Sex differences in molecular and cellular substrates of stress

Women are twice as likely as men to suffer from stress-related psychiatric disorders, like unipolar depression and post-traumatic stress disorder. Although the underlying neural mechanisms are not well characterized, the pivotal role of stress in the onset and severity of these diseases has led to the idea that sex differences in stress responses account for this sex bias. Corticotropin-releasing factor (CRF) orchestrates stress responses by acting both as a neurohormone to initiate the hypothalamic-pituitary-adrenal (HPA) axis and as a neuromodulator in the brain. One target of CRF modulation is the locus coeruleus (LC)-norepinephrine system, which coordinates arousal components of the stress response. Hypersecretion of CRF and dysregulation of targets downstream from CRF, such as the HPA axis and LC-norepinephrine system, are characteristic features of many stress-related psychiatric diseases, suggesting a causal role for CRF and its targets in the development of these disorders. This review will describe sex differences in CRF and the LC-norepinephrine system that can increase stress sensitivity in females, making them vulnerable to stress-related disorders. Evidence for gonadal hormone regulation of hypothalamic CRF is discussed as an effect that can lead to increased HPA axis activity in females. Sex differences in the structure of LC neurons that create the potential for hyperarousal in response to emotional stimuli are described. Finally, sex differences at the molecular level of the CRF(1) receptor that make the LC-norepinephrine system more reactive in females are reviewed. The implications of these sex differences for the treatment of stress-related psychiatric disorders also will be discussed.     

In vivo catechol activity in the rat locus coeruleus following different nociceptive stimuli and naloxone.

The nucleus locus coeruleus (LC) has been implicated in the processing of spinal reflexes following noxious stimuli. It has been demonstrated that noxious stimuli activate LC neuronal firing, but little is known about the neurochemical changes that might occur following such activation. To determine the effects of different noxious stimuli on LC neuronal activity, anaesthetized rats were exposed to mechanical (tail pinch), thermal (55 degrees C water), and chemical (5% Formalin injected in the hind paw) stimuli; the catechol oxidation current (CA.OC), an index of noradrenergic neuronal activity, in the locus coeruleus was monitored using differential normal pulse voltammetry. In addition, the effect of the opioid antagonist naloxone on the CA.OC in the LC was examined. Exposure to both mechanical and chemical stimuli significantly increased CA.OC indicating an increase in LC noradrenergic neuronal activity, while the thermal stimulus had no effect. Treatment with naloxone (1 mg/kg i.v.) had no effect on CA.OC in the LC. The results show a differential responsiveness of LC noradrenergic neurons to different modes of noxious stimuli and fail to demonstrate a tonic opioid regulation of these neurons in the anaesthetized rat.     

Neuronal Activation in the Central Nervous System of Rats in the Initial Stage of Chronic Kidney Disease-Modulatory Effects of Losartan and Moxonidine

The effect of mild chronic renal failure (CRF) induced by 4/6-nephrectomy (4/6NX) on central neuronal activations was investigated by c-Fos immunohistochemistry staining and compared to sham-operated rats. In the 4/6 NX rats also the effect of the angiotensin receptor blocker, losartan, and the central sympatholyticum moxonidine was studied for two months. In serial brain sections Fos-immunoreactive neurons were localized and classified semiquantitatively. In 37 brain areas/nuclei several neurons with different functional properties were strongly affected in 4/6NX. It elicited a moderate to high Fos-activity in areas responsible for the monoaminergic innervation of the cerebral cortex, the limbic system, the thalamus and hypothalamus (e.g. noradrenergic neurons of the locus coeruleus, serotonergic neurons in dorsal raphe, histaminergic neurons in the tuberomamillary nucleus). Other monoaminergic cell groups (A5 noradrenaline, C1 adrenaline, medullary raphe serotonin neurons) and neurons in the hypothalamic paraventricular nucleus (innervating the sympathetic preganglionic neurons and affecting the peripheral sympathetic outflow) did not show Fos-activity. Stress- and pain-sensitive cortical/subcortical areas, neurons in the limbic system, the hypothalamus and the circumventricular organs were also affected by 4/6NX. Administration of losartan and more strongly moxonidine modulated most effects and particularly inhibited Fos-activity in locus coeruleus neurons. In conclusion, 4/6NX elicits high activity in central sympathetic, stress- and pain-related brain areas as well as in the limbic system, which can be ameliorated by losartan and particularly by moxonidine. These changes indicate a high sensitivity of CNS in initial stages of CKD which could be causative in clinical disturbances.     

Effects of negative air ions on activity of neural substrates involved in autonomic regulation in rats

The neural mechanism by which negative air ions (NAI) mediate the regulation of autonomic nervous system activity is still unknown. We examined the effects of NAI on physiological responses, such as blood pressure (BP), heart rate (HR), and heart rate variability (HRV) as well as neuronal activity, in the paraventricular nucleus of the hypothalamus (PVN), locus coeruleus (LC), nucleus ambiguus (NA), and nucleus of the solitary tract (NTS) with c-Fos immunohistochemistry in anesthetized, spontaneously breathing rats. In addition, we performed cervical vagotomy to reveal the afferent pathway involved in mediating the effects of NAI on autonomic regulation. NAI significantly decreased BP and HR, and increased HF power of the HRV spectrum. Significant decreases in c-Fos positive nuclei in the PVN and LC, and enhancement of c-Fos expression in the NA and NTS were induced by NAI. After vagotomy, these physiological and neuronal responses to NAI were not observed. These findings suggest that NAI can modulate autonomic regulation through inhibition of neuronal activity in PVN and LC as well as activation of NA neurons, and that these effects of NAI might be mediated via the vagus nerves.     

辣椒素引起脑干内心血管活动相关核团中c-fos的表达

在１６只切断两侧缓冲神经的大鼠,观察颈总动脉注射辣椒素对脑干内心血管活动相关核团ｃ－ｆｏｓ原癌基因表达的影响。在剂对照组大鼠脑干,仅见少数Ｆｏｓ蛋白样免疫反应（ＦＬＩ）神经元。与对照组相比,颈总动脉注射辣椒素（１０μｍｏｌ,０．１ｍｌ）时,脑干内巨细胞旁外侧核（ＰＧＬ）、蓝斑（ＬＣ）、最后区（ＡＰ）和孤束核（ＮＴＳ）等部位的ＦＬＩ神经元显著增加,而中脑中央灰质（ＰＡＧ）和中缝核群（ＲＮ）的ＦＬＩ神     

Effects of chlordiazepoxide on footshock- and corticotropin-releasing factor-induced increases in cortical and hypothalamic norepinephrine secretion in rats

Noradrenergic and corticotropin-releasing factor (CRF) neuronal systems within the brain have been implicated in stress and anxiety. Synaptic release of cerebral norepinephrine (NE) is increased during stress, and following intracerebral CRF administration. Benzodiazepines are commonly used anxiolytic drugs but information on their effects on the stress- and CRF-related release of NE is limited. We have used in vivo microdialysis to test the effects of the benzodiazepine, chlordiazepoxide (CDP) on the noradrenergic responses to footshock and intracerebroventricular CRF in the medial hypothalamus and the medial prefrontal cortex (PFM) of freely moving rats. Footshock (60 x 0.1-0.2 mA shocks in 20 min) significantly increased microdialysate concentrations of NE in the first sample collected after initiating the footshock. In the hypothalamus, microdialysate NE was augmented 64% above baseline. A second footshock session (100 min after the first footshock) increased microdialysate NE to 313% of the baseline. Thus the noradrenergic responses to footshock were enhanced by preceding footshocks. CRF (100 ng) administered into the locus coeruleus (LC) almost tripled microdialysate concentrations of NE in the PFM. CDP (5mg/kg, i.p.) had no statistically significant effects on the basal dialysate concentrations of NE, but it significantly attenuated both footshock- and CRF-induced increases in dialysate NE. CDP may exert a direct inhibitory effect on the noradrenergic neurons, alter the input to LC noradrenergic neurons, or alter the ability of CRF to activate the LC noradrenergic system.     

虎斑颈槽蛇脑一氧化氮合酶NADPH-d组织化学定位研究

运用NADPH—d组织化学方法,对虎斑颈槽蛇脑的一氧化氮合酶进行了定位研究。结果表明：阳性反应主要位于脑的大脑半球、视上核、室旁核、联合下器官、内侧纵束、室旁灰质、蓝斑以及网状结构等区域,比较讨论了与其他脊椎动物的异同。分析一氧化氮作为神经信号分子除了参与各类动物共有的一些机能的调节以外,不同类群或种类中还参与某些特定生理功能的调节。     

Increased expression of corticotropin-releasing factor receptor mRNA in the locus coeruleus of stress-induced rat model of depression

Hypersecretion of corticotropin-releasing factor (CRF) has been hypothesized to occur in depression. To investigate CRF receptor (CRFR) response to the increased production of CRF in chronically stressed rats, we measured by in situ hybridization the expression of CRFR mRNA in the locus coeruleus (LC) concomitant with measuring plasma adrenocorticotropin (ACTH). The expression of both CRFR mRNA in the LC and the plasma level of ACTH increased significantly in "depression-model rats" which exhibit reduced activity following exposure to 14 days forced walking stress (FWS), but not in "spontaneous recovery rats" whose activity was restored after the long-term stress. These results suggest that the LC neurons continue to be stimulated by CRF, and that the hypothalamic-pituitary-adrenal (HPA) axis is hyperfunctioning in the depression-model rats.     

Brain and pituitary receptors for corticotropin releasing factor: Localization and differential regulation after adrenalectomy

Specific receptors for corticotropin releasing factor (CRF) were identified in two functionally distinct systems within the brain, the cortex and the limbic system. Autoradiographic mapping of the CRF receptors in the brain revealed high binding density throughout the neocortex and cerebellar cortex, subiculum, lateral septum, olfactory tract, bed nucleus of the stria terminalis, interpeduncular nucleus and superior colliculus. Moderate to low binding was found in the hippocampus, nucleus accumbens, claustrum, nucleus periventricularis thalamus, mammillary bodies, subthalamic nucleus, periaqueductal grey, locus coeruleus and nucleus of the spinal trigeminal tract. As in the anterior pituitary gland, CRF receptors in the brain were shown to be coupled to adenylate cyclase. However, in contrast to the marked decrease in CRF receptors observed after adrenalectomy in the anterior pituitary gland, CRF receptor concentration in the brain and pars intermedia of the pituitary was unchanged. The presence of CRF receptors in areas involved in the control of hypothalamic and autonomic nervous system functions is consistent with the major role of CRF in the integrated response to stress.     

Efferent ACTH-IR opiocortin projections from nucleus tractus solitarius: a hypothalamic deafferentation study

The distribution of opiocortin (OR-ir) immunoreactive fibers was examined immunocytochemically throughout the brain in rats following surgical isolation of the arcuate opiocortin-ir neuronal pool in the medial basal hypothalamus (MBH). Fibers which emanate from this pool were completely severed and thus eliminated from the rest of the brain, leaving intact those which can be identified immunocytochemically as opiocortin-ir projections from the medullary pool located in the nucleus tractus solitarius (NTS). These studies reveal a unique organizational pattern of proopiomelanocortin (POMC) peptidergic neuronal systems and demonstrate that several pontine and medullary regions receive projections from both the hypothalamic (arcuate) and medullary (NTS) opiocortin-ir perikarya. Comparative analyses of deafferented and control brains reveal that certain brainstem autonomic centers such as parabrachial (PB), locus coeruleus (LC), nucleus paragiganticellularis (PGi) are recipients of fibers which emanate from both arcuate and NTS opiocortin-ir perikarya. Areas which receive projections from arcuate opiocortin-ir neurons alone include forebrain and hypothalamic nuclei as well as the periaqueductal grey.     

中缝背核参与下丘脑弓状核对蓝斑伤害性反应的抑制

实验在56只水合氯醛麻醉的成年雄性大鼠上进行。实验结果表明:电刺激中缝背核(DR)能减慢蓝斑(LC)大多数神经元自发放电频率;而损毁DR则增加大多数LC神经元的自发放电频率。电刺激下丘脑弓状核(ARC)能抑制LC神经元对外周坐骨神经伤害性刺激的反应。刺激DR可增强此种抑制作用;相反,损毁DR能部分减弱此种抑制效应。结果提示,DR对LC神经元有紧张性抑制作用,并对刺激ARC抑制LC神经元伤害性反应起着调制作用。     

Coordinate Regulation of the Cyclic AMP System with Firing Rate and Expression of Tyrosine Hydroxylase in the Rat Locus Coeruleus: Effects of Chronic Stress and Drug Treatments

Recent studies have demonstrated that chronic stress increases the firing rate and expression of tyrosine hydroxylase (TH) in neurons of the locus coeruleus (LC), the major noradrenergic nucleus in brain. The present study was undertaken to examine the influence of chronic stress and other treatments known to influence the activity of LC neurons on the cyclic AMP (cAMP) second messenger system in these neurons. Chronic (5 days) cold exposure significantly increased levels of TH immunoreactivity in the LC, as previously reported, but not in substantia nigra (SN) or ventral tegmentum (VT), two dopaminergic nuclei studied for comparison. Chronic cold exposure increased levels of cAMP-dependent protein kinase activity in soluble, but not particulate, fractions of the LC, and increased basal and GTP- and forskolin-stimulated adenylate cyclase activity in this brain region. In contrast, levels of the protein kinase and adenylate cyclase in VT, SN, and frontal cortex were not significantly influenced by cold exposure. To study further the relationship between regulation of LC firing rate, TH expression, and the cAMP system in the LC, other treatments known to influence TH were examined. Reserpine treatment, shown previously to increase levels of TH, was found to increase both LC firing rate and levels of soluble cAMP-dependent protein kinase activity in the LC. 6-Hydroxydopamine, shown previously to increase levels of TH and firing rate of LC neurons, also increased soluble levels of protein kinase activity. Other treatments known to either increase (adrenalectomy) or decrease (chronic imipramine) levels of TH in the LC were also found to increase or decrease, respectively, levels of cAMP-dependent protein kinase activity in this brain region.(ABSTRACT TRUNCATED AT 250 WORDS)     

Afferent pathway from locus coeruleus to the nucleus solitarius

After the destruction of the nucleus tractas solitarii, just caudally to the writing pen by means of a stereotaxic instrument, the system of afferent fibres to the nucleus in question was investigated by the methods of Nauta and Fink--Heimer. The fibre terminals were revealed near locus coeruleus. Investigation of the locus coeruleus by Golgi method demonstrated that it usually has neurons of reticular type and transitional ones which resemble by their form the neurons specific for sensory formations. It is possible to conclude that locus coeruleus posesses connections of visceral origin which may play a part in the afferent influence of locus coeruleus on the brain cortex.     

Low nNOS protein in the locus coeruleus in major depression

Disruptions of glutamatergic and noradrenergic signaling have been postulated to occur in depressive disorders. Glutamate provides excitatory input to the noradrenergic locus coeruleus (LC). In this study, the location of immunoreactivity against neuronal nitric oxide synthase (nNOS), an intracellular mediator of glutamate receptor activation, was examined in the normal human LC, and potential changes in nNOS immunoreactivity that might occur in major depression were evaluated. Tissue containing LC, and a non-limbic, LC projection area (cerebellum) was obtained from 11 to 12 matched pairs of subjects with major depression and control subjects lacking major psychiatric diagnoses. In the LC region, nNOS immunoreactivity was found in large neuromelanin-containing neurons, small neurons lacking neuromelanin, and glial cells. Levels of nNOS immunoreactivity were significantly lower in the LC (- 44%, p < 0.05), but not in the cerebellum, when comparing depressed with control subjects. nNOS levels were positively correlated with brain pH values in depressed, but not control, subjects in both brain regions. Low levels of nNOS in the LC may reflect altered excitatory input to this nucleus in major depression. However, pH appears to effect preservation of nNOS immunoreactivity in subjects with depression. This factor may contribute, in part, to low levels of nNOS in depression.     

Ultrastructural Correlates of Enhanced Norepinephrine and Neuropeptide Y Cotransmission in the Spontaneously Hypertensive Rat Brain

The spontaneously hypertensive rat (SHR) replicates many clinically relevant features of human essential hypertension and also exhibits behavioral symptoms of attention-deficit/hyperactivity disorder and dementia. The SHR phenotype is highly complex and cannot be explained by a single genetic or physiological mechanism. Nevertheless, numerous studies including our own work have revealed striking differences in central catecholaminergic transmission in SHR such as increased vesicular catecholamine content in the ventral brainstem. Here, we used immunolabeling followed by confocal microscopy and electron microscopy to quantify vesicle sizes and populations across three catecholaminergic brain areas—nucleus tractus solitarius and rostral ventrolateral medulla, both key regions for cardiovascular control, and the locus coeruleus. We also studied colocalization of neuropeptide Y (NPY) in norepinephrine and epinephrine-containing neurons as NPY is a common cotransmitter with central and peripheral catecholamines. We found significantly increased expression and coexpression of NPY in norepinephrine and epinephrine-positive neurons of locus coeruleus in SHR compared with Wistar rats. Ultrastructural analysis revealed immunolabeled vesicles of 150 to 650 nm in diameter (means ranging from 250 to 300 nm), which is much larger than previously reported. In locus coeruleus and rostral ventrolateral medulla, but not in nucleus tractus solitarius, of SHR, noradrenergic and adrenergic vesicles were significantly larger and showed increased NPY colocalization when compared with Wistar rats. Our morphological evidence underpins the hypothesis of hyperactivity of the noradrenergic and adrenergic system and increased norepinephrine and epinephrine and NPY cotransmission in specific brain areas in SHR. It further strengthens the argument for a prohypertensive role of C1 neurons in the rostral ventrolateral medulla as a potential causative factor for essential hypertension.     

Role of the lateral paragigantocellular nucleus in the network of paradoxical (REM) sleep: an electrophysiological and anatomical study in the rat

The lateral paragigantocellular nucleus (LPGi) is located in the ventrolateral medulla and is known as a sympathoexcitatory area involved in the control of blood pressure. In recent experiments, we showed that the LPGi contains a large number of neurons activated during PS hypersomnia following a selective deprivation. Among these neurons, more than two-thirds are GABAergic and more than one fourth send efferent fibers to the wake-active locus coeruleus nucleus. To get more insight into the role of the LPGi in PS regulation, we combined an electrophysiological and anatomical approach in the rat, using extracellular recordings in the head-restrained model and injections of tracers followed by the immunohistochemical detection of Fos in control, PS-deprived and PS-recovery animals. With the head-restrained preparation, we showed that the LPGi contains neurons specifically active during PS (PS-On neurons), neurons inactive during PS (PS-Off neurons) and neurons indifferent to the sleep-waking cycle. After injection of CTb in the facial nucleus, the neurons of which are hyperpolarized during PS, the largest population of Fos/CTb neurons visualized in the medulla in the PS-recovery condition was observed in the LPGi. After injection of CTb in the LPGi itself and PS-recovery, the nucleus containing the highest number of Fos/CTb neurons, moreover bilaterally, was the sublaterodorsal nucleus (SLD). The SLD is known as the pontine executive PS area and triggers PS through glutamatergic neurons. We propose that, during PS, the LPGi is strongly excited by the SLD and hyperpolarizes the motoneurons of the facial nucleus in addition to local and locus coeruleus PS-Off neurons, and by this means contributes to PS genesis.     

Expression of the immediate-early gene cFos in brain of the rats exposed to repeated 2 G influence

Early gene c-Fos expression was studied by means of c-Fos protein immunostaining in brain locus coeruleus (LC) of the rats exposed to primary and repeated hypergravity. One-hour 2 G influence on rats induced in LC cells expression of c-Fos protein, pointing out early gene c-Fos expression and synaptic activation of LC neurons. After repeated 1-hour 2 G, postponed for 35 days after primary 30-day 2 G influence, expression of c-Fos protein in LC neurons was not found. This fact is considered as a sign of memorizing of primary hypergravity influence.     

Identification of a Group of GABAergic Neurons in the Dorsomedial Area of the Locus Coeruleus

The locus coeruleus (LC)-norepinephrine (NE) system in the brainstem plays a critical role in a variety of behaviors is an important target of pharmacological intervention to several neurological disorders. Although GABA is the major inhibitory neurotransmitter of LC neurons, the modulation of LC neuronal firing activity by local GABAergic interneurons remains poorly understood with respect to their precise location, intrinsic membrane properties and synaptic modulation. Here, we took an optogenetic approach to address these questions. Channelrhodopsin (ChR2) in a tandem with the yellow fluorescent protein (YFP) was expressed in GABAergic neurons under the control of glutamic acid decarboxylase 2 (GAD2) promoter. Immediately dorsomedial to the LC nucleus, a group of GABAergic neurons was observed. They had small soma and were densely packed in a small area, which we named the dorsomedial LC or dmLC nucleus. These GABAergic neurons showed fast firing activity, strong inward rectification and spike frequency adaptation. Lateral inhibition among these GABAergic neurons was observed. Optostimulation of the dmLC area drastically inhibited LC neuronal firing frequency, expanded the spike intervals, and reset their pacemaking activity. Analysis of the light evoked inhibitory postsynaptic currents (IPSCs) indicated that they were monosynaptic. Such light evoked IPSCs were not seen in slices where this group of GABAergic neurons was absent. Thus, an isolated group of GABAergic neurons is demonstrated in the LC area, whose location, somatic morphology and intrinsic membrane properties are clearly distinguishable from adjacent LC neurons. They interact with each and may inhibit LC neurons as well as a part of local neuronal circuitry in the LC.