Shi Z, Cassaglia PA, Pelletier NE, Brooks VL.
PMID: PMID: 30628058 | DOI: DOI:10.1113/JP277517
KEY POINTS: ICV insulin increased SNA and baroreflex control of SNA and HR dramatically more in obese male rats; in obese females, the responses were abolished. In obese males, the enhanced LSNA responses were associated with reduced tonic inhibition of LSNA by NPY in the PVN. Yet, PVN NPY injection decreased LSNA similarly in OP/OR/CON rats. Collectively, these results suggest that NPY inputs were decreased. In obese females, NPY inhibition in the PVN was maintained. Moreover, NPY neurons in the ArcN became resistant to the inhibitory effects of insulin. A HFD did not alter arcuate NPY neuronal InsR expression in males or females. Obesity-induced "selective sensitization" of the brain to the sympathoexcitatory effects of insulin and leptin may contribute to elevated basal SNA, and therefore hypertension development, in males with obesity. These data may explain in part why obesity increases SNA less in women compared to men. ABSTRACT: Obesity increases sympathetic nerve activity (SNA) in men, but not women; however, the mechanisms are unknown. We tested if intracerebroventricular insulin infusion increases SNA more in obese male than female rats and if sex differences are mediated by changes in tonic inhibition of SNA by Neuropeptide Y (NPY) in the paraventricular nucleus (PVN). When consuming a high fat diet, obesity prone (OP) rats accrued excess fat, whereas obesity resistant (OR) rats maintained adiposity as in rats eating a control (CON) diet. Insulin increased lumbar SNA (LSNA) similarly in CON/OR males and females under urethane-anesthesia. The LSNA response was magnified in OP males, but abolished in OP females. In males, blockade of PVN NPY Y1 receptors with BIBO3304 increased LSNA in CON/OR rats, but not OP rats. Yet, PVN nanoinjections of NPY decreased LSNA similarly between groups. Thus, tonic PVN NPY inhibition of LSNA may be lost in obese males, due to a decrease in NPY inputs. In contrast, in females, PVN BIBO3304 increased LSNA similarly in OP, OR and CON rats. After insulin, PVN BIBO3304 failed to increase LSNA in CON/OR females, but increased LSNA in OP females, suggesting that with obesity NPY neurons become resistant to the inhibitory effects of insulin. These sex differences were not associated with changes in arcuate NPY neuronal insulin receptor expression. Collectively, these data reveal a marked sex difference in the impact of obesity on insulin's sympathoexcitatory actions and implicate sexually dimorphic changes in NPY inhibition of SNA in the PVN as one mechanism.
Shiers, S;Elahi, H;Hennen, S;Price, T;
| DOI: 10.1016/j.ynpai.2021.100081
The anterior cingulate cortex (ACC) is a critical region of the brain for the emotional and affective components of pain in rodents and humans. Hyperactivity in this region has been observed in neuropathic pain states in both patients and animal models and ablation of this region from cingulotomy, or inhibition with genetics or pharmacology can diminish pain and anxiety. Two adenylyl cyclases (AC), AC1 and AC8 play an important role in regulating nociception and anxiety-like behaviors through an action in the ACC, as genetic and pharmacological targeting of these enzymes reduces mechanical hypersensitivity and anxiety-like behavior, respectively. However, the distribution of these ACs in the ACC has not been studied in the context of neuropathic pain. To address this gap in knowledge, we conducted RNAscope in situ hybridization to assess AC1 and AC8 mRNA distribution in mice with spared nerve injury (SNI). Given the key role of AC1 in nociception in neuropathic, inflammatory and visceral pain animal models, we hypothesized that AC1 would be upregulated in the ACC of mice following nerve injury. This hypothesis was also founded on data showing increased AC1 expression in the ACC of mice with zymosan-induced visceral inflammation. We found that AC1 and AC8 are widely expressed in many regions of the mouse brain including the hippocampus, ACC, medial prefrontal cortex and midbrain regions, but AC1 is more highly expressed. Contrary to our hypothesis, SNI causes an increase in AC8 mRNA expression in NMDAR-2B (Nr2b) positive neurons in the contralateral ACC but does not affect AC1 mRNA expression. Our findings show that changes in Adcy1 mRNA expression in the ACC are insufficient to explain the important role of this AC in mechanical hypersensitivity in mice following nerve injury and suggest a potential unappreciated role of AC8 in regulation of ACC synaptic changes after nerve injury.
Schneider JS.
PMID: 29902255 | DOI: 10.1371/journal.pone.0199189
Reduced expression of GM1 and other major brain gangliosides GD1a, GD1b and GT1b have been reported in Parkinson's disease (PD) brain. Mechanisms underlying these changes are unclear but may be due to a deficit in the ganglioside biosynthetic process. The present study examined the extent to which deficits in gene expression of key biosynthetic enzymes involved in synthesis of GM1 and GD1b (B3galt4) and GD1a and GT1b (St3gal2) exist in neuromelanin-containing neurons in the PD substantia nigra (SN). In situ hybridization histochemistry was used to examine gene expression of B3GALT4 and ST3GAL2 in neuromelanin-containing neurons in the SN in 8 normal controls (61-92 yrs.) and 7 PD subjects (77-95 yrs). There was a significant decrease in both B3GALT4 and ST3GAL2 gene expression in residual neuromelanin-containing cells in the SN of PD patients compared to age-matched neurologically normal controls. These changes appeared to be cell-type specific as abundant B3GALT4 and ST3GAL2 gene expression was observed in non-neuromelanin containing neurons located outside of the SN in the PD brain. These data show that residual neuromelanin-containing neurons in the PD SN have decreased expression of the ganglioside biosynthetic genes B3GALT4 and ST3GAL2, consistent with previous reports of decreased levels of gangliosides GM1, GD1a, GD1b and GT1b in the PD SN. These changes may increase the vulnerability of these neurons to degeneration in response to a variety of potential stressors.
Zhang N, Zhang HY, Bi SA, Moran TH and Bi S
PMID: 30902570 | DOI: 10.1016/j.neulet.2019.03.030
Thyrotropin-releasing hormone (TRH) plays an important role in the regulation of energy balance. While the regulation of TRH in the paraventricular nucleus (PVN) in response to changes of energy balance has been well studied, how TRH is regulated in the dorsomedial hypothalamus (DMH) in maintaining energy homeostasis remains unclear. Here, we assessed the effects of food restriction and exercise on hypothalamic Trh expression using Otsuka Long-Evens Tokushima Fatty (OLETF) rats. Sedentary ad lib fed OLETF rats (OLETF-SED) became hyperphagic and obese. These alterations were prevented in OLETF rats with running wheel access (OLETF-RW) or food restriction in which their food was pair-fed (OLETF-PF) to the intake of lean control rats (LETO-SED). Evaluation of hypothalamic gene expression revealed that Trh mRNA expression was increased in the PVN of OLETF-SED rats and normalized in OLETF-RW and OLETF-PF rats compared to LETO-SED rats. In contrast, the expression of Trh in the DMH was decreased in OLETF-SED rats relative to LETO-SED rats. This alteration was reversed in OLETF-RW rats as seen in LETO-SED rats, but food restriction resulted in a significant increase in DMH Trh expression in OLETF-PF rats compared to LETO-SED rats. Strikingly, while Trh mRNA expression was decreased in the PVN of intact rats in response to acute food deprivation, food deprivation resulted in increased expression of Trh in the DMH. Together, these results demonstrate the differential regulation of Trh expression in the PVN and DMH in OLETF rats and suggest that DMH TRH also contributes to hypothalamic regulation of energy balance.
Herath, M;Cho, E;Marklund, U;Franks, A;Bornstein, J;Hill-Yardin, E;
| DOI: 10.3390/biom13071063
Mutations in the Neuroligin-3 (Nlgn3) gene are implicated in autism spectrum disorder (ASD) and gastrointestinal (GI) dysfunction, but cellular Nlgn3 expression in the enteric nervous system remains to be characterised. We combined RNAScope in situ hybridization and immunofluorescence to measure Nlgn3 mRNA expression in cholinergic and VIP-expressing submucosal neurons, nitrergic and calretinin-containing myenteric neurons and glial cells in both WT and Nlgn3R451C mutant mice. We measured Nlgn3 mRNA neuronal and glial expression via quantitative three-dimensional image analysis. To validate dual RNAScope/immunofluorescence data, we interrogated available single-cell RNA sequencing (scRNASeq) data to assess for Nlgn3, Nlgn1, Nlgn2 and their binding partners, Nrxn1-3, MGDA1 and MGDA2, in enteric neural subsets. Most submucosal and myenteric neurons expressed Nlgn3 mRNA. In contrast to other Nlgns and binding partners, Nlgn3 was strongly expressed in enteric glia, suggesting a role for neuroligin-3 in mediating enteric neuron-glia interactions. The autism-associated R451C mutation reduces Nlgn3 mRNA expression in cholinergic but not in VIPergic submucosal neurons. In the myenteric plexus, Nlgn3 mRNA levels are reduced in calretinin, nNOS-labelled neurons and S100 β -labelled glia. We provide a comprehensive cellular profile for neuroligin-3 expression in ileal neuronal subpopulations of mice expressing the R451C autism-associated mutation in Nlgn3, which may contribute to the understanding of the pathophysiology of GI dysfunction in ASD.
Van Gulick ER, Marquis TJ, Sower SA.
PMID: 28782533 | DOI: 10.1016/j.ygcen.2017.08.004
RNA expression of lamprey gonadotropin-releasing hormone (lGnRH)-I, -II, and -III was demonstrated in the brains of larval, parasitic phase and adult sea lampreys, Petromyzon marinus, using a highly sensitive triple-label in situ hybridization technique. In female larval lampreys, lGnRH-I and-II were co-expressed in the same neurons throughout the olfactory bulbs, preoptic area (POA), and rhombencephalon (hindbrain); lGnRH-I, -II and -III were triple co-expressed in the hypothalamus and in the paranuclear region of neuronal somas in the rhombencephalon. In female parasitic phase lampreys, lGnRH-I and -II were co-expressed in the POA, thalamus, and preoptico-neurohypophyseal tract (PNT); lGnRH-III was minimally triple co-expressed with lGnRH-I and -II in the hypothalamus. In adult female lampreys, lGnRH-I and -III were co-expressed in the hypothalamus; lGnRH-I was also expressed in the neurohypophysis (NH). In adult male lampreys, lGnRH-I and-III were co-expressed in the primordial hippocampus, POA, thalamus, hypothalamus, NH, and PNT; lGnRH-I was also expressed in the epithalamus. In summary, we provide the first study using in situ hybridization of all three lGnRHs (lGnRH-I, -II, and -III) at three major life stages (larval, parasitic, and adult) of lampreys, which strongly supports previous immunohistological studies and suggests that lGnRH-I and -II are the predominant lGnRHs in larval and parasitic phase lampreys, and that lGnRH-I and -III are the predominant lGnRHs in adult female and male lampreys. Therefore, our results show that lGnRH-I, -II, and -III have different localization and co-expression in the development and sexual maturation of lampreys, which may suggest unique physiological roles at each life stage and sex in the developing and mature lamprey brain.
Ding, CY;Ding, YT;Ji, H;Wang, YY;Zhang, X;Yin, DM;
PMID: 37147705 | DOI: 10.1186/s13578-023-01032-4
Where the gene is expressed determines the function of the gene. Neuregulin 1 (Nrg1) encodes a tropic factor and is genetically linked with several neuropsychiatry diseases such as schizophrenia, bipolar disorder and depression. Nrg1 has broad functions ranging from regulating neurodevelopment to neurotransmission in the nervous system. However, the expression pattern of Nrg1 at the cellular and circuit levels in rodent brain is not full addressed.Here we used CRISPR/Cas9 techniques to generate a knockin mouse line (Nrg1Cre/+) that expresses a P2A-Cre cassette right before the stop codon of Nrg1 gene. Since Cre recombinase and Nrg1 are expressed in the same types of cells in Nrg1Cre/+ mice, the Nrg1 expression pattern can be revealed through the Cre-reporting mice or adeno-associated virus (AAV) that express fluorescent proteins in a Cre-dependent way. Using unbiased stereology and fluorescence imaging, the cellular expression pattern of Nrg1 and axon projections of Nrg1-positive neurons were investigated.In the olfactory bulb (OB), Nrg1 is expressed in GABAergic interneurons including periglomerular (PG) and granule cells. In the cerebral cortex, Nrg1 is mainly expressed in the pyramidal neurons of superficial layers that mediate intercortical communications. In the striatum, Nrg1 is highly expressed in the Drd1-positive medium spiny neurons (MSNs) in the shell of nucleus accumbens (NAc) that project to substantia nigra pars reticulata (SNr). In the hippocampus, Nrg1 is mainly expressed in granule neurons in the dentate gyrus and pyramidal neurons in the subiculum. The Nrg1-expressing neurons in the subiculum project to retrosplenial granular cortex (RSG) and mammillary nucleus (MM). Nrg1 is highly expressed in the median eminence (ME) of hypothalamus and Purkinje cells in the cerebellum.Nrg1 is broadly expressed in mouse brain, mainly in neurons, but has unique expression patterns in different brain regions.
Journal of Neuroendocrinology
Prengel, T;Brunne, B;Habiballa, M;Rune, G;
| DOI: 10.1111/jne.13276
Microglia have been shown to sculpt postnatal circuitry from birth, up to adulthood due to their role in both synapse formation and synaptic pruning, the elimination of weak, redundant synapses. Microglia are differentiated in a sex-dependent manner. In this study, we tested whether sexual differentiation of microglia results in sex-dependent postnatal reorganization of CA1 synaptic connectivity in the hippocampus. The stereological counting of synapses in mice in the electron microscope showed a continuous rise in synapse density until the fourth week, followed by a plateau phase and loss of synapses from the eighth week onwards, with no difference between sexes. This course of alteration in synapse numbers did not differ between sexes. But selectively, on postnatal day (P) 14 the density of synapses was significantly higher in the female than in the male hippocampus. Higher synapse density in females was paralleled by higher activity of microglia, as indicated by morphological changes, CD68 expression, and proximity of microglia to synaptic sites. In Thy1-GFP mice, consistent with increased synapse numbers, bouton density was also clearly increased in females at P14. At this time point, CD47 expression, the “don't eat me” signal of neurons, was similar in males and females. The decrease in bouton density thereafter in conjuction with increased synapse numbers argues for a role of microglia in the formation of multispine boutons (MSB). Our data in females at P14 support the regulatory role of microglia in synapse density. Sexual differentiation of microglia, however, does not substantially affect long-term synaptic reorganization in the hippocampus.
Wang, G;Woods, C;Johnson, M;Milner, T;Glass, M;
| DOI: 10.1016/j.neuroscience.2021.12.041
The hypothalamic paraventricular nucleus (PVN) plays a key role in hypertension, however the signaling pathways that contribute to the adaptability of the PVN during hypertension are uncertain. We present evidence that signaling at the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) GluA1 receptor subunit contributes to increased blood pressure in a model of neurogenic hypertension induced by 14-day slow-pressor angiotensin II (AngII) infusion in male mice. It was found that AngII hypertension was associated with an increase in plasma membrane affiliation of GluA1, but decreased GluA2, in dendritic profiles of PVN neurons. The increased plasma membrane GluA1 was paralleled by the results of whole-cell current clamping experiments, which showed heightened AMPA currents in PVN neurons from AngII-infused male mice. The inhibition of heightened AMPA currents was blocked by 1-Naphthyl acetyl spermine trihydrochloride, pointing to the involvement of GluA2-lacking GluA1 receptors in the elevated AMPA signaling in PVN neurons. A further functional role for GluA1 signaling in the PVN was demonstrated by the attenuated hypertensive response following silencing of GluA1 in the PVN of AngII-infused mice. In female mice, AngII-infusion did not impact blood pressure. In addition, AngII was not associated with alterations in transcription or plasma membrane localization of GluA1 in females. Posttranslational modifications that increase the plasma membrane localization of AMPA GluA1 and heighten the rapid signaling actions of glutamate in PVN neurons may serve as a molecular substrate underlying sex differences in hypertension.
Buhidma, Y;Hobbs, C;Malcangio, M;Duty, S;
PMID: 37100804 | DOI: 10.1038/s41531-023-00510-3
Pain is a key non-motor feature of Parkinson's disease (PD) that significantly impacts on life quality. The mechanisms underlying chronic pain in PD are poorly understood, hence the lack of effective treatments. Using the 6-hydroxydopamine (6-OHDA) lesioned rat model of PD, we identified reductions in dopaminergic neurons in the periaqueductal grey (PAG) and Met-enkephalin in the dorsal horn of the spinal cord that were validated in human PD tissue samples. Pharmacological activation of D1-like receptors in the PAG, identified as the DRD5+ phenotype located on glutamatergic neurons, alleviated the mechanical hypersensitivity seen in the Parkinsonian model. Downstream activity in serotonergic neurons in the Raphé magnus (RMg) was also reduced in 6-OHDA lesioned rats, as detected by diminished c-FOS positivity. Furthermore, we identified increased pre-aggregate α-synuclein, coupled with elevated activated microglia in the dorsal horn of the spinal cord in those people that experienced PD-related pain in life. Our findings have outlined pathological pathways involved in the manifestation of pain in PD that may present targets for improved analgesia in people with PD.
Philosophical transactions of the Royal Society of London. Series B, Biological sciences
Frehner, SS;Dooley, KT;Palumbo, MC;Smith, AL;Goodman, MM;Bales, KL;Freeman, SM;
PMID: 35858098 | DOI: 10.1098/rstb.2021.0118
Oxytocin is an endogenous neuropeptide hormone that influences social behaviour and bonding in mammals. Variations in oxytocin receptor (OXTR) expression may play a role in the social deficits seen in autism spectrum disorder. Previous studies from our laboratory found a dense population of OXTR in the human substantia nigra (SN), a basal ganglia structure in the midbrain that is important in both movement and reward pathways. Here, we explore whether differences in OXTR can be identified in the dopaminergic SN pars compacta of individuals with autism. Postmortem human brain tissue specimens were processed for OXTR autoradiography from four groups: males with autism, females with autism, typically developing (TD) males and TD females. We found that females with autism had significantly lower levels of OXTR than the other groups. To examine potential gene expression differences, we performed in situ hybridization in adjacent slides to visualize and quantify OXTR mRNA as well as mRNA for tyrosine hydroxylase. We found no differences in mRNA levels for either gene across the four groups. These results suggest that a dysregulation in local OXTR protein translation or increased OXTR internalization/recycling may contribute to the differences in social symptoms seen in females with autism. This article is part of the theme issue 'Interplays between oxytocin and other neuromodulators in shaping complex social behaviours'.
Garrido-Gil, P;Pedrosa, MA;Garcia-Garrote, M;Pequeño-Valtierra, A;Rodríguez-Castro, J;García-Souto, D;Rodríguez-Pérez, AI;Labandeira-Garcia, JL;
PMID: 35943203 | DOI: 10.1002/glia.24255
There are sex differences in microglia, which can maintain sex-related gene expression and functional differences in the absence of circulating sex steroids. The angiotensin type 2 (AT2) receptors mediate anti-inflammatory actions in different tissues, including brain. In mice, we performed RT-PCR analysis of microglia isolated from adult brains and RNA scope in situ hybridization from males, females, ovariectomized females, orchiectomized males and brain masculinized females. We also compared wild type and AT2 knockout mice. The expression of AT2 receptors in microglial cells showed sex differences with much higher AT2 mRNA expression in females than in males, and this was not dependent on circulating gonadal hormones, as observed using ovariectomized females, brain masculinized females and orchiectomized males. These results suggest genomic reasons, possibly related to sex chromosome complement, for sex differences in AT2 expression in microglia, as the AT2 receptor gene is located in the X chromosome. Furthermore, sex differences in expression of AT2 receptors were associated to sex differences in microglial expression of key anti-inflammatory cytokines such as interleukin-10 and pro-inflammatory cytokines such as interleukin-1β and interleukin-6. In conclusion, sex differences in microglial AT2 receptor expression appear as a major factor contributing to sex differences in the neuroinflammatory responses beyond the effects of circulating steroids.
