganglia (NG), immunoreactivity for other NMDA receptor subunits (NR2B, NR2C and NR2D) are expressed in more limited subpopulations of vagal afferents. To determine whether vagal afferent neurons that project to the stomach or duodenum exhibit distinct NMDA receptor subunit phenotypes, we examined immunoreactivity (IR) for NMDA receptor NR1, NR2B, NR2C and NR2D subunits in NG neurons that were labeled by injections of the retrograde tracer Fast Blue (FB) into the wall of the stomach or duodenum. FB injections into the fundus or corpus of the stomach labeled comparable numbers of neurons in both the left and right NG, while proximal duodenal injections labeled only neurons of left NG. NR1-IR expression was observed in most neurons innervating the upper GI tract (fundus, 97%; corpus, 95%; duodenum, 98%). Likewise, most neurons that innervated the upper GI tract expressed NR2B-IR (fundus, 98%; corpus, 85%; duodenum, 81%). NR2C-IR was observed in only 52%, 46% and 32% of FB-positive neurons projecting to the fundus, corpus or duodenum respectively, while NR2D-IR occurred in an even more restricted FB-labeled subpopulation (fundus, 13%; corpus, 26%; and duodenum, 18%). Our observations indicate that different subpopulations of vagal afferents express distinct NMDA receptor subunit phenotypes. However, the neuronal distribution of NMDA receptor subunits is not correlated with innervation of either the stomach or duodenum.

Changes in microglial activation within the hindbrain, nodose ganglia, and the spinal cord following subdiaphragmatic vagotomy

Damage to peripheral nerve branches triggers activation of microglia in CNS areas containing motor neuron soma and primary afferent terminals of the damaged fibers. Furthermore, microglial activation occurs in areas containing the soma and terminals of spared nerve branches of a damaged nerve. Because the abdominal viscera are innervated by spinal afferents as well as vagal afferents and efferents, we speculated that spinal nerves might respond like spared nerve branches following damage to vagal fibers. Therefore, we tested the hypothesis that damage to the abdominal vagus would result in microglial activation in vagal structures-the nucleus of the solitary tract (NTS), dorsal motor nucleus of the vagus nerve (DMV), and nodose ganglia (NG)-as well as spinal cord (SC) segments that innervate the abdominal viscera. To test this hypothesis, rats underwent subdiaphragmatic vagotomy or sham surgery and were treated with saline or the microglial inhibitor, minocycline. Microglial activation was determined by quantifying changes in the intensity of fluorescent staining with a primary antibody against ionizing calcium adapter binding molecule 1 (Iba1). We found that subdiaphragmatic vagotomy significantly activated microglia in the NTS, DMV, and NG two weeks post-vagotomy. Microglial activation remained significantly increased in the NG and DMV for at least 42 days. Surprisingly, vagotomy significantly decreased microglial activation in the SC. Minocycline treatment attenuated microglial activation in all studied areas. Our results indicate that microglial activation in vagal structures following abdominal vagal damage is accompanied by suppression of microglial activation in associated areas of the spinal cord.

Sleeve Gastrectomy and Roux-en-Y Gastric Bypass Alter the Gut-Brain Communication

This study investigated the anatomical integrity of vagal innervation of the gastrointestinal tract following vertical sleeve gastrectomy (VSG) and Roux-en-Y gastric bypass (RYGB) operations. The retrograde tracer fast blue (FB) was injected into the stomach to label vagal neurons originating from nodose ganglion (NG) and dorsal motor nucleus of the vagus (DMV). Microglia activation was determined by quantifying changes in the fluorescent staining of hindbrain sections against an ionizing calcium adapter binding molecule 1 (Iba1). Reorganization of vagal afferents in the hindbrain was studied by fluorescent staining against isolectin 4 (IB4). The density of Iba1- and IB4-immunoreactivity was analyzed using Nikon Elements software. There was no difference in the number of FB-labeled neurons located in NG and DMV between VSG and VSG-sham rats. RYGB, but not RYGB-sham rats, showed a dramatic reduction in number of FB-labeled neurons located in the NG and DMV. VSG increased, while the RYGB operation decreased, the density of vagal afferents in the nucleus tractus solitarius (NTS). The RYGB operation, but not the VSG procedure, significantly activated microglia in the NTS and DMV. Results of this study show that the RYGB, but not the VSG procedure, triggers microglia activation in vagal structures and remodels gut-brain communication.

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. J. Comp. Neurol. 521:3584‐3599, 2013.