Rat Brain Nuclei Research Articles

The anticonvulsant effect of saiga horn on febrile seizures by regulating brain serotonin content and inhibiting neuroinflammation

Ethnopharmacological relevanceSaiga antelope horn (SAH) is derived from the Saiga antelope (Saiga tatarica Linnaeus) of the bovidae family. SAH has been used for the treatment of febrile seizures (FS) in children for thousands of years in China. Due to the biological protection of Saiga antelope and its ethical reasons, the application of SAH has been widely restricted. Unfortunately, the field of artificial manufacturing of SAH is still blank at present. The mechanism of SAH in the treatment of FS is still unclear, which seriously hinders the further development of artificial antelope horns and the search for substitutes for SAH. At present, there is an urgent need to determine the mechanism of SAH in the treatment of FS, so as to provide a theoretical basis for artificial antelope horn and its substitutes. Aim of the studyTo explore the anti-FS effect of natural SAH on FS rat model and its possible mechanism, and to provide a theoretical basis for the subsequent manufacture of artificial antelope horns and the search for the best substitutes. Materials and methodsFS was induced by a warm water bath (48 ± 0.5 °C). The latency and seizure grade of FS were observed and recorded. Hematoxylin-eosin (HE) staining was used to observe the functional defect of hippocampal cells. The contents of tryptophan (TRP), serotonin (5-HT), IL-1β and TNF-a in rat brain tissue were determined by ELISA. qRT-PCR and Western blot were used to detect the expression of 5-HT synthesis related neurotransmitter receptors, catalytic enzymes and inflammatory factors in hippocampus. Immunofluorescence was used to observe the expression of TPH2 protein in the dorsal raphe nucleus of rat brain. ResultsAfter pretreating rats with SAH, the seizure grade of FS was significantly reduced and the latency was prolonged. SAH can reduce the histological damage of hippocampal tissue induced by FS in rats. Further analysis of ELISA results showed that SAH significantly increased the levels of TRP and 5-HT in the brain of FS rats, and significantly decreased the levels of IL-1β and TNF-a. The results of QPCR showed that SAH could up-regulate the expression of ER-β and TPH2 mRNA and down-regulate the expression of IL-1β and TNF-ɑ mRNA in the hippocampus of rats. In addition, WB and immunofluorescence results showed that SAH could significantly up-regulate the expression of ER-β/TPH2/5-HT pathway in the hippocampus of FS rats and the expression of TPH2 protein in the raphe nucleus, but had no significant effect on SERT protein in the hippocampus of FS rats. In addition, ER-β protein inhibitor PHTPP significantly inhibited the therapeutic effect of SAH on FS rats. ConclusionsThe present study demonstrates that SAH has a significant anticonvulsant effect on the FS rat model. The mechanism may be related to the increase of TRP content and up-regulation the expression of ER-β/TPH2/5-HT signaling pathway in the brain of FS rats, thereby increasing the content of 5-HT in the brain and reducing the content of IL-1β and TNF-a in the brain.

Orthodontic pain: c-Fos expression in rat brain nuclei after rapid maxillary expansion

The aim of this in vivo study was to quantitatively evaluate pain after rapid maxillary expansion (RME) in young rats by analyzing the activation of nociception-related structures, that is, the caudalis, interpolaris, and oralis subnuclei, according to the Fos expression. A total of 65 Wistar rats were assigned to three groups: control group (n=15) with no treatment, positive control group (n=25), and experimental group (n=25) with RME. The experimental animals were euthanized at 6, 12, 24, 48, and 72 hours after RME, and the brain was later carefully collected. Coronal sections through the spinal trigeminal caudalis, spinal trigeminal interpolaris, and spinal trigeminal oralis were cut (thickness of 40 µm) on a cryostat and processed for Fos immunohistochemistry. Images from the sections were captured under light microscopy, and ImageJ software was used to count Fos-like immunoreactive neurons. The Analysis of variance (ANOVA) and Tukey test were used for statistical analysis, and the significance level was set at 5%. RME induced incisor distalization and opening of the midpalatal suture, as well as neuronal activation of the spinal trigeminal nucleus. The experimental group demonstrated significantly more Fos-positive neurons in subnuclei caudalis and subnuclei interpolaris 6 hours after the maxillary expansion. The Fos immunoreactivity significantly decreased at 12 hours and increased again at 24 and 48 hours (P < 0.001). The RME increases the neural activation of brain regions involved in the nociception region, as determined by the Fos expression. The most intense Fos-like immunoreactive expression was detected in the brain 6 hours after the start of the palatal expansion.

Inflammatory Stress Induced by Intraperitoneal Injection of LPS Increases Phoenixin Expression and Activity in Distinct Rat Brain Nuclei.

Due to phoenixin’s role in restraint stress and glucocorticoid stress, as well as its recently shown effects on the inflammasome, we aimed to investigate the effects of lipopolysaccharide (LPS)-induced inflammatory stress on the activity of brain nuclei-expressing phoenixin. Male Sprague Dawley rats (n = 6/group) were intraperitoneally injected with either LPS or control (saline). Brains were processed for c-Fos and phoenixin immunohistochemistry and the resulting slides were evaluated using ImageJ software. c-Fos was counted and phoenixin was evaluated using densitometry. LPS stress significantly increased c-Fos expression in the central amygdaloid nucleus (CeM, 7.2-fold), supraoptic nucleus (SON, 34.8 ± 17.3 vs. 0.0 ± 0.0), arcuate nucleus (Arc, 4.9-fold), raphe pallidus (RPa, 5.1-fold), bed nucleus of the stria terminalis (BSt, 5.9-fold), dorsal motor nucleus of the vagus nerve (DMN, 89-fold), and medial part of the nucleus of the solitary tract (mNTS, 121-fold) compared to the control-injected group (p < 0.05). Phoenixin expression also significantly increased in the CeM (1.2-fold), SON (1.5-fold), RPa (1.3-fold), DMN (1.3-fold), and mNTS (1.9-fold, p < 0.05), leading to a positive correlation between c-Fos and phoenixin in the RPa, BSt, and mNTS (p < 0.05). In conclusion, LPS stress induces a significant increase in activity in phoenixin immunoreactive brain nuclei that is distinctively different from restraint stress.

Neuromodulation Effect of Very Low Intensity Transcranial Ultrasound Stimulation on Multiple Nuclei in Rat Brain.

ObjectiveLow-intensity transcranial ultrasound stimulation (TUS) is a non-invasive neuromodulation technique with high spatial resolution and feasible penetration depth. To date, the mechanisms of TUS modulated neural oscillations are not fully understood. This study designed a very low acoustic intensity (AI) TUS system that produces considerably reduced AI Ultrasound pulses (ISPTA < 0.5 W/cm2) when compared to previous methods used to measure regional neural oscillation patterns under different TUS parameters.MethodsWe recorded the local field potential (LFP) of five brain nuclei under TUS with three groups of simulating parameters. Spectrum estimation, time-frequency analysis (TFA), and relative power analysis methods have been applied to investigate neural oscillation patterns under different stimulation parameters.ResultsUnder PRF, 500 Hz and 1 kHz TUS, high-amplitude LFP activity with the auto-rhythmic pattern appeared in selected nuclei when ISPTA exceeded 12 mW/cm2. With TFA, high-frequency energy (slow gamma and high gamma) was significantly increased during the auto-rhythmic patterns. We observed an initial plateau in nuclei response when ISPTA reached 16.4 mW/cm2 for RPF 500 Hz and 20.8 mW/cm2 for RPF 1 kHz. The number of responding nuclei started decreasing while ISPTA continued increasing. Under 1.5 kHz TUS, no auto-rhythmic patterns have been observed, but slow frequency power was increased during TUS. TUS inhibited most of the frequency band and generated obvious slow waves (theta and delta band) when stimulated at RPF = 1.5 kHz, ISPTA = 8.8 mW/cm2.ConclusionThese results demonstrate that very low intensity Transcranial Ultrasound Stimulation (VLTUS) exerts significant neuromodulator effects under specific parameters in rat models and may be a valid tool to study neuronal physiology.

Label-free lipidome study of paraventricular thalamic nucleus (PVT) of rat brain with post-traumatic stress injury by Raman imaging.

Post-traumatic stress disorder (PTSD) is a widespread psychiatric injury that develops serious life-threatening symptoms like substance abuse, severe depression, cognitive impairments, and persistent anxiety. However, the mechanisms of post-traumatic stress injury in brain are poorly understood due to the lack of practical methods to reveal biochemical alterations in various brain regions affected by this type of injury. Here, we introduce a novel method that provides quantitative results from Raman maps in the paraventricular nucleus of the thalamus (PVT) region. By means of this approach, we have shown a lipidome comparison in PVT regions of control and PTSD rat brains. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was also employed for validation of the Raman results. Lipid alterations can reveal invaluable information regarding the PTSD mechanisms in affected regions of brain. We have showed that the concentration of cholesterol, cholesteryl palmitate, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, ganglioside, glyceryl tripalmitate and sulfatide changes in the PVT region of PTSD compared to control rats. A higher concentration of cholesterol suggests a higher level of corticosterone in the brain. Moreover, concentration changes of phospholipids and sphingolipids suggest the alteration of phospholipase A2 (PLA2) which is associated with inflammatory processes in the brain. Our results have broadened the understanding of biomolecular mechanisms for PTSD in the PVT region of the brain. This is the first report regarding the application of Raman spectroscopy for PTSD studies. This method has a wide spectrum of applications and can be applied to various other brain related disorders or other regions of the brain.

Restraint stress increases the expression of phoenixin immunoreactivity in rat brain nuclei

Phoenixin is a recently discovered peptide, which has been associated with reproduction, anxiety and food intake. Based on a considerable co-localization it has been linked to nesfatin-1, with a possible antagonistic mode of action. Since nesfatin-1 is known to play a role in anxiety and the response to stress, this study aims to investigate the effects of a well-established psychological stress model, restraint stress, on phoenixin-expressing brain nuclei and phoenixin expression in rats. Male Sprague-Dawley rats were subjected to restraint stress (n = 8) or left undisturbed (control, n = 6) and the brains processed for c-Fos- and phoenixin immunohistochemistry. The number of c-Fos expressing cells was counted and phoenixin expression assessed semiquantitatively. Restraint stress significantly increased c-Fos expression in the dorsal motor nucleus of vagus nerve (DMN, 52-fold, p < 0.001), raphe pallidus (RPa, 15-fold, p < 0.001), medial part of the nucleus of the solitary tract (mNTS, 16-fold, p < 0.001), central amygdaloid nucleus, medial division (CeM, 9-fold, p = 0.01), supraoptic nucleus (SON, 9-fold, p < 0.001) and the arcuate nucleus (Arc, 2.5-fold, p < 0.03) compared to control animals. Also phoenixin expression significantly increased in the DMN (17-fold, p < 0.001), RPa (2-fold, p < 0.001) and mNTS (1.6-fold, p < 0.001) with positive correlations between c-Fos and phoenixin (r = 0.74–0.85; p < 0.01) in these nuclei. This pattern of activation suggests an involvement of phoenixin in response to restraint stress. Whether phoenixin mediates stress effects or is activated in a counterbalancing fashion will have to be further investigated.

Role of TACE in LPS‐induced Production of Soluble Tumor Necrosis Factor‐α in Astrocytes

The pro‐inflammatory cytokine tumor necrosis factor alpha (TNF‐α) in the brain has been implicated in the neurohumoral activation in cardiovascular diseases like heart failure and hypertension. TNF‐α is originally synthesized as a membrane bound protein, the transmembrane TNF‐α (tmTNF‐α). TNF‐α converting enzyme (TACE) proteolytically cleaves the extracellular domain of tmTNF‐α) to release the fully functional form of soluble TNF‐α (sTNF‐α), the major form that contributes to the sympathetic excitation‐induced by cytokine TNF‐α. Our previous work has demonstrated a significant contribution of cytokine synthesis within the central nervous system in mediating neurohumoral excitation and the progression of heart failure in rats, and that both TNF‐α and TACE are increased within cardiovascular regulatory brain nuclei in rats with heart failure. However, the molecular mechanisms regulating TNF‐α and TACE expression within the brain and the specific cell types contributing to this process are currently unknown. The present study sought to determine whether astrocytes might contribute to central nervous system inflammation as a significant source of TNF‐α and TACE. We isolated and cultured astrocytes from male and female neonatal rat brains. Cells were maintained in culture for 28 days prior to treatment with lipopolysaccharide (LPS) alone or in combination with a matrix metalloproteinase inhibitor. Cells and conditioned media were collected after 8 hours to measure RNA expression as well as TNF‐α secretion. Primary astrocytes expressed TACE mRNA at baseline, and LPS increased mRNA levels of TNF‐α (2.8±0.6 vs 3.8±2.1‐fold change), interleukin‐1β (IL‐1β) (19.9±12.3 vs 22.2±7.6‐fold change), and TACE (1.8±0.2 vs 2.2±0.3‐fold change) to a similar extent in astrocytes from male and female rats. Neither the specific TACE inhibitor TAPI‐1 nor the broad range metalloproteinase inhibitor GM6001 significantly attenuated the LPS‐induced increase in TNF‐α at the mRNA level, and in fact we observed a trend towards increased TNF‐α expression specifically in astrocytes from male animals co‐treated with LPS and either inhibitor. This was in contrast to protein expression of sTNF‐α. LPS significantly increased TNF‐α secretion into the media in astrocytes from male (677±28 vs 5±1pg/ml) and female (658±42 vs 6±4pg/ml) animals to a similar extent. Interestingly, this was attenuated by treatment with TAPI‐1 (169±14 male and 108±11pg/ml female) but not GM6001 (526±51 male and 667±44pg/ml female). However, the LPS‐induced increase in TACE expression was attenuated in cells treated with GM6001 but not those treated with TAPI‐1. These data suggest that astrocytes in the brain may contribute to the production of cytokines and TACE in pathophysiological conditions, and TACE plays an important role in releasing the inflammatory sTNF‐α from its precursor tmTNF‐α.Support or Funding InformationSupported by R01‐HL139521 and T32 HL007121.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of vitamin D3 supplementation on cognition and blood lipids: a 12-month randomised, double-blind, placebo-controlled trial

Alzheimer’s disease (AD) is the leading cause of dementia and loss of autonomy and independency in the elderly.1 Currently, no effective pharmacological interventions change the onset or progression of AD.2...

Sushi repeat-containing protein X-linked 2: A novel phylogenetically conserved hypothalamo-pituitary protein.

Sushi repeat-containing protein X-linked 2 (SRPX2) is a novel protein associated with language development, synaptic plasticity, tissue remodeling, and angiogenesis. We investigated the expression and spatial localization of SRPX2 in normal mouse, rat, monkey, and human brain using in situ hybridization and immunohistochemistry. Antibody specificity was determined using in vitro siRNA based silencing of SRPX2. Cell type-specific expression was verified by double-labeling with oxytocin or vasopressin. Western blot was used to detect SRPX2 protein in rat and human plasma and cerebrospinal fluid. Unexpectedly, SRPX2 mRNA expression levels were strikingly higher in the hypothalamus as compared to the cortex. All SRPX2 immunoreactive (ir) neurons were localized in the hypothalamic paraventricular, periventricular, and supraoptic nuclei in mouse, rat, monkey, and human brain. SRPX2 colocalized with vasopressin or oxytocin in paraventricular and supraoptic neurons. Hypothalamic SRPX2-ir positive neurons gave origin to dense projections traveling ventrally and caudally toward the hypophysis. Intense axonal varicosities and terminal arborizations were identified in the rat and human neurohypophysis. SRPX2-ir cells were also found in the adenohypophysis. Light SRPX2-ir projections were observed in the dorsal and ventral raphe, locus coeruleus, and the nucleus of the solitary tract in mouse, rat and monkey. SRPX2 protein was also detected in plasma and CSF. Our data revealed intense phylogenetically conserved expression of SRPX2 protein in distinct hypothalamic nuclei and the hypophysis, suggesting its active role in the hypothalamo-pituitary axis. The presence of SRPX2 protein in the plasma and CSF suggests that some of its functions depend on secretion into body fluids.

Activity-based anorexia activates nesfatin-1 immunoreactive neurons in distinct brain nuclei of female rats

Activity-based anorexia (ABA) is an established animal model for the eating disorder anorexia nervosa (AN). The pathophysiology of AN and the involvement of food intake-regulatory peptides is still poorly understood. Nesfatin-1, an anorexigenic peptide also involved in the mediation of stress, anxiety and depression might be a likely candidate involved in the pathogenesis of AN. Therefore, activation of nesfatin-1 immunoreactive (ir) brain nuclei was investigated under conditions of ABA. Female Sprague-Dawley rats were used and divided into four groups (n=6/group): activity-based anorexia (ABA), restricted feeding (RF), activity (AC) and ad libitum fed (AL). After the 21-day experimental period and development of ABA, brains were processed for c-Fos/nesfatin-1 double labeling immunohistochemistry. ABA increased the number of nesfatin-1 immunopositive neurons in the paraventricular nucleus, arcuate nucleus, dorsomedial hypothalamic nucleus, locus coeruleus and in the rostral part of the nucleus of the solitary tract compared to AL and AC groups (p<0.05) but not to RF rats (p>0.05). Moreover, we observed significantly more c-Fos and nesfatin-1 ir double-labeled cells in ABA rats compared to RF, AL and AC in the supraoptic nucleus (p<0.05) and compared to AL and AC in the paraventricular nucleus, arcuate nucleus, dorsomedial hypothalamic nucleus, dorsal raphe nucleus and the rostral raphe pallidus (p<0.05). Since nesfatin-1 plays a role in the inhibition of food intake and the response to stress, we hypothesize that the observed changes of brain nesfatin-1 might play a role in the pathophysiology and symptomatology under conditions of ABA and potentially also in patients with AN.

Water apparent diffusion coefficient correlates with gamma oscillation of local field potentials in the rat brain nucleus accumbens following alcohol injection

Ethanol is a vasoactive agent as well as psychoactive drug. The neurovascular response, coupled with neuronal activity, can be disturbed by alcohol intake. Hence, blood oxygenation level-dependent (BOLD) fMRI, which relies on neurovascular coupling, might not be reliable to reflect alcohol-induced neuronal responses. Recently, diffusion fMRI has been shown to be more sensitive to neural activity than BOLD fMRI even when neurovascular coupling is disrupted. Especially, the apparent diffusion coefficient (ADC) is sensitive to changes occurring in the cellular tissue structure upon activation. In the present study, we compared BOLD fMRI signals, ADC, and local field potentials (LFPs) in the nucleus accumbens (NAc) following injection of an ethanol solution (0.4 g/kg body weight) in rats under medetomidine anesthesia. An increase in the gamma oscillation power of LFP and an ADC decrease were observed 5 min after the injection of EtOH. The BOLD signals showed a negative slow drift, similar to mean arterial pressure with a peak approximately 10 min after the injection. These results confirm that DfMRI can be a better marker of the neuronal activity than BOLD fMRI, especially when the brain hemodynamic status is changed by vasoactive drugs such as ethanol.

Caracterización de la estructura secundaria de subtipos de la histona H1 por dicroísmo circular

Introducción: Las histonas H1 modulan la estructura y la función de la cromatina. Las células somáticas de mamífero contienen los subtipos H1°, H1a, H1b, H1c, H1d y H1e; en células germinales de testículo y en ovocito, se encuentran respectivamente H1t y H1oo. Su estructura está conformada por un dominio central globular flanqueado por los dominios N-Terminal (DNT) y C-Terminal (DCT). Objetivo: Caracterizar la estructura secundaria de subtipos de la histona H1 mediante dicroísmo circular (DC). Materiales y Métodos: La histona H1 total se extrajo de núcleos de cerebro de rata por cromatografía de intercambio catiónico; la H1° se purificó por filtración en gel y las H1a, H1b, H1c y H1e por cromatografía líquida de alta resolución de fase reversa (RF-HPLC). Los espectros de DC se realizaron en tampón fosfato 10 mM; tampón fosfato 10 mM, 20% TFE (trifluoroetanol); tampón fosfato 10 mM, 40% TFE; tampón fosfato 10 mM, 60% TFE; tampón fosfato 10 mM, 150 mM NaCl y tampón fosfato 10 mM, 1 M NaCl. El análisis de los espectros se realizó con el programa Standard Analysis. Resultados: El porcentaje de hélice-alfa se calculó por diferentes métodos matemáticos teniendo en cuenta elipticidad molar a 193 nm y a 222 nm; con programa de deconvolución K2D y con relaciones cualitativas R1 y R2. El TFE induce la estructura en hélice-alfa en cada uno de los subtipos, mientras que NaCl no induce ningún cambio importante. Conclusión: Los subtipos con mayor contenido de hélice-alfa son H1a y H1c. Las diferencias observadas en el porcentaje de hélice-alfa entre los diferentes subtipos puede ser importante para su diferenciación funcional.

Leptin in the Hindbrain Facilitates Phosphorylation of STAT3 in the Hypothalamus

Leptin receptors (ObRb) in the forebrain and hindbrain are each independently recognized as important mediators of leptin‐induced changes in energy balance. We recently used sub‐threshold doses of leptin to show that chronic activation of both hypothalamic and brainstem ObRb is required to reduce body fat. The objective of this study was to identify selectively activated brain nuclei in rats receiving chronic infusions of sub‐threshold leptin in both forebrain and hindbrain. Male Sprague Dawley rats were fitted with 3rd and 4th ventricle cannulas. Alzet pumps were used to infuse either saline (S) or leptin (L) (0.1 µg/24h in 3rd, 0.6 µg/24h in 4th) for 6 days, resulting in four treatment groups SS, SL, LS, LL (3th‐4th). There was no effect of leptin in single ventricle infused groups (LS, SL) compared to controls (SS). Double ventricle infused rats (LL) showed a 50% inhibition of food intake, 30% loss in body fat, and an increase in ObRb activation marker phosphorylated STAT3 (pSTAT3‐ir) in the arcuate nucleus (Arc), ventromedial (VMH), dorsomedial (DMH) and posterior hypothalamus compared to other groups. No differences in pSTAT3‐ir were observed in midbrain or brainstem nuclei despite a 6 fold higher infusion of leptin into the 4th ventricle than the 3rd. Delta Fos B‐ir, a chronic neuronal activation marker, showed that multiple brain nuclei were chronically activated due to the process of infusion, but only the Arc, VMH, DMH and ventral tuberomammillary nucleus showed a significant increase in LL rats compared to other groups. These data imply that the simultaneous activation of both forebrain and hindbrain ObRb is required to reduce body fat and that leptin in the hindbrain facilitates phosphorylation of STAT3 in hypothalamic areas involved in the control of energy balance.

Establishment of C6 brain glioma models through stereotactic technique for laser interstitial thermotherapy research.

Objective:To establish C6 brain glioma models using stereotactic technique, and to study effects of laser interstitial thermotherapy (LITT) in rat models of glioma.Methods:C6 glioma cells were cultured in dulbecco's minimum essential medium (DMEM) cell culture medium. The in vitro C6 cell cultures were stereotaxically implanted into the right caudate nucleus of rat brain. Presence of tumor was confirmed with Factor VIII R, hematoxylin–eosin stain, staining of glial fibrillary acid protein, and S-100 immunohistochemistry. After magnetic resonance (MR) scanning and correction of tumor location, the models were divided into groups according to the treating time and laser power (2–10 W). Semiconductor laser optical fibers were inserted in tumors for LITT. Cortex's temperature conducted from the center target was measured using infrared thermograph, and deep-tissue temperature around the target was measured using a thermocouple.Results:Rat C6 gliomas were inoculated with optimized stereotactic technique. These gliomas resembled human glioma in terms of histopathological features. Such models are more reliable and reproducible, with 100% yield of intracranial tumor and no extracranial growth extension. The difference between cortex temperature conducted from center target and deep-tissue temperature around target was not statistically significant.Conclusion:The rat C6 brain glioma model established in the study was a perfect model to study LITT of glioma. Infrared thermograph technique measured temperature conveniently and effectively. The technique is noninvasive, and the obtained data could be further processed using software used in LITT research. To measure deep-tissue temperature, combining thermocouple with infrared thermograph technique would present better results.

Galanin activated Gi/o-proteins in human and rat central nervous systems

The neuropeptide galanin (GAL) is involved in the control of hormone secretion, nociception, feeding behavior, attention, learning and memory. The anatomical localization of galanin receptors in the brain has been described using autoradiography and immunohistochemistry, but both techniques are limited by the availability of specific radioligands or antibodies. Functional autoradiography provides an alternative method by combining anatomical resolution and information of the activity mediated by G-protein coupled receptors. The present study analyzes the functional GAL receptors coupled to Gi/o-proteins in human and rat brain nuclei using [35S]GTPγS autoradiography.The results show the anatomical distribution of Gi/o-proteins activated by GAL receptors that trigger intracellular signaling mechanisms. The activity mediated by GAL receptors in human and rat brain showed a good correlation of the net stimulation in areas such as spinal cord, periaqueductal gray, putamen, CA3 layers of hippocampus, substantia nigra and diverse thalamic nuclei.The functional GAL receptors coupled to Gi/o-proteins showed a similar pattern for both species in most of the areas analyzed, but some discrete nuclei showed differences in the activity mediated by GAL, such as the ventroposteromedial thalamic nucleus, or areas that regulate learning and memory processes in the hippocampus. Taken into consideration the present results, the rat could be used as an experimental model for the study of the physiological role of GAL-mediated neurotransmission and the modulation of GAL receptors activity in the human CNS.

Acute effects on brain cholecystokinin-like concentration and anxiety-like behaviour in the female rat upon a single injection of 17β-estradiol

BackgroundThe neuropeptide cholecystokinin (CCK) has been implicated in the neurobiology of anxiety and panic disorders, as well as in dopamine-related behaviours. Anxiety and panic-disorders are twice as common in females compared to males, but studies of females are rare, although increasing in number. Limited studies have found that CCK fluctuates in limbic regions during the estrous cycle, and that CCK and its receptors are sensitive to estrogen. Aim/PurposeThe aim of the present work was to study the acute effects of 17β-estradiol on anxiety-like behaviour and on CCK-like immunoreactivity (LI) in the female rat brain (amygdala, hippocampus, nucleus accumbens, and cingulate cortex). MethodsFour groups of female Sprague–Dawley rats were used: ovariectomized, ovariectomized+17β-estradiol-replacement, sham, and sham+17β-estradiol-replacement. The effect of 17β-estradiol-replacement on anxiety-related behaviour was measured in all animals on the elevated plus maze 2–24h after injection. CCK-LI concentration was measured in punch biopsies by means of radioimmunoassay. Results17β-estradiol decreased anxiety-like behaviour 2h after administration in ovariectomized and sham-operated animals, as demonstrated by increased exploration of the open arms compared to respective sesame oil-treated controls. This effect was not present when testing occurred 24h post-treatment. The rapid behavioural effect of 17β-estradiol was accompanied by changes in CCK-LI concentrations in regions of the limbic system including cingulate cortex, hippocampus, amygdala and nucleus accumbens. ConclusionAlthough the interpretation of these data requires caution since the data were collected from two different experiments, our results suggest that estrogen-induced anxiolytic effects may be associated with changes of the CCK-system in brain regions controlling anxiety-like behaviour.

The rat dorsal column nuclei contain a region homologous to the human Locus K

Locus K is a newly identified region within the territory of the human nucleus cuneatus that shares neurochemical and histological features with protopathic second order sensory nuclei (Del Fiacco et al., 2013; Serra et al., 2013; SIAI 2014). This work is aimed at examining the rat dorsal column nuclei in order to ascertain whether a structure homologous to the human Locus K occurs in the rat brain. Rat brainstem sections were observed by means of ABC and fluorescence immunohistochemistry for neuropeptides calcitonin gene-related peptide (CGRP) and substance P (SP), Kluver-Barrera and Nissl staining. Results of the peptide immunoreactive structures in the rat dorsal column nuclei are in general good agreement with findings in previous studies (Hoeflinger et al., 1993). However, at caudalmost levels of the complex, in the territory of the cuneate fascicle and dorsal to the caudal pole of the cuneate nucleus, a small column of gray matter area can be identified that contains a dense plexus of varicose labelled nerve fibres. The observed discrete region has never distinctly described beforehand. Both its position and aspect at neuropeptide-immunoreactivity resemble those of the Locus K we detected in the human dorsal column nuclei, allowing the possibility that it represents the its homologous nucleus in the rat brain. Work funded by Fondazione Banco di Sardegna.

Glutamate microinjection into the hypothalamic paraventricular nucleus attenuates ulcerative colitis in rats

To investigate the effects of glutamate microinjection into hypothalamic paraventricular nucleus (PVN) on ulcerative colitis (UC) in rats and to explore the relevant mechanisms. 2,4,6-Trinitrobenzenesulfonic acid (100 mg/kg in 50% ethanol) was instilled into the colon of adult male SD rats to induce UC. A colonic damage score (CDS) was used to indicate the severity of the colonic mucosal damage. The pathological changes in the colonic mucosa were evaluated using immunohistochemistry, Western blotting, biochemical analyses or ELISA. Ten minutes before UC induction, drugs were microinjected into the relevant nuclei in rat brain to produce chemical stimulation or chemical lesion. Microinjection of glutamate (3, 6 and 12 μg) into the PVN dose-dependently decreased the CDS in UC rats. This protective effect was eliminated after kainic acid (0.3 μg) was microinjected into PVN or into the nucleus tractus solitarius (NTS) that caused chemical lesion of these nuclei. This protective effect was also prevented when the AVP-V1 receptor antagonist DPVDAV (200 ng) was microinjected into the NTS. The discharge frequency of the vagus was markedly decreased following microinjection of glutamate into the PVN. Microinjection of glutamate into the PVN in UC rats significantly increased the cell proliferation and anti-oxidant levels, and decreased the apoptosis and Bax and caspase 3 expression levels and reduced the pro-inflammatory factors in the colonic mucosa. The activation of hypothalamic PVN exerts protective effects against UC, which is mediated by the NTS and vagus. The effects may be achieved via anti-oxidative, anti-apoptotic, and anti-inflammatory factors.

Adeno-associated Virus-mediated, Mifepristone-regulated Transgene Expression in the Brain

Gene therapy, in its current configuration, is irreversible and does not allow control over transgene expression in case of side effects. Only few regulated vector systems are available, and none of these has reached clinical applicability yet. The mifepristone (Mfp)-regulated Gene Switch (GS) system is characterized by promising features such as being composed of mainly human components and an approved small-molecule drug as an inducer. However, it has not yet been evaluated in adeno-associated virus (AAV) vectors, neither has it been tested for applicability in viral vectors in the central nervous system (CNS). Here, we demonstrate that the GS system can be used successfully in AAV vectors in the brain, and that short-term induced glial cell line-derived neurotrophic factor (GDNF) expression prevented neurodegeneration in a rodent model of Parkinson's disease (PD). We also demonstrate repeated responsiveness to the inducer Mfp and absence of immunological tissue reactions in the rat brain. Human equivalent dosages of Mfp used in this study were lower than those used safely for treatment of psychiatric threats, indicating that the inducer could be safely applied in patients. Our results suggest that the GS system in AAV vectors is well suited for further development towards clinical applicability.

MicroRNA expression profiling of hypothalamic arcuate and paraventricular nuclei from single rats using Illumina sequencing technology

MicroRNAs (miRNAs) finely tune messenger RNA (mRNA) expression. As the brain is a highly heterogeneous tissue, physiologically relevant miRNA expression profiling greatly benefits from sampling brain regions or nuclei. MiRNA expression profiling from individual samples is also important for investigating potential differences between animals according to their physiological and pathophysiological status. We have punched the arcuate (ARC) and paraventricular (PVN) nuclei from the hypothalamus of seven male Wistar rats and used them to establish a novel method for the characterization of the miRNA expression profile of individual rat brain nuclei. The identity of the ARC and PVN samples was checked for proopiomelanocortin and arginine vasopressin mRNA expression, respectively. Individual cDNA libraries were constructed from purified RNAs between 16 and 26 bases, using barcoded adapters. Libraries were multiplexed and sequenced using Illumina technology to a read depth >105. The ARC and PVN profiles displayed similar expression from a set of more than 210 miRNA genes. Expression was high or moderate for about twenty miRNAs that may be used to define a common ARC/PVN prototype profile of male Wistar rats. These miRNAs included seven of the eight genes of the let-7 family, the two miR-7 genes, miR-9 gene and 5′ copy of the three miR-30 loci. Our method shows that the ARC and PVN from a single rat are accessible for miRNA digital characterization. This method will allow miRNA transcriptome characterization for any rat brain substructure or nuclei that can be microdissected.