Corrigendum: Glutamatergic and GABAergic neurons in the preoptic area of the hypothalamus play key roles in menopausal hot flashes.

[This corrects the article DOI: 10.3389/fnagi.2022.993955.].

Mapping the 5-HTergic neural pathways in perimenopausal mice and elucidating the role of oestrogen receptors in 5-HT neurotransmission.

Perimenopausal syndrome (PMS) encompasses neuropsychiatric symptoms, such as hot flashes and depression, which are associated with alterations in the 5-HTergic neural pathway in the brain. However, the specific changes and mechanisms underlying these alterations remain unclear. In this study, ovariectomized mice were used to successfully establish a perimenopause model, and the changes in the expression of 5-HT and its receptors (5-HT1AR and 5-HT2AR) across 72 brain regions in these ovariectomized mice were assessed by immunohistochemistry. Although both 5-HT and 5-HT1AR were widely expressed throughout the brain, only a limited number of regions expressed 5-HT2AR. Notably, decreased expression of 5-HT was observed across almost all brain regions in the ovariectomy (OVX) group compared with the Sham group. Altered expression of both receptors was found within areas related to hot flashes (the preoptic area) or mood disorders (the amygdala). Additionally, reduced oestrogen receptor (ER)α/ß expression was detected in cells in the raphe nucleus (RN), an area known to regulate body temperature. Results showed that ERα/ß positively regulate the transcriptional activity of the enzymes TPH2/MAOA, which are involved in serotonin metabolism during perimenopause. This study revealed the changes in 5-HT neuropathways (5-HT, 5-HT1AR and 5-HT2AR) in perimenopausal mice, mainly in brain regions related to regulation of the body temperature, mood, sleep and memory. This study clarified that the expression of oestrogen receptor decreased in perimenopause, which regulated the transcription levels of TPH2 and MAOA, and ultimately led to the reduction of 5-HT content, providing a new target for clinical diagnosis and treatment of perimenopausal diseases.

Glutamatergic and GABAergic neurons in the preoptic area of the hypothalamus play key roles in menopausal hot flashes.

During menopause, when estrogen levels are low, abnormalities in the hypothalamic preoptic area (POA) of the thermoregulatory center can cause hot flashes. However, the involved neural population has not been identified. Proteomics showed that under low estrogen, differentially expressed proteins in the hypothalamus were associated with glutamatergic and GABAergic synapses. RNAscope, Western blotting and qRT-PCR indicated that the number of glutamatergic neurons in the POA was decreased, while the number of GABAergic neurons was increased. Chemogenetics showed that the rat body temperature decreased slowly after glutamatergic neurons were activated and increased quickly after glutamatergic neurons were inhibited, while it increased quickly after GABAergic neurons were activated and decreased slowly after GABAergic neurons were inhibited. RNAscope, immunofluorescence, Western blotting and qRT-PCR further showed that glutamate decarboxylase (GAD) 1 expression in the POA was increased, while GAD2 expression in the POA was decreased; that thermosensitive transient receptor potential protein (ThermoTRP) M (TRPM) 2 expression in glutamatergic neurons was decreased, while TRPM8 expression in GABAergic neurons was increased; and that estrogen receptor (ER) α and ß expression in the POA was decreased, and ERα and ERß expressed in both glutamatergic and GABAergic neurons. Estrogen therapy corrected these abnormalities. In addition, CUT&Tag and Western blot after injection of agonists and inhibitors of ERs showed that ERα and ERß were both transcription factors in glutamatergic and GABAergic synapses. Mechanistically, during menopause, estrogen may regulate the transcription and expression of GADs and ThermoTRPs through ERs, impacting the number and function of glutamatergic and GABAergic neurons, resulting in unbalanced heat dissipation and production in the POA and ultimately triggering hot flashes.

Morphological properties of medial amygdala-projecting retinal ganglion cells in the Mongolian gerbil.

The amygdala is a limbic structure that is involved in many brain functions, including emotion, learning and memory. It has been reported that melanopsin-expressing retinal ganglion cells (ipRGCs) innervate the medial amygdala (MeA). However, whether conventional RGCs (cRGCs) project to the MeA remains unknown. The goal of this study was to determine if cRGCs project to the MeA and to determine the morphological properties of MeA-projecting RGCs (MeA-RGCs). Retrogradely labeled RGCs in whole-mount retinas were intracellularly injected to reveal their dendritic morphologies. Immunohistochemical staining was performed to selectively label ipRGCs (MeA-ipRGCs) and cRGCs (MeA-cRGCs). The results showed that 95.7% of the retrogradely labeled cells were cRGCs and that the rest were ipRGCs. Specifically, MeA-cRGCs consist of two morphological types. The majority of them exhibit small but dense dendritic fields and diffuse ramification patterns as previously reported in RGB2 (95%), while the rest exhibit small but sparse dendritic branching patterns resembling those of RGB3 cells (5%). MeA-ipRGCs consist of M1 and M2 subtypes. The MeA-RGCs showed an even retinal distribution patterns. The soma and dendritic field sizes of the MeA-RGCs did not vary with eccentricity. In conclusion, the present results suggest that MeA-RGCs are structurally heterogeneous. These direct RGCs that input to the MeA could be important for regulating amygdala functions.

Orexin gene transfer into the amygdala suppresses both spontaneous and emotion-induced cataplexy in orexin-knockout mice.

Narcolepsy is a chronic sleep disorder linked to the loss of orexin-producing neurons in the hypothalamus. Cataplexy, a sudden loss of muscle tone during waking, is an important distinguishing symptom of narcolepsy and it is often triggered by strong emotions. The neural circuit underlying cataplexy attacks is not known, but is likely to involve the amygdala, a region implicated in regulating emotions. In mice models of narcolepsy, transfer of the orexin gene into surrogate neurons has been successful in ameliorating narcoleptic symptoms. However, it is not known whether this method also blocks cataplexy triggered by strong emotions. To examine this possibility, the gene encoding mouse prepro-orexin was transferred into amygdala neurons of orexin-knockout (KO) mice (rAAV-orexin; n = 8). Orexin-KO mice that did not receive gene transfer (no-rAAV; n = 7) or received only the reporter gene (rAAV-GFP; n = 7) served as controls. Three weeks later, the animal's sleep and behaviour were recorded at night (no-odour control night), followed by another recording at night in the presence of predator odour (odour night). Orexin-KO mice given the orexin gene transfer into surrogate amygdala neurons had significantly less spontaneous bouts of cataplexy, and predator odour did not induce cataplexy compared with control mice. Moreover, the mice with orexin gene transfer were awake more during the odour night. These results demonstrate that orexin gene transfer into amygdala neurons can suppress both spontaneous and emotion-induced cataplexy attacks in narcoleptic mice. It suggests that manipulating amygdala pathways is a potential strategy for treating cataplexy in narcolepsy.

Post-hypoxic and ischemic neuroprotection of BMP-7 in the cerebral cortex and caudate-putamen tissue of rat.

Previous reports have indicated that exogenous bone morphogenetic protein-7 (BMP-7) has a neuroprotective effect after cerebral ischemia injury and promotes motor function recovery, but the appropriate BMP-7 concentration and time course are unclear. Here, we assessed endogenous BMP-7 expression in hypoxia and ischemia-damaged brain tissues and investigated the effects of different BMP-7 concentrations in pre- and post-hypoxic primary rat neurons. The results showed that BMP-7 expression was significantly higher in the ischemic hemisphere. The expressions of BMP-7 and caspase-3 were localized in the cytoplasm of the primary cerebral cortical and caudate-putamen neurons 24h after hypoxia/reoxygenation. After BMP-7 treatment, the number of caspase-3 positive neurons began to decrease with increasing BMP-7 concentrations up to 80ng/ml, but not beyond. Although the numbers of caspase-3-positive neurons between pre- and post-hypoxia/reoxygenation were not significantly different, more dendrites were observed in the groups treated prior to hypoxia/reoxygenation. These results suggest that increased BMP-7 expression can be induced in the cerebral cortex and caudate-putamen both in vivo and in vitro in hypoxic-ischemic states. The neuroprotective mechanism of BMP-7 may include apoptosis suppression, and its effect was enhanced from 40 to 80ng/ml. Pre-hypoxic BMP-7 treatment may be useful to stimulate dendrite sprouting in non-injured neurons.

Direct retino-raphe projection alters serotonergic tone and affective behavior.

Light is a powerful modulator of higher-order cognitive processes such as mood but it remains unclear which neural circuits mediate the impact of light on affective behavior. We found that light deprivation produces a depressive-like behavioral state that is reversed by activation of direct retinal signals to the serotonergic dorsal raphe nucleus (DRN) in a manner equivalent to treatment with the selective serotonin reuptake inhibitor fluoxetine. Surprisingly, the DRN-projecting retinal ganglion cells (RGCs) are indistinguishable from the classic alpha/Y-like RGC type that contributes to image-forming visual pathways. Silencing RGC firing or specific immunotoxin ablation of DRN-projecting RGCs increased depressive-like behavior and reduced serotonin levels in the DRN. Serotonin has a key role in the pathophysiology of depression, and these results demonstrate that retino-raphe signals modulate DRN serotonergic tone and affective behavior.

Y-like retinal ganglion cells innervate the dorsal raphe nucleus in the Mongolian gerbil (Meriones unguiculatus).

BACKGROUND: The dorsal raphe nucleus (DRN) of the mesencephalon is a complex multi-functional and multi-transmitter nucleus involved in a wide range of behavioral and physiological processes. The DRN receives a direct input from the retina. However little is known regarding the type of retinal ganglion cell (RGC) that innervates the DRN. We examined morphological characteristics and physiological properties of these DRN projecting ganglion cells. METHODOLOGY/PRINCIPAL FINDINGS: The Mongolian gerbils are highly visual rodents with a diurnal/crepuscular activity rhythm. It has been widely used as experimental animals of various studies including seasonal affective disorders and depression. Young adult gerbils were used in the present study. DRN-projecting RGCs were identified following retrograde tracer injection into the DRN, characterized physiologically by extracellular recording and morphologically after intracellular filling. The result shows that DRN-projecting RGCs exhibit morphological characteristics typical of alpha RGCs and physiological response properties of Y-cells. Melanopsin was not detected in these RGCs and they show no evidence of intrinsic photosensitivity. CONCLUSIONS/SIGNIFICANCE: These findings suggest that RGCs with alpha-like morphology and Y-like physiology appear to perform a non-imaging forming function and thus may participate in the modulation of DRN activity which includes regulation of sleep and mood.

Pifithrin-alpha reduces cerebral vasospasm by attenuating apoptosis of endothelial cells in a subarachnoid haemorrhage model of rat.

BACKGROUND: The mechanism of cerebral vasospasm following subarachnoid haemorrhage (SAH) is not understood. Here, we hypothesized that apoptosis of endothelial cells induced by p53 and its target gene em dash p53 upregulated modulator of apoptosis (PUMA) played an important role in development of cerebral vasospasm. We also observed the effects of a p53 inhibitor, pifithrin-alpha (PFT-alpha), on reducing the expression of p53 and PUMA, consequently decreasing the apoptosis of endothelial cells and alleviating cerebral vasospasm. METHODS: Male Sprague-Dawley rats weighing 300-350 g were randomly divided into five groups: a control group (sham surgery), a SAH group, a SAH+dimethyl sulfoxide (DMSO) group, a SAH + PFT-alpha (0.2 mg/kg) group and a SAH + PFT-alpha (2.0 mg/kg) group. PFT-alpha was injected intraperitoneally immediately after SAH. Rats were sacrificed 24 hours after SAH. Western blot and immunohistochemical staining were used to detect the levels of p53, PUMA and caspase-3 protein. In addition, mortality and neurological scores were assessed for each group. Statistical significance was assured by analysis of variance performed in one way ANOVA followed by the Tukey test. The neurological and mortality scores were analyzed by Dunn's method and Fisher exact test, respectively. RESULTS: After SAH, Western blot and immunohistochemical staining showed the levels of p53, PUMA and caspase-3 in the endothelial cells and the numbers of TdT mediated dUTP nick end labelling (TUNEL) positive endothelial cells were all significantly increased in the basilar arteries (P<0.05), but significantly reduced by PFT-alpha (P<0.05). These changes were accompanied by increasing diameters and declining wall thickness of basilar arteries (P<0.05), as well as reduced mortality and neurological deficits of the rats (P<0.05). CONCLUSIONS: PFT-alpha could protect cerebral vessels from development of vasospasm and improve neurological outcome as well as reduce the mortality via suppressing apoptosis induced by p53 in the endothelial cells of cerebral vessels.

Multiple effects of 2ME2 and D609 on the cortical expression of HIF-1alpha and apoptotic genes in a middle cerebral artery occlusion-induced focal ischemia rat model.

Despite 2-methoxyestradiol (2ME2) and tricyclodecan-9-yl-xanthogenate (D609) having multiple effects on cancer cells, mechanistically, both of them down-regulate hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF). We hypothesize HIF-1alpha plays an essential role in cerebral ischemia as a pro-apoptosis regulator; 2ME2 and D609 decrease the levels of HIF-1alpha and VEGF, that might contribute to protecting brain from ischemia injury. A total of 102 male Sprague-Dawley rats were split into five groups: sham, middle cerebral artery occlusion (MCAO), MCAO + dimethyl sulfoxide, MCAO + 2ME2, and MCAO + D609. 2ME2 and D609 were injected intraperitoneally 1 h after reperfusion. Rats were killed at 24 h and 7 days. At 24 h, 2ME2 and D609 reduce the levels of HIF-1alpha and VEGF (enzyme-linked immunosorbent assay), depress the expression of HIF-1alpha, VEGF, BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) and cleaved caspase 3 (western blot and immunohistochemistry) in the brain infarct area. Double fluorescence labeling shows HIF-1alpha positive immunoreactive materials are co-localized with BNIP3 and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling inside the nuclei of neurons. At 7 days, 2ME2 and D609 reduce the infarct volume (2,3,7-triphenyltetrazolium chloride) and blood-brain barrier extravasation, decrease the mortality and improve the neurological deficits. In conclusion, 2ME2 and D609 are powerful agents to protect brain from cerebral ischemic injury by inhibiting HIF-1alpha expression, attenuating the superfluous expression of VEGF to avoid blood-brain barrier disruption and suppressing neuronal apoptosis via BNIP3 pathway.