Double-labeled Neurons Research Articles

Significant Quantitative Differences in Orexin Neuronal Activation After Pain Assessments in an Animal Model of Sickle Cell Disease.

Sickle cell disease is a hemoglobinopathy that causes sickling of red blood cells, resulting in vessel blockage, stroke, anemia, inflammation, and extreme pain. The development and treatment of pain, in particular, neuropathic pain in sickle cell disease patients is poorly understood and impedes our progress toward the development of novel therapies to treat pain associated with sickle cell disease. The orexin/hypocretin system offers a novel approach to treat chronic pain and hyperalgesia. These neuropeptides are synthesized in three regions: perifornical area (PFA), lateral hypothalamus (LH), and dorsomedial hypothalamus (DMH). Data suggest that orexin–A neuropeptide has an analgesic effect on inflammatory pain and may affect mechanisms underlying the maintenance of neuropathic pain. The purpose of this study was to determine whether there are neuronal activation differences in the orexin system as a result of neuropathic pain testing in a mouse model of sickle cell disease. Female transgenic sickle mice that express exclusively (99%) human sickle hemoglobin (HbSS-BERK) and age-/gender-matched controls (HbAA-BERK mice; n = 10/group, 20–30 g) expressing normal human hemoglobin A were habituated to each test protocol and environment before collecting baseline measurements and testing. Four measures were used to assess pain-related behaviors: thermal/heat hyperalgesia, cold hyperalgesia, mechanical hyperalgesia, and deep-tissue hyperalgesia. Hypothalamic brain sections from HbAA-BERK and HbSS-BERK mice were processed to visualize orexin and c-Fos immunoreactivity and quantified. The percentage of double labeled neurons in the PFA was significantly higher than the percentage of double labeled neurons in the LH orexin field of HbAA-BERK mice (*p < 0.05). The percentages of double labeled neurons in PFA and DMH orexin fields are significantly higher than those neurons in the LH of HbSS-BERK mice (*p < 0.05). These data suggest that DMH orexin neurons were preferentially recruited during neuropathic pain testing and a more diverse distribution of orexin neurons may be required to produce analgesia in response to pain in the HbSS-BERK mice. Identifying specific orexin neuronal populations that are integral in neuropathic pain processing will allow us to elucidate mechanisms that provide a more selective, targeted approach in treating of neuropathic pain in sickle cell disease.

The blockade of oxytocin receptors in the paraventricular thalamus reduces maternal crouching behavior over pups in lactating mice

Oxytocin (OT) systems contribute to the elicitation of stereotypic maternal behaviors. OT peptide-expressing neurons are predominantly localized in the hypothalamus, whereas OT receptor (OTR)-expressing neurons are widely distributed throughout the brain. Among those OTR-expressing regions, the paraventricular thalamus (PVT) consists of heterogeneous neuropeptide-responsive neurons critical for appetitive motivation, food intake control, and social behaviors; however, the precise distribution of OTR-expressing neurons within the PVT and whether these neurons are involved in maternal behaviors in mice are unknown. The distribution of OTR-expressing neurons was examined in an OTR-Venus transgenic line expressing a fluorescent protein controlled by the OTR promoter. The number of Venus expressing neurons was higher in the posterior PVT (pPVT) than in the anterior PVT (aPVT). When OTR-Venus dams were exposed to pups, the number of double-labelled neurons expressing both OTR-Venus and a marker of neuronal activity (c-Fos) was increased in the pPVT compared to non-exposed dams, while the aPVT remained unchanged. To investigate whether OT signaling in the pPVT is essential for maternal behaviors, an OT antagonist (OTA) was transiently or chronically infused into the pPVT of lactating dams during the postpartum period. Although the transient OTR blockade did not affect maternal behaviors, a chronic OTR blockade specifically reduced the duration of crouching behavior over pups. Taken together, these findings suggest that OTR-expressing neurons in the pPVT are involved in maternal crouching behavior.

Targeted recombination in active populations as a new mouse genetic model to study sleep-active neuronal populations: Demonstration that Lhx6+ neurons in the ventral zona incerta are activated during paradoxical sleep hypersomnia.

The cFos immunostaining allowed the identification of multiple populations of neurons involved in the generation of paradoxical sleep. We adopted the transgenic (targeted recombination in active populations) mouse model, which following injection of tamoxifen, allows expression of Cre-dependent reporter constructs (i.e., mCherry) in neurons expressing cFos during waking or paradoxical sleep hypersomnia following automatic paradoxical sleep deprivation. Three groups of mice were subjected to two periods of waking, one period of waking and one of paradoxical sleep hypersomnia, or two periods of paradoxical sleep hypersomnia. A high percentage of double-labelled neurons was observed in the lateral hypothalamic area and zona incerta of two periods of waking and two periods of paradoxical sleep hypersomnia in mice, but not in those of one period of waking and one of paradoxical sleep hypersomnia in animals. Melanin-concentrating hormone neurons in the lateral hypothalamic area and Lhx6+ cells in the zona incerta constituted 5.7±1.5% and 8.8±2.3% of all mCherry+ cells and 20.6±4.8% and 24.6±5.9% of all cFos+ neurons in two periods of paradoxical sleep hypersomnia in animals. In addition, melanin-concentrating hormone cells as well as Lhx6+ neurons rarely expressed mCherry (or cFos) in the waking condition, in contrast to orexin neurons, which constituted approximately 30% of mCherry+ and cFos+ neurons. Our results validate the TRAP methodology and open the way to use it for identifying the neurons activated during waking and paradoxical sleep hypersomnia. Furthermore, they indicate for the first time that Lhx6+ neurons in the zona incerta, like melanin-concentrating hormone cells in the lateral hypothalamic area, are activated during paradoxical sleep hypersomnia but not during waking. These results indicate that Lhx6+ neurons might play a role in the control of paradoxical sleep, like the melanin-concentrating hormone cells.

Abstract P3063: Reduced 5-ht3r Signaling In The Nodose Ganglia Neurons Of Adult Shr

Introduction: Gut dysbiosis is linked to hypertension (HTN), but the precise mechanisms of host-microbiota interactions are not known. Gut and brain communicate via the vagal axis, of which serotonin (5-HT) is an important mediator. 5-HT acts via vagal 5-HT3a receptor (5-HT3aR), stimulation of which causes a reflex bradycardia similar to activation of the baroreflex in the nucleus of the solitary tract (NTS). As the microbiota can exert powerful effects on gut 5-HT production, we investigated 5-HT3aR-dependent signaling in the spontaneously hypertensive rats (SHR). Methods: WKY and SHR were randomly injected with AAV-hSyn-gCAMP in the left or the right nodose ganglia. All rats also received an injection of CTB 594 (1ul of 1% solution) to the proximal colon and/or small intestine, to retrogradely label NG. In vivo Ca 2+ imaging was performed in all anesthetized rats using a two-photon microscope. High definition Z-stack images of the whole NG followed by a video of real time neuronal activity were made (~30 3um stacks every 2 seconds for 5 minutes). During this time, a bolus i.v. injection of 5-HT3aR agonist 1-Phenylbigunide (35ug) was delivered while simultaneously recording the real time activity of NG neurons and blood pressure/heart rate by telemetry. Data were analyzed using Fiji and Python script. qPCR for 5-HT3aRs was performed in RNA isolated from the proximal colon, nodose ganglia (NG) and NTS of adult male SHR and WKY rats. Results: Injection of 5-HT3aR agonist produced activation of the same number of gCAMP-labeled neurons in the SHR and WKY; however, a number of double-labeled gut-projecting neurons that were activated by the 5-HT3aR agonist were slightly lower in the SHR (11%) vs the WKY (13.5%). SHR NG neurons exhibited slower rate of activation compared to WKY NG neurons following the injection, which was associated with dampened effects of 5-HT3aR agonist on BP and HR in the SHR. Expression of 5-HT3aRs was completely diminished in the gut and NG (P&lt;0.001) and significantly downregulated in the NTS (by ~40%, P&lt;0.01) of SHR compared to WKY. Conclusions: Reduced 5-HT3aR NG signaling may be a consequence of gut dysbiosis in the SHR. Future studies will elucidate the functional significance of this pathway in HTN.

Peripheral nerve injury attenuates stress-induced Fos-family expression in the Locus Coeruleus of male Sprague-Dawley rats

The ability to cope with acute stressors is impaired in people with chronic neuropathic injuries. The regulation of stress coping responses depends critically on several parallel interconnected neural circuits, one of which originates in the Locus Coeruleus. In rats, chronic constriction injury (CCI) and acute stress each modulate noradrenergic activity of the Locus Coeruleus (LC) although with different temporal patterns. This study investigated the effects of CCI on the neuronal activity of the LC to acute restraint stress using the immunohistochemical detection of Fos-family protein expression. Male Sprague-Dawley rats underwent CCI surgery and 11 days later were restrained for 15 min. The number and location of single-labelled neurons (c-Fos, FosB/ΔFosB and tyrosine hydroxylase (TH) immunoreactive) neurons and double labelled neurons (c-Fos, or FosB/ΔFosB with TH) were quantified for the LC and surrounding regions. Comparisons were made with rats that underwent sham surgery or anaesthesia (20 min). Restraint triggered a struggling response in all rats. CCI attenuated restraint-induced Fos expression in LC neurons. A significant proportion (30–50%) of these LC Fos positive neurons did not contain TH. These data suggest that nerve injury might impair the ordinary cellular response of the LC to an acute stress. The association of stress-related disorders in people with neuropathic injuries suggests that the observations made in this study may reflect a part of the mechanism underlying these clinical comorbidities.

Collateral Projections from the Medullary Dorsal Horn to the Ventral Posteromedial Thalamic Nucleus and the Parafascicular Thalamic Nucleus in the Rat

Medullary dorsal horn (MDH), the homolog of spinal dorsal horn, plays essential roles in processing of nociceptive signals from orofacial region toward higher centers, such as the ventral posteromedial thalamic nucleus (VPM) and parafascicular thalamic nucleus (Pf), which belong to the sensory-discriminative and affective aspects of pain transmission systems at the thalamic level, respectively. In the present study, in order to provide morphological evidence for whether neurons in the MDH send collateral projections to the VPM and Pf, a retrograde double tracing method combined with immunofluorescence staining for substance P (SP), SP receptor (SPR) and Fos protein was used. Fluoro-gold (FG) was injected into the VPM and the tetramethylrhodamine-dextran (TMR) was injected into the Pf. The result revealed that both FG- and TMR-labeled projection neurons were observed throughout the entire extent of the MDH, while the FG/TMR double-labeled neurons were mainly located in laminae I and III. It was also found that some of the FG/TMR double-labeled neurons within lamina I expressed SPR and were in close contact with SP-immunoreactive (SP-ir) terminals. After formalin injection into the orofacial region, 41.4% and 34.3% of the FG/TMR double-labeled neurons expressed Fos protein in laminae I and III, respectively. The present results provided morphological evidence for that some SPR-expressing neurons within the MDH send collateral projections to both VPM and Pf and might be involved in sensory-discriminative and affective aspects of acute orofacial nociceptive information transmission.

Lateral parabrachial neurons innervate orexin neurons projecting to brainstem arousal areas in the rat

Orexin (ORX) neurons in the hypothalamus send their axons to arousal-promoting areas. We have previously shown that glutamatergic neurons in the lateral parabrachial nucleus (LPB) innervate ORX neurons. In this study, we examined potential pathways from the LPB to ORX neurons projecting to arousal-promoting areas in the brainstem by a combination of tract-tracing techniques in male Wistar rats. We injected the anterograde tracer biotinylated dextranamine (BDA) into the LPB and the retrograde tracer cholera toxin B subunit (CTb) into the ventral tegmental area, dorsal raphe nucleus, pedunculopontine tegmental nucleus, laterodorsal tegmental area, or locus coeruleus (LC). We then analyzed the BDA-labeled fibers and ORX-immunoreactive neurons in the hypothalamus. We found that double-labeled ORX and CTb neurons were the most abundant after CTb was injected into the LC. We also observed prominently overlapping distribution of BDA-labeled fibers, arising from neurons located in the lateral-most part of the dorsomedial nucleus and adjacent dorsal perifornical area. In these areas, we confirmed by confocal microscopy that BDA-labeled synaptophysin-immunoreactive axon terminals were in contiguity with cell bodies and dendrites of CTb-labeled ORX-immunoreactive neurons. These results suggest that the LPB innervates arousal-promoting areas via ORX neurons and is likely to promote arousal responses to stimuli.

Bladder overactivity and afferent hyperexcitability induced by prostate-to-bladder cross-sensitization in rats with prostatic inflammation.

There is clinical evidence showing that prostatic inflammation contributes to overactive bladder symptoms in male patients; however, little is known about the underlying mechanisms In this study, we investigated the mechanism that prostatic inflammation causes detrusor overactivity by using a rat model of chemically induced prostatic inflammation. We observed a significant number of dorsal root ganglion neurons with dichotomized afferents innervating both prostate and bladder. We also found that prostatic inflammation induces bladder overactivity and urothelial NGF overexpression in the bladder, both dependent on activation of the pelvic nerve, as well as changes in ion channel expression and hyperexcitability of bladder afferent neurons. These results indicate that the prostate-to-bladder cross-sensitization through primary afferent pathways in the pelvic nerve, which contain dichotomized afferents, could be an important mechanism contributing to bladder overactivity and afferent hyperexcitability induced by prostatic inflammation. Prostatic inflammation is reportedly an important factor inducing lower urinary tract symptoms (LUTS) including urinary frequency, urgency and incontinence in patients with benign prostatic hyperplasia (BPH). However, the underlying mechanisms inducing bladder dysfunction after prostatic inflammation are not well clarified. We therefore investigated the effects of prostatic inflammation on bladder activity and afferent function using a rat model of non-bacterial prostatic inflammation. We demonstrated that bladder overactivity, evident as decreased voided volume and shorter intercontraction intervals in cystometry, was observed in rats with prostatic inflammation versus controls. Tissue inflammation, evident as increased myeloperoxidase activity, and IL-1α, IL-1β, and IL-6 levels inside the prostate, but not in the bladder, following intraprostatic formalin injection induced an increase in NGF expression in the bladder urothelium, which depended on activation of the pelvic nerve. A significant proportion (18-19%) of dorsal root ganglion neurons were double labelled by dye tracers injected into either bladder or prostate. In rats with prostatic inflammation, TRPV1, TRPA1 and P2X2 increased, and Kv1.4, a potassium channel α-subunit that can form A-type potassium (KA ) channels, decreased at mRNA levels in bladder afferent and double-labelled neurons vs. non-labelled neurons, and slow KA current density decreased in association with hyperexcitability of these neurons. Collectively, non-bacterial inflammation localized in the prostate induces bladder overactivity and enhances bladder afferent function. Thus, prostate-to-bladder afferent cross-sensitization through primary afferents in the pelvic nerve, which contain dichotomized afferents, could underlie storage LUTS in symptomatic BPH with prostatic inflammation.

Collateral Projections from the Lateral Parabrachial Nucleus to the Central Amygdaloid Nucleus and the Ventral Tegmental Area in the Rat.

ABSTRACTLateral parabrachial nucleus (LPB) is a critical region in the integration and transmission of peripheral nociceptive information. The parabrachio‐amygdaloid (P‐Amy) pathway and parabrachio‐ventral tegmental area (P‐VTA) pathway is thought to be significant in regulation of pain‐related negative emotions. In present study, retrograde tract tracers Fluoro‐gold (FG) and tetramethylrhodramine‐dextran (TMR) were stereotaxically injected into the right central amygdaloid nucleus (CeA) and right VTA, respectively. Then, part of these rats were performed with the spare nerve injury (SNI) in the controlateral side of FG and TMR injection. Afterwards, double‐ or triple‐immunofluorescent histochemistry was used to examine FG/TMR double‐ and FG/TMR/FOS or FG/TMR/CGRP triple‐labeled neurons in the LPB. The results showed that all of FG, TMR single‐ and FG/TMR double‐labeled neurons were distributed in the LPB bilaterally with an ipsilateral predominance. The proportion of FG/TMR double‐labeled neurons to the total number of FG‐ and TMR‐labeled neurons was 10.78% and 13.07%, respectively. Nearly all of the FG/TMR double‐labeled neurons (92.67%) showed calcitonin gene‐related peptide (CGRP) immunopositive. On the other hand, in the SNI rats, about 89.49% and 77.87% of FG‐ and TMR‐labeled neurons were FG/FOS‐ and TMR/FOS‐positive neurons; about 93.33% of the FG/TMR double‐labeled neurons were FOS‐LI. Our results suggest that the part of CGRP immunopositive neurons in the LPB send projection fibers to both the CeA and VTA by the way of axon collaterals, which are activated by the nociceptive stimulation in the SNI condition, and may play an important role in the transmission of peripheral nociceptive information. Anat Rec, 302:1178–1186, 2019. © 2018 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Cortical projections to the two retinotopic maps of primate pulvinar are distinct.

Comprised of at least five distinct nuclei, the pulvinar complex of primates includes two large visually driven nuclei; one in the dorsal (lateral) pulvinar and one in the ventral (inferior) pulvinar, that contain similar retinotopic representations of the contralateral visual hemifield. Both nuclei also appear to have similar connections with areas of visual cortex. Here we determined the cortical connections of these two nuclei in galagos, members of the stepsirrhine primate radiation, to see if the nuclei differed in ways that could support differences in function. Injections of different retrograde tracers in each nucleus produced similar patterns of labeled neurons, predominately in layer 6 of V1, V2, V3, MT, regions of temporal cortex, and other visual areas. More complete labeling of neurons with a modified rabies virus identified these neurons as pyramidal cells with apical dendrites extending into superficial cortical layers. Importantly, the distributions of cortical neurons projecting to each of the two nuclei were highly overlapping, but formed separate populations. Sparse populations of double-labeled neurons were found in both V1 and V2 but were very low in number (<0.1%). Finally, the labeled cortical neurons were predominately in layer 6, and layer 5 neurons were labeled only in extrastriate areas. Terminations of pulvinar projections to area 17 was largely in superficial cortical layers, especially layer 1.

Transsynaptic tracing to dissect supraspinal serotonergic input regulating the bladder reflex in rats.

This study was designed to determine specific cell groups of the raphe nuclei (RN) that give rise to supraspinal serotonergic projections regulating the bladder reflex. Anesthetized rats underwent surgery to open the abdomen and expose the bladder. A total of 6 µL transsynaptic neuronal tracer pseudorabies virus (PRV-152), encoding for green fluorescent protein (GFP), was injected into the bladder detrusor. After 72 or 96 h, animals were perfused and the brain was dissected for processing transverse and sagittal sections. Subsequently, fluorescent immunohistochemistry for GFP and Serotonin (5-hydroxytryptamine [5-HT]) was performed in the brain sections. Under the microscope, each RN subset was characterized individually from caudal to rostral according to the atlas. GFP+ or GFP/5-HT double labeled neurons in each subset were quantified for statistical analysis. At 72-h post-infection, very few GFP+ or GFP/5-HT double-labeled neurons appeared in the brainstem and beyond. In contrast, many labeled neurons were found at these levels after 96 h. Quantitative analysis showed that the majority of infected 5-HT+ neurons were located in the pallidus, obscurus, and magnus nuclei. Conversely, very few infected neurons were found in other raphe subsets, that is the pontis, median, dorsal, or linear nuclei. Overall, the raphe magnus had the highest number of GFP-labeled and GFP/5-HT double-labeled cells. The caudal subsets of RN, especially the raphe magnus, are the main sources of serotonergic input to the lower spinal cord controlling bladder activity.

Anatomic Evidence for Information Exchange between Primary Afferent Sensory Neurons Innervating the Anterior Eye Chamber and the Dura Mater in Rat.

Our previous studies suggested that mechanosensitive trigeminal ganglion (TG) nerve endings innervating the inner wall of the anterior eye chamber (IWAEC) might play a role in baroreception of the IOP. It has been reported that mechanosensitive TG nerve endings also innervate the dura mater. An acute IOP elevation evokes eye pain accompanied by an ipsilateral headache, suggesting that information exchange may occur between the primary afferent neurons (PANs) innervating the IWAEC and the dura mater. To verify the information exchange between PANs of the two locations, we investigated the anatomic connection between them. Non-trans-synaptic tracers, 1,1'-dilinoleyl-3,3,3',3'-tetramethylindo-carbocyanine, 4-chlorobenzenesulfonate (FAST Dil) and cholera toxin subunit-B with a 488-nm fluorescent tag (CTB-488), were applied to the dura of the anterior cranial fossa (DACF) and the anterior eye chamber (AEC) to label the PANs. A trans-synaptic tracer, GFP-expressing pseudorabies virus (PRV152), was injected into the AEC while FAST Dil was applied to the DACF to explore the connection between PANs. Fluorescent localization in the TG was studied with a confocal fluorescent microscope. Nine days after rats were treated with CTB-488 in the AEC and FAST Dil on the DACF, FAST Dil-labeled (red), and CTB-488-labeled (green) TG neurons were observed in the medial part of the TG, while double-labeled neurons were absent. If PRV152 was used to substitute CTB-488, then FAST Dil (red) and PRV152 (green) double-labeled TG neurons and axons were observed 3 days later. Our results indicate that synapses exist between PANs of the IWAEC and the DACF, providing anatomic evidence for information exchange between them.

Neuroanatomical connections between kisspeptin neurones and somatostatin neurones in female and male rat hypothalamus: a possible involvement of SSTR1 in kisspeptin release.

Somatostatin (SST) a neuropeptide involved in the central modulation of several physiological functions, is co-distributed in the same hypothalamic areas as kisspeptin (KP), the most potent secretagogue of the gonadotropin-releasing hormone (GnRH) secretion known to date. As SST infused intracerebroventricularly (icv) evoked a potent inhibition of GnRH release, we explored neuroanatomical relationships between KP and SST populations in male and female rats. For that, intact males and ovariectomised oestradiol-replaced females were killed and their brains processed in order to simultaneously detect KP, SST and synapsin, a marker for synapses. We observed numerous appositions of KP on SST neurones both in female and male arcuate nucleus (ARC) and ventromedial hypothalamus. A large association between SST terminals and KP neurones at the level of the pre-optic area (POA) was also observed in female rats and in a more limited frame in males. Finally, most KP neurones from the ARC showed SST appositions in both sexes. To determine whether SST could affect KP cell activity, we assessed whether SST receptors (SSTR) were present on KP neurones in the ARC. We also looked for the presence of SSTR1 and SSTR2A in the brain of male rats. Brains were processed through a sequential double immunocytochemistry in order to detect KP and SSTR1 or KP and SSTR2A. We observed overlapping distributions of immunoreactive neurones for SSTR1 and KP and counted approximately one third of KP neurones with SSTR1. In contrast, neurones labelled for SSTR2A or KP were often juxtaposed in the ARC and the occurrence of double-labelled neurones was sporadic (<5%). These results suggest that SST action on KP neurones would pass mainly through SSTR1 at the level of the ARC. This article is protected by copyright. All rights reserved.

Dynamic Quantitative Assessment of Motor Axon Sprouting after Direct Facial–Hypoglossal End-To-Side Neurorrhaphy in Rats

End-to-side (ETS) neurorrhaphy is a promising procedure for peripheral nerve repair, yet controversies regarding the efficacy of this repair in facial nerve anastomosis for facial paralysis still exist. Thirty rats were divided into three groups: intact control group, direct facial-hypoglossal ETS neurorrhaphy, and end-to-end (ETE) neurorrhaphy. Nerve regeneration was assessed with vibrissae motor performance, electrophysiological tests, retrograde labeling, and histomorphological analysis at 4 and 8 months postoperatively. Both ETS and ETE neurorrhaphies resulted in axonal regeneration and functional recovery of the recipient nerve but did not reach the level of intact controls. Significantly higher numbers of myelinated axons and labeled neurons giving regenerating fibers were found in group ETE compared with group ETS at both time points, consistent with the functional and electrophysiological recovery. Group ETS showed significantly smaller fiber diameter and thinner myelin thickness than group ETE at 4 months, but the difference became nonsignificant at 8 months. ETS neurorrhaphy had a very slight effect on the donor nerve, as determined electrophysiologically and histomorphologically. Sparsely distributed double-labeled neurons and relatively large amounts of single-labeled neurons contributing to reinnervation were found through double retrograde neuronal labeling in group ETS. Further quantitative analysis of the percentage of double-labeled neurons showed a pronounced tendency to decline from 19.8% at 4 months to 6.0% at 8 months postoperatively. Successful reinnervation after ETS neurorrhaphy could be achieved through both collateral sprouting and terminal sprouting, with the latter seeming to be the principal origin of motor nerve sprouting.

Central Norepinephrine‐Kisspeptin Pathway as Part of the Vaso‐motor Effects of Estrogen in an Animal Model of Menopausal Hot Flashes

Menopausal transition includes the vasomotor disturbance hot flash as a significant symptom. Thus, the lack of estrogen has pronounced effects on thermoregulation, but how estrogen modulates thermoregulation remains poorly understood. Both norepinephrine (NE) and kisspeptin (Kp) have been shown to be involved in the central control of body temperature. In this work, we investigated the role of NE in the activation of hypothalamic Kp neurons and vasomotor effects caused by the lack of 17β‐estradiol (E2) in ovariectomized (OVX) rats. The first experiment was designed to determine the effects of E2 on tail skin temperature (TST) and correlate neuronal activation. Adult female rats were OVX and implanted with subcutaneous Silastic capsules containing oil (OVX) or E2 (6 ug/capsule/rat; OVX+E2). The TST was recorded for 2 hours on days 3, 7 and 14 after surgery. Rats were perfused on the morning of day 14 and the brains were processed for immunohistochemistry. OVX+E2 rats displayed greater uterine weight than OVX rats. The TST was higher in OVX compared to OVX+E2 rats at 7 and 14 days after ovariectomy. On day 14, OVX rats displayed an increased percentage of tyrosine hydroxylase (TH)‐immunoreactive (ir) neurons expressing Fos in the A1, A2 and locus coeruleus (LC), which was positively correlated with TST. The number of Fos‐ir neurons was also higher in the anteroventral periventricular (AVPV), median preoptic (MnPO), medial preoptic (MPO), and paraventricular (PVN) nuclei of OVX rats compared with OVX+E2. In the arcuate nucleus (ARC), the numbers of Kp single‐labeled and Fos/Kp double‐labeled neurons were markedly higher in OVX rats. In a second experiment, we investigated whether NE projections to the hypothalamus would drive the activity of Kp neurons in OVX rats. The α2‐adrenergic agonist clonidine (CLO) was used to reduce central norepinephrine release and TST and Kiss1 gene expression were measured. OVX and OVX+E2 rats received daily intracerebroventricular injections of CLO (10 ug/3 μL/rat; OVX+CLO) or vehicle (OVX+V and OVX+E2+V) on days 12, 13 and 14 after ovariectomy. OVX+V rats displayed higher TST than OVX+E2+V, and this response was completely restored in OVX+CLO rats, whose TST was similar to that of OVX+E2+V rats. The same response was found for Kiss1 mRNA in the ARC, with higher expression in OVX than in OVX+E 2, and levels fully restored in OVX+CLO rats. On the other hand, CLO significantly increased Kiss1 expression in the AVPV of OVX+CLO rats compared to OVX+V and OVX+E2+V rats, and this response was associated with an increased secretion of luteinizing hormone (LH) in OVX + CLO rats. Thus, the increase in TST caused by the absence of E2 in OVX rats is associated with an increased activity of noradrenergic and hypothalamic nuclei and seems to depend on the NE activation of Kp neurons in the ARC. These findings provide evidence of the involvement of a central NE‐Kp pathway as part of the vasomotor effects of estrogen, with possible implication in origin of postmenopausal hot flash.Support or Funding InformationCNPq; FAPEMIGThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Activity-based anorexia activates CRF immunoreactive neurons in female rats

Activity-based anorexia (ABA) is a well-established animal model mimicking the eating disorder anorexia nervosa (AN). Since the pathophysiology of AN is yet poorly understood and specific drug treatments are lacking so far, animal models might be useful to further understand this disease. ABA consists of time-restricted access to food for 1.5 h/day and the possibility to exercise in a running wheel for 24 h/day. This combination leads to robust body weight loss as observed in AN. Here, we investigated the activation of brain corticotropin-releasing factor (CRF) neurons, a transmitter involved in the response to stress, emotional processes and also food intake. After development of ABA, rat brains were processed for c-Fos and CRF double immunohistochemistry. ABA increased the number of c-Fos/CRF double labeled neurons in the paraventricular nucleus (PVN) and the dorsomedial hypothalamic nucleus (DMH) compared to the ad libitum (AL, ad libitum fed, no running wheel) and activity (AC, ad libitum fed, running wheel, p < 0.05) but not to the restricted feeding (RF, food for 1.5 h/day, no running wheel, p > 0.05) group. Also the number of CRF neurons was increased in the DMH of ABA rats compared to AL and AC (p < 0.05). In the Edinger-Westphal nucleus (EW) the number of c-Fos positive neurons was increased in ABA and RF compared to AC (p < 0.05), while the number of double labeled neurons was not different (p > 0.05). Taken together, brain CRF activated under conditions of ABA might play a role in the development and maintenance of this animal model and possibly also in human AN.

The effects of FK1706 on nerve regeneration and bladder function recovery following an end-to-side neurorrhaphy in rats.

BackgroundImmunophilin ligands are neuroregenerative agents binding to FK506 binding proteins, by which stimulate recovery of neurons in a variety of injury nerves. FK1706 is a novel immunophilin ligand which has neuroprotective and neuroregenerative effects but without immunosuppressive activity. At present, most reports about FK1706 in ameliorating nerve injury and functional recovery are limited to cavernous nerve injury and erectile function recovery. This study aimed to demonstrate the effects of FK1706 on nerve regeneration and bladder function recovery following an end-to-side neurorrhaphy in rat models.MethodThe numbers of regenerated myelinated axons of the pelvic parasympathetic nerve (PPN) in the three groups’ rats (FK1706 + ETS, ETS and control groups) were evaluated. Their intravesical pressure (IVP), S100β and growth associated protein 43 (GAP43) expressions were also compared.ResultsIn FK1706 + ETS group, 90% the rats showed that the frequency of FG labeled neurons was larger than the 3.5 cutoff value, 100% the rats showed that the frequency of FG-FB double-labeled neurons was larger than the 5.5 cutoff value. The average maximum of IVP in FK1706 + ETS group reached 76.3% of the value in control group. Their average number of myelinated axons of regenerated PPN reached 80% of the amount in control group. The nerve regeneration-associated markers data indicated that the expression level of S100β and GAP43 in FK1706 + ETS group was approximately 2-fold higher than that of ETS group (P < 0.05).ConclusionsAfter end-to-side neurorrhaphy, FK1706 effectively enhanced the nerve regeneration and bladder function recovery.

Neuronal connections of the central amygdalar nucleus with refeeding-activated brain areas in rats

Following fasting, satiety is accompanied by neuronal activation in brain areas including the central amygdalar nucleus (CEA). Since CEA is known to inhibit food intake, we hypothesized that CEA contributes to the termination of meal during refeeding. To better understand the organization of this satiety-related circuit, the interconnections of the CEA with refeeding-activated neuronal groups were elucidated using retrograde (cholera toxin-β subunit, CTB) and anterograde (phaseolus vulgaris leucoagglutinin, PHA-L) tracers in male rats. C-Fos-immunoreactivity was used as marker of neuronal activation. The refeeding-activated input of the CEA primarily originated from the paraventricular thalamic, parasubthalamic and parabrachial nuclei. Few CTB-c-Fos double-labeled neurons were detected in the prefrontal cortex, lateral hypothalamic area, nucleus of the solitary tract (NTS) and the bed nuclei of the stria terminalis (BNST). Only few refeeding-activated proopiomelanocortin-producing neurons of the arcuate nucleus projected to the CEA. Anterograde tract tracing revealed a high density of PHAL-labeled axons contacted with refeeding-activated neurons in the BNST, lateral hypothalamic area, parasubthalamic, paraventricular thalamic and parabrachial nuclei and NTS; a low density of labeled axons was found in the paraventricular hypothalamic nucleus. Chemogenetic activation of the medial CEA (CEAm) inhibited food intake during the first hour of refeeding, while activation of lateral CEA had no effect. These data demonstrate the existence of reciprocal connections between the CEA and distinct refeeding-activated hypothalamic, thalamic and brainstem nuclei, suggesting the importance of short feedback loops in the regulation of satiety and importance of the CEAm in the regulation of food intake during refeeding.

Distinct recruitment of basolateral amygdala-medial prefrontal cortex pathways across Pavlovian appetitive conditioning

Associative learning can enable environmental cues to signal food and stimulate feeding, independent of physiological hunger. Two forebrain regions necessary in cue driven feeding, the basolateral area of the amygdala and the medial prefrontal cortex, communicate via extensive, topographically organized connections. The basolateral nucleus (BLA) sends extensive projections to the prelimbic cortex (PL), and our aim here was to determine if this pathway was selectively recruited during cue-food associative learning. The anterior and posterior basolateral nuclei are recruited during different phases of cue-food learning, and thus we examined whether distinct pathways that originate in these nuclei and project to the PL are differently recruited during early and late stages of learning. To accomplish this we used neuroanatomical tract tracing combined with the detection of Fos induction. To identify projecting neurons within the BLA, prior to training, rats received a retrograde tracer, Fluoro-Gold (FG) into the PL. Rats were given either one or ten sessions of tone-food presentations (Paired group) or tone-only presentations (Control group). The Paired group learned the tone-food association quickly and robustly and had greater Fos induction within the anterior and posterior BLA during early and late learning compared to the Control group. Notably, the Paired group had more double-labeled neurons (FG + Fos) during late training compared to the Control group, specifically in the anterior BLA. This demonstrates selective recruitment of the anterior BLA-PL pathway by late cue-food learning. These findings indicate plasticity and specificity in the BLA-PL pathways across cue-food associative learning.

The effects of venous ensheathment on facial nerve repair in the rat.

To investigate the protective effect of autologous venous ensheathment on sutured rat facial nerve and to test whether the ensheathment could improve the functional recovery of repaired nerve and accuracy of axonal growth. In vivo study. Forty-six rats were examined, with six rats serving as normal controls and 40 receiving facial nerve transection and suture repair (SR) or transection and suture repair with an additional venous ensheathment (VE). The rats were then subjected to functional testing, histological assessment of nerve specimens, or retrograde tracing, respectively. At the postoperative day (POD) 60, the venous ensheathment showed no adhesion at the surrounding tissues. No significant difference in neuroma formation was found between the two surgical manipulations (SR and VE groups) (P < 0.05). Retrogradely labeled motoneurons in facial nuclei were extremely disorganized after the facial nerve undertook surgical manipulation. In all manipulated groups, double retrogradely labeled neurons, indicative of aberrant axonal branching during regeneration, could be observed after peripheral manipulation across all time points. With the two facial surgical manipulations, the average count of double-labeled neurons at POD 60 was significantly less than at POD 21 (P < 0.05). Autologous venous ensheathment could not help with the functional recovery of facial nerve or improve the accuracy of axonal regeneration. Further studies are warranted to elucidate the effects of venous ensheathment in other motor and sensory nerve models. NA. Laryngoscope, 127:1558-1564, 2017.