Presynaptic inputs to vasopressin neurons in the hypothalamic supraoptic nucleus and paraventricular nucleus in mice.

Arginine vasopressin (AVP) neurons in the hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN) are involved in important physiological behaviors, such as controling osmotic stability and thermoregulation. However, the presynaptic input patterns governing AVP neurons have remained poorly understood due to their heterogeneity, as well as intermingling of AVP neurons with other neurons both in the SON and PVN. In the present study, we employed a retrograde modified rabies-virus system to reveal the brain areas that provide specific inputs to AVP neurons in the SON and PVN. We found that AVP neurons of the SON and PVN received similar input patterns from multiple areas of the brain, particularly massive afferent inputs from the diencephalon and other brain regions of the limbic system; however, PVNAVP neurons received relatively broader and denser inputs compared to SONAVP neurons. Additionally, SONAVP neurons received more projections from the median preoptic nucleus and organum vasculosum of the lamina terminalis (a circumventricular organ), compared to PVNAVP neurons, while PVNAVP neurons received more afferent inputs from the bed nucleus of stria terminalis and dorsomedial nucleus of the hypothalamus, both of which are thermoregulatory nuclei, compared to those of SONAVP neurons. In addition, both SONAVP and PVNAVP neurons received direct afferent projections from the bilateral suprachiasmatic nucleus, which is the master regulator of circadian rhythms and is concomitantly responsible for fluctuations in AVP levels. Taken together, our present results provide a comprehensive understanding of the specific afferent framework of AVP neurons both in the SON and PVN, and lay the foundation for further dissecting the diverse roles of SONAVP and PVNAVP neurons.

Whole-brain monosynaptic inputs and outputs of glutamatergic neurons of the vestibular nuclei complex in mice.

Vestibular nuclei complex (VN) glutamatergic neurons play a critical role in the multisensory and multimodal processing. The dysfunction of VN leads to a series of vestibular concurrent symptoms, such as disequilibrium, spatial disorientation, autonomic disorders and even emotion disorders. However, the reciprocal neural connectivity in the whole brain of VN glutamatergic neurons was incompletely understood. Here, we employed a cell-type-specific, cre-dependent, modified virus vector to retrogradely and anterogradely trace VN glutamatergic neurons in the VGLUT2-IRES-Cre mouse line. We identified and analyzed statistically the afferents and efferents of VN glutamatergic neurons in the whole brain, and also reconstructed monosynaptic inputs distribution of VN glutamatergic neurons at the three-dimensional level with the combination of a fluorescence micro-optical sectioning tomography system (fMOST). We found that VN glutamatergic neurons primarily received afferents from 57 nuclei and send efferents to 59 nuclei in the whole brain, intensively located in the brainstem and cerebellum. Projections from nuclei in the cerebellum targeting VN glutamatergic neurons mainly performed the balance control - the principal function of the vestibular system. In addition, VN glutamatergic neurons sent projections to oculomotor nucleus, trochlear nucleus and abducens nucleus dominating the eye movement. Except for the maintenance of balance, VN glutamatergic neurons were also directly connected with other functional regions, such as sleep-wake state (locus coeruleus, dorsal raphe nucleus, and laterodorsal tegmental nucleus, gigantocellular reticular nucleus, lateral paragigantocellular nucleus, periaqueductal gray, subcoeruleus nucleus, parvicellular reticular nucleus, paramedian raphe nucleus), and emotional regulation (locus coeruleus and dorsal raphe nucleus). Hence, this study revealed a comprehensive whole-brain neural connectivity of VN glutamatergic neurons and provided with a neuroanatomic foundation to further study on central vestibular circuits.

Hypnotic activities of Zao Ren An Shen capsule, a traditional Chinese medicine, in an anxiety-like mouse model.

PURPOSE: Zao Ren An Shen capsule (ZRASC) which is composed of three kinds of traditional Chinese herbs is a popular Chinese medicine for the treatment of insomnia. This study investigated the hypnotic effect of ZRASC in an anxiety-like mouse model. METHODS: We determined the role of ZRASC in anxiety and co-morbid insomnia using electroencephalogram and electromyogram recordings. Anxiety-like behaviors were tested by using the open-field, light/dark box, or elevated plus-maze in mice. Immunohistochemical techniques were employed to reveal the mechanism by which ZRASC regulated anxiety and insomnia. RESULTS: ZRASC at 680 mg/kg prolonged the time spent in the central area, open arms area, and light box by 1.9, 2.3, and 1.7-fold respectively, compared with the vehicle control group in immobilization stress (IMS) mice. ZRASC at 680 mg/kg given at 08:00 h increased the amount of non-rapid eye movement sleep by 1.4-fold in a 2-h period after dosing in IMS mice. However, it did not alter the sleep-wake behaviors in normal mice. Immunohistochemistry showed that IMS increased c-Fos expression in the neurons of the stria terminalis and tuberomammillary nucleus by 1.8 and 1.6-fold, respectively. In addition, ZRASC (680 mg/kg) reversed the IMS-induced c-Fos expression. CONCLUSIONS: Our results suggest that ZRASC is an effective therapeutic strategy for both anxiety disorder and sleep disturbances in an anxiety-like mouse model.

Fructose-1,6-bisphosphate aldolase is involved in Mycoplasma bovis colonization as a fibronectin-binding adhesin.

Mycoplasma bovis is a common pathogenic microorganism of cattle and represents an important hazard on the cattle industry. Adherence to host cells is a significant component of mycoplasma-pathogenesis research. Fibronectin (Fn), an extracellular matrix protein, is a common host cell factor that can interact with the adhesions of pathogens. The aims of this study were to investigate the Fn-binding properties of M. bovis fructose-1,6-bisphosphate aldolase (FBA) and evaluate its role as a cell adhesion factor during mycoplasma colonization. The fba (MBOV_RS00435) gene of M. bovis was cloned and expressed, with the resulting recombinant protein used to prepare rabbit polyclonal antibodies. The purified recombinant FBA (rFBA) was shown to have fructose bisphosphate aldolase activity. Western blot indicated that FBA was an antigenically conserved protein in several M. bovis strains. Western blot combined with immunofluorescent assay (IFA) revealed that FBA was dual-localized to both cytoplasm and membrane in M. bovis. IFA showed that rFBA was able to adhere to embryonic bovine lung (EBL) cells. Meanwhile, an adhesion inhibition assay demonstrated that anti-rFBA antibodies could significantly block the adhesion of M. bovis to EBL cells. Moreover, a dose-dependent binding of rFBA to Fn was found by dot blotting and enzyme-linked immunosorbent assays. Together these results provided evidence that FBA is a surface-localized and antigenic protein of M. bovis, suggesting that it may function as a virulence determinant through interacting with host Fn.

Activation of Parabrachial Nucleus Glutamatergic Neurons Accelerates Reanimation from Sevoflurane Anesthesia in Mice.

BACKGROUND: The parabrachial nucleus (PBN), which is a brainstem region containing glutamatergic neurons, is a key arousal nucleus. Injuries to the area often prevent patient reanimation. Some studies suggest that brain regions that control arousal and reanimation are a key part of the anesthesia recovery. Therefore, we hypothesize that the PBN may be involved in regulating emergence from anesthesia. METHODS: We investigated the effects of specific activation or inhibition of PBN glutamatergic neurons on sevoflurane general anesthesia using the chemogenetic "designer receptors exclusively activated by designer drugs" approach. Optogenetic methods combined with polysomnographic recordings were used to explore the effects of transient activation of PBN glutamatergic neuron on sevoflurane anesthesia. Immunohistochemical techniques are employed to reveal the mechanism by which PBN regulated sevoflurane anesthesia. RESULTS: Chemogenetic activation of PBN glutamatergic neurons by intraperitoneal injections of clozapine-N-oxide decreased emergence time (mean ± SD, control vs. clozapine-N-oxide, 55 ± 24 vs. 15 ± 9 s, P = 0.0002) caused by sevoflurane inhalation and prolonged induction time (70 ± 15 vs. 109 ± 38 s, n = 9, P = 0.012) as well as the ED50 of sevoflurane (1.48 vs. 1.60%, P = 0.0002), which was characterized by a rightward shift of the loss of righting reflex cumulative curve. In contrast, chemogenetic inhibition of PBN glutamatergic neurons slightly increased emergence time (56 ± 26 vs. 87 ± 26 s, n = 8, P = 0.034). Moreover, instantaneous activation of PBN glutamatergic neurons expressing channelrhodopsin-2 during steady-state general anesthesia with sevoflurane produced electroencephalogram evidence of cortical arousal. Immunohistochemical experiments showed that activation of PBN induced excitation of cortical and subcortical arousal nuclei during sevoflurane anesthesia. CONCLUSIONS: Activation of PBN glutamatergic neurons is helpful to accelerate the transition from general anesthesia to an arousal state, which may provide a new strategy in shortening the recovery time after sevoflurane anesthesia.

Whole-Brain Monosynaptic Afferent Projections to the Cholecystokinin Neurons of the Suprachiasmatic Nucleus.

The suprachiasmatic nucleus (SCN) is the principal pacemaker driving the circadian rhythms of physiological behaviors. The SCN consists of distinct neurons expressing neuropeptides, including arginine vasopressin (AVP), vasoactive intestinal polypeptide (VIP), gastrin-releasing peptide (GRP), cholecystokinin (CCK), and so on. AVP, VIP, and GRP neurons receive light stimulation from the retina to synchronize endogenous circadian clocks with the solar day, whereas CCK neurons are not directly innervated by retinal ganglion cells and may be involved in the non-photic regulation of the circadian clock. To better understand the function of CCK neurons in non-photic circadian rhythm, it is vital to clarify the direct afferent inputs to CCK neurons in the SCN. Here, we utilized a recently developed rabies virus- and Cre/loxP-based, cell type-specific, retrograde tracing system to map and quantitatively analyze the whole-brain monosynaptic inputs to SCN CCK neurons. We found that SCN CCK neurons received direct inputs from 29 brain nuclei. Among these nuclei, paraventricular nucleus of the hypothalamus (PVH), paraventricular nucleus of the thalamus (PVT), supraoptic nucleus (SON), ventromedial nucleus of the hypothalamus, and seven other nuclei sent numerous inputs to CCK neurons. Moderate inputs originated from the zona incerta, periventricular hypothalamic nucleus, and five other nuclei. A few inputs to CCK neurons originated from the orbital frontal cortex, prelimbic cortex, cingulate cortex, claustrum, and seven other nuclei. In addition, SCN CCK neurons were preferentially innervated by AVP neurons of the ipsilateral PVH and SON rather than their contralateral counterpart, whereas the contralateral PVT sent more projections to CCK neurons than to its ipsilateral counterpart. Taken together, these results expand our knowledge of the specific innervation to mouse SCN CCK neurons and provide an important indication for further investigations on the function of CCK neurons.