Corticotropin-releasing factor overexpression gives rise to sex differences in Alzheimer's disease-related signaling.

Several neuropsychiatric and neurodegenerative disorders share stress as a risk factor and are more prevalent in women than in men. Corticotropin-releasing factor (CRF) orchestrates the stress response, and excessive CRF is thought to contribute to the pathophysiology of these diseases. We previously found that the CRF1 receptor (CRF1) is sex biased whereby coupling to its GTP-binding protein, Gs, is greater in females, whereas ß-arrestin-2 coupling is greater in males. This study used a phosphoproteomic approach in CRF-overexpressing (CRF-OE) mice to test the proof of principle that when CRF is in excess, sex-biased CRF1 coupling translates into divergent cell signaling that is expressed as different brain phosphoprotein profiles. Cortical phosphopeptides that distinguished female and male CRF-OE mice were overrepresented in unique pathways that were consistent with Gs-dependent signaling in females and ß-arrestin-2 signaling in males. Notably, phosphopeptides that were more abundant in female CRF-OE mice were overrepresented in an Alzheimer's disease (AD) pathway. Phosphoproteomic results were validated by demonstrating that CRF overexpression in females was associated with increased tau phosphorylation and, in a mouse model of AD pathology, phosphorylation of ß-secretase, the enzyme involved in the formation of amyloid ß. These females exhibited increased formation of amyloid ß plaques and cognitive impairments relative to males. Collectively, the findings are consistent with a mechanism whereby the excess CRF that characterizes stress-related diseases initiates distinct cellular processes in male and female brains, as a result of sex-biased CRF1 signaling. Promotion of AD-related signaling pathways through this mechanism may contribute to female vulnerability to AD.

Increased vulnerability of the brain norepinephrine system of females to corticotropin-releasing factor overexpression.

Stress-related psychiatric disorders are more prevalent in women than men. As hypersecretion of the stress neuromediator, corticotropin-releasing factor (CRF) has been implicated in these disorders, sex differences in CRF sensitivity could underlie this disparity. Hyperarousal is a core symptom that is shared by stress-related disorders and this has been attributed to CRF regulation of the locus ceruleus (LC)-norepinephrine arousal system. We recently identified sex differences in CRF(1) receptor (CRF(1)) signaling and trafficking that render LC neurons of female rats more sensitive to CRF and potentially less able to adapt to excess CRF compared with male rats. The present study used a genetic model of CRF overexpression to test the hypothesis that females would be more vulnerable to LC dysregulation by conditions of excess CRF. In both male and female CRF overexpressing (CRF-OE) mice, the LC was more densely innervated by CRF compared with wild-type controls. Despite the equally dense CRF innervation of the LC in male and female CRF-OE mice, LC discharge rates recorded in slices in vitro were selectively elevated in female CRF-OE mice. Immunoelectron microscopy revealed that this sex difference resulted from differential CRF(1) trafficking. In male CRF-OE mice, CRF(1) immunolabeling was prominent in the cytoplasm of LC neurons, indicative of internalization, a process that would protect cells from excessive CRF. However, in female CRF-OE mice, CRF(1) labeling was more prominent on the plasma membrane, suggesting that the compensatory response of internalization was compromised. Together, the findings suggest that the LC-norepinephrine system of females will be particularly affected by conditions resulting in elevated CRF because of differences in receptor trafficking. As excessive LC activation has been implicated in the arousal components of stress-related psychiatric disorders, this may be a cellular mechanism that contributes to the increased incidence of these disorders in females.

Sex differences in corticotropin-releasing factor receptor signaling and trafficking: potential role in female vulnerability to stress-related psychopathology.

Although the higher incidence of stress-related psychiatric disorders in females is well documented, its basis is unknown. Here, we show that the receptor for corticotropin-releasing factor (CRF), the neuropeptide that orchestrates the stress response, signals and is trafficked differently in female rats in a manner that could result in a greater response and decreased adaptation to stressors. Most cellular responses to CRF in the brain are mediated by CRF receptor (CRFr) association with the GTP-binding protein, G(s). Receptor immunoprecipitation studies revealed enhanced CRFr-G(s) coupling in cortical tissue of unstressed female rats. Previous stressor exposure abolished this sex difference by increasing CRFr-G(s) coupling selectively in males. These molecular results mirrored the effects of sex and stress on sensitivity of locus ceruleus (LC)-norepinephrine neurons to CRF. Differences in CRFr trafficking were also identified that could compromise stress adaptation in females. Specifically, stress-induced CRFr association with beta-arrestin2, an integral step in receptor internalization, occurred only in male rats. Immunoelectron microscopy confirmed that stress elicited CRFr internalization in LC neurons of male rats exclusively, consistent with reported electrophysiological evidence for stress-induced desensitization to CRF in males. Together, these studies identified two aspects of CRFr function, increased cellular signaling and compromised internalization, which render CRF-receptive neurons of females more sensitive to low levels of CRF and less adaptable to high levels of CRF. CRFr dysfunction in females may underlie their increased vulnerability to develop stress-related pathology, particularly that related to increased activity of the LC-norepinephrine system, such as depression or post-traumatic stress disorder.

Evidence for functional release of endogenous opioids in the locus ceruleus during stress termination.

Endogenous opioids target noradrenergic locus ceruleus (LC) neurons and potently inhibit LC activity. Nonetheless, it has been difficult to demonstrate functional regulation of the LC-noradrenergic system by endogenous opioids because of the lack of effect of opiate antagonists. The present findings provide evidence that endogenous opioids regulate LC neuronal activity during the termination of a stressor. LC neuronal discharge was recorded from halothane-anesthetized rats before, during, and after hypotensive stress elicited by intravenous nitroprusside infusion. In naive rats, mean arterial blood pressure was temporally correlated with LC activity such that hypotension was associated with increased LC discharge and a return to the normotensive state was associated with a decrease in LC discharge below pre-stress values. After microinfusion of an antagonist of the stress neuropeptide corticotropin-releasing factor (CRF) into the LC, the increase in LC discharge associated with hypotension was prevented, whereas LC inhibition associated with termination of the challenge occurred at an earlier time and was of a greater magnitude. In contrast, microinfusion of naloxone into the LC completely abolished LC inhibition associated with termination of the stressor. Naloxone microinfusion did not prevent LC inhibition associated with hypertension produced by intravenous vasopressin administration, suggesting that endogenous opioids may be selectively engaged during the termination of hypotensive stress. These results provide evidence for a functional release of endogenous opioids within the LC. This action of endogenous opioids may serve to counterbalance excitatory effects of CRF on the LC-norepinephrine system, thereby limiting its activation by stress.

Pontine regulation of pelvic viscera: pharmacological target for pelvic visceral dysfunctions.

The pathophysiology and pharmacological targets of disorders of the bladder and colon have focused predominantly on the periphery. However, these viscera are regulated by the CNS, which, in turn, must integrate their functions with compatible behaviours. This review focuses on the role of the pontine micturition centre, Barrington's nucleus, as a key to this integration. Through its efferent network this pontine centre links parasympathetic preganglionic neurones with forebrain-projecting nuclei, providing an anatomical substrate for coregulation of pelvic visceral and forebrain activity. Disorders characterized by multiple pelvic visceral symptoms and comorbidity with psychiatric disorders (for example functional bowel disorders) might have their roots in dysfunctions of this circuit, which could provide a novel target for pharmacological treatment.

Peptides that fine-tune the serotonin system.

The dorsal raphe nucleus (DR) contains serotonin (5-HT) neurons that innervate the cortex and limbic system and through these projections is thought to regulate cognition and behavior. Clinical and pharmacological findings implicate dysfunctions in the DR-5-HT system in affective disorders, including anxiety, depression and suicide. Although the DR is often considered in light of its 5-HT neurons, recent studies underscore the complexity of this nucleus and its heterogeneous nature. Of particular interest, are peptides that are either present within neurons in the DR, innervate DR-5-HT neurons or act upon local circuitry within the DR to indirectly impact on this 5-HT system. These peptides are positioned to fine-tune the activity of selective groups of serotonergic neurons within the DR and thereby 5-HT release in different terminal fields. This review will focus on substance P and corticotropin-releasing factor as two peptides that act independently and interdependently to influence DR-5-HT function. The role of non-serotonergic components of the DR in translating the effect of each of these peptides is discussed. This synthesis refines our views on the regulation of the DR-5-HT system and importantly, gives insight into mechanisms of endogenous control of DR function, the dysregulation of which may contribute to pathophysiology.

A.E. Bennett Research Award. Anatomic basis for differential regulation of the rostrolateral peri-locus coeruleus region by limbic afferents.

BACKGROUND: Neurochemical and electrophysiological studies indicate that the locus coeruleus (LC)-norepinephrine system is activated by physiological and external stressors. This activation is mediated in part by corticotropin-releasing factor (CRF), the hypothalamic neurohormone that initiates the endocrine response to stress. We have previously shown that the central nucleus of the amygdala (CNA) provides CRF afferents to noradrenergic processes in the peri-LC area that may serve to integrate emotional and cognitive responses to stress. The bed nucleus of the stria terminalis (BNST) shares many anatomical and neurochemical characteristics with the CNA, including a high density of CRF-immunoreactive cells and fibers; however, recent studies have suggested that the CNA and the BNST may differentially regulate responses to conditioned and unconditioned fear, respectively, suggesting divergent neuroanatomical circuits underlying these processes. METHODS: In the present study, neuroanatomical substrates subserving regulation of the LC by the BNST were examined. Anterograde tract-tracing was combined with immunoelectron microscopy to test the hypotheses that BNST efferents target noradrenergic neurons of the LC and that these efferents exhibit immunolabeling for CRF. RESULTS: Ultrastructural analysis of sections that were dually labeled for the anterograde tracer biotinylated dextran amine (BDA) injected into the BNST and tyrosine hydroxylase (TH)-immunoreactivity demonstrated that BDA-labeled axon terminals formed synaptic specializations (primarily inhibitory) with TH-labeled dendrites and dendrites that lacked TH immunoreactivity. In contrast to CNA efferents that exhibited substantial immunolabeling for CRF, far fewer BDA-labeled terminals from the BNST in the rostrolateral peri-LC contained CRF. CONCLUSIONS: The present results indicate that the BNST may provide distinct neurochemical regulation of the peri-LC as compared to other limbic afferents such as the CNA. These data are interesting in light of behavioral studies showing that the CNA and BNST may be differentially involved in fear versus anxiety, respectively.

Evidence for corticotropin-releasing hormone projections from Barrington's nucleus to the periaqueductal gray and dorsal motor nucleus of the vagus in the rat.

The present study used anterograde and retrograde tract tracing and immunohistochemistry to determine the efferent projections of corticotropin-releasing hormone (CRH) neurons of Barrington's nucleus in the rat. Injections of Phaseolus vulgaris-leucoagglutinin into Barrington's nucleus resulted in anterograde labeling in the dorsal motor nucleus of the vagus, periaqueductal gray, medial thalamic nuclei, lateral hypothalamus, paraventricular nucleus of the hypothalamus, lateral preoptic area, and lateral septum. The retrograde tract tracer, fluorogold, injected into the lumbosacral spinal cord labeled many, but not all, CRH-immunoreactive neurons in Barrington's nucleus. Moreover, some Barrington's neurons that were retrogradely labeled from the spinal cord were not CRH-immunoreactive. Several CRH-immunoreactive Barrington's neurons were retrogradely labeled by fluorogold injections into the periaqueductal gray, and these were located predominantly in the dorsal part of the nucleus. Additionally, some CRH-immunoreactive Barrington's neurons were retrogradely labeled from fluorogold injections into the dorsal motor nucleus of the vagus. In contrast, fluorogold injections into the lateral hypothalamus, lateral preoptic area, or lateral septum did not result in double labeling of CRH-immunoreactive neurons in Barrington's nucleus. These results suggest that many, but not all, CRH-containing neurons of Barrington's nucleus project to the lumbosacral spinal cord. In addition to their previously documented projections to the spinal cord, these neurons may be a source of CRH in the periaqueductal gray and dorsal motor nucleus of the vagus. CRH projections of Barrington's nucleus may play a role in behavioral or autonomic aspects of stress responses, in addition to their proposed role in micturition.

Corticotropin-releasing factor-containing axon terminals synapse onto catecholamine dendrites and may presynaptically modulate other afferents in the rostral pole of the nucleus locus coeruleus in the rat brain.

Physiological and immunohistochemical studies have suggested that corticotropin-releasing factor (CRF), the hypophysiotropic peptide that initiates endocrine responses to stress, may serve as a neurotransmitter to activate noradrenergic neurons in the nucleus locus coeruleus (LC). We combined immunoperoxidase labeling for CRF and immunogold-silver localization of the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) in single sections through the rat LC to determine potential substrates for interactions between these two transmitters. Light microscopic analysis indicated that CRF processes are dense and highly varicose in the rostral LC region in the vicinity of noradrenergic dendrites. Electron microscopy of this rostral region revealed that immunoperoxidase labeling for CRF was mainly restricted to axons and axon terminals and was rarely seen in somata or dendrites. Axon terminals containing CRF immunoreactivity varied in size, content of synaptic vesicles, and formation of synaptic specializations. The postsynaptic targets of the CRF-labeled axon terminals consisted of both TH-labeled dendrites and dendrites lacking detectable TH-immunoreactivity. Of 113 CRF-immunoreactive axon terminals, approximately 70% were in direct contact with TH-labeled and unlabeled dendrites. Of the CRF-labeled axon terminals forming synapses with TH-labeled and unlabeled dendrites, they were either of the asymmetric (excitatory type; 19%) or symmetric (inhibitory type; 11%) variety or did not form identifiable contacts in the plane of section analyzed. Unlabeled axon terminals and glial processes were also commonly located adjacent to the plasma membranes of CRF-labeled axon terminals. These results provide the first direct ultrastructural evidence that axon terminals containing CRF-immunoreactivity 1) directly contact catecholamine-containing dendrites within the rostral pole of the LC, 2) may presynaptically modulate other afferents, and 3) are often enveloped by astrocytic processes.

Evidence for regional heterogeneity in corticotropin-releasing factor interactions in the dorsal raphe nucleus.

The dorsal raphe nucleus (DR) is innervated by fibers containing the stress-related neurohormone corticotropin-releasing factor (CRF), which alters DR neuronal activity and serotonin release in rats. This study examined the relative distribution of CRF-immunoreactive fibers in the rat DR by using light level densitometry. Additionally, CRF-immunoreactive processes within specific subregions of the DR were examined at the ultrastructural level by using electron microscopy. CRF-immunoreactive fibers were organized within the DR along a caudal-rostral gradient, such that proceeding rostrally, innervation shifted from dorsolateral to ventromedial. Numerous CRF-immunoreactive axon terminals containing dense-core vesicles were found in both the caudal dorsolateral region and the rostral ventromedial/interfascicular region. These formed synaptic specializations with unlabeled dendrites and frequently contacted nonlabeled axon terminals. Semiquantitative analysis revealed certain differences between the two regions with respect to the types of associations made by CRF-immunoreactive terminals. Associations with dendrites were more frequent in the dorsolateral vs. ventromedial region (65% of 171 terminals vs. 39% of 233 terminals, respectively), whereas associations with axon terminals were more frequent in the ventromedial/interfascicular vs. the dorsolateral region (72% of 233 terminals vs. 57% of 171 terminals, respectively). Additionally, synaptic specializations between CRF-immunoreactive terminals and dendrites were more frequently asymmetric in the dorsolateral region (60%) and symmetric (49%) in the ventromedial/interfascicular region. Regional differences in CRF terminal interactions in the DR could account for the reported heterogeneous effects of CRF on DR neuronal activity and forebrain serotonin release. Importantly, the present results provide anatomical substrates for regulation of the DR by endogenous CRF.

Transneuronal labeling from the rat distal colon: anatomic evidence for regulation of distal colon function by a pontine corticotropin-releasing factor system.

Neural circuits that are positioned to regulate rat distal colon function were identified by immunohistochemical detection of pseudorabies virus (PRV) and corticotropin-releasing factor (CRF). The distribution of PRV-immunoreactive neurons was examined in spinal cord and brain at increasing times (72-118 hours) after distal colon injection. At 72-80 hours, PRV-labeling was confined to the spinal cord, in the parasympathetic preganglionic column in the lumbosacral spinal cord and in the intermediolateral column of the thoracic spinal cord. At longer survival times (88 hours), PRV-immunolabeled neurons in the lumbosacral spinal cord were also distributed in superficial layers of the dorsal horn, the dorsal commissure, and around the central canal. Trans-synaptic labeling was identified in the medullary raphe nuclei, parapyramidal region, A5, Barrington's nucleus, A7, and the dorsal cap of the paraventricular nucleus of the hypothalamus after longer survival times (88-91 hours). Substantial labeling of the locus coeruleus, periaqueductal gray and forebrain regions occurred at later survival times (> or = 96 hours). In dual-labeled sections, CRF terminal labeling surrounded PRV-labeled neurons in the parasympathetic preganglionic column of the lumbosacral spinal cord. Additionally, many neurons in Barrington's nucleus, but not other CRF-containing nuclei, were double labeled for CRF and PRV. These results, taken with previous studies, support a convergence in transneuronal labeling from different pelvic viscera that may be related to coordination of overall pelvic visceral functions. Importantly, they provide an anatomic substrate for an impact of CRF from Barrington's nucleus in normal and pathophysiological functions of the distal colon.

Cellular interactions between axon terminals containing endogenous opioid peptides or corticotropin-releasing factor in the rat locus coeruleus and surrounding dorsal pontine tegmentum.

Recent evidence suggests that certain stressors release both endogenous opioids and corticotropin-releasing factor (CRF) to modulate activity of the locus coeruleus (LC)-norepinephrine (NE) system. In ultrastructural studies, axon terminals containing methionine(5)-enkephalin (ENK) or CRF have been shown to target LC dendrites. These findings suggested the hypothesis that both neuropeptides may coexist in common axon terminals that are positioned to have an impact on the LC. This possibility was examined by using immunofluorescence and immunoelectron microscopic analysis of the rat LC and neighboring dorsal pontine tegmentum. Ultrastructural analysis indicated that CRF- and ENK-containing axon terminals were abundant in similar portions of the neuropil and that approximately 16% of the axon terminals containing ENK were also immunoreactive for CRF. Dually labeled terminals were more frequently encountered in the "core" of the LC vs. its extranuclear dendritic zone, which included the medial parabrachial nucleus (mPB). Triple labeling for ENK, CRF, and tyrosine hydroxylase (TH) showed convergence of opioid and CRF axon terminals with noradrenergic dendrites as well as evidence for inputs to TH-labeled dendrites from dually labeled opioid/CRF axon terminals. One potential source of ENK and CRF in the dorsal pons is the central nucleus of the amygdala (CNA). To determine the relative contribution of ENK and CRF terminals from the CNA, the CNA was electrolytically lesioned. Light-level densitometry revealed robust decreases in CRF immunoreactivity in the LC and mPB on the side ipsilateral to the lesion but little or no change in ENK immunoreactivity, confirming previous studies of the mPB. Degenerating terminals from the CNA in lesioned rats were found to be in direct contact with TH-labeled dendrites. Together, these data indicate that ENK and CRF may be colocalized to a subset of individual axon terminals in the LC "core." The finding that the CNA provides, to dendrites in the area examined, a robust CRF innervation, but little or no opioid innervation, suggests that ENK and CRF axon terminals impacting LC neurons originate from distinct sources and that terminals that colocalize ENK and CRF are not from the CNA.

Effects of corticotropin-releasing factor on neuronal activity in the serotonergic dorsal raphe nucleus.

The present study examined the regional localization of corticotropin-releasing factor (CRF)- and 5-hydroxytryptamine (5-HT)-immunoreactive (IR) fibers within the rat dorsal raphe nucleus (DRN) using immunohistochemistry. Additionally, the effects of CRF, administered intracerebroventricularly (0.1-3.0 micrograms) or intraraphe (0.3-30 ng), on discharge rates of putative 5-HT DRN neurons were quantified using in vivo single unit recording in halothane-anesthetized rats. CRF-IR fibers were present at all rostrocaudal levels of the DRN and exhibited a topographical distribution. CRF produced predominantly inhibitory effects on DRN discharge at lower doses and these effects diminished or became excitatory at higher doses. Inhibition of DRN discharge by CRF was attenuated by the nonselective CRF antagonist, DPheCRF12-41 and the CRF-R1-selective antagonist, antalarmin, implicating the CRF-R1 receptor subtype in these electrophysiological effects. The present findings provide anatomical and physiological evidence for an impact of CRF on the DRN-5HT system.

Effects of corticotropin-releasing factor on brain serotonergic activity.

The serotonergic dorsal raphe nucleus is innervated by corticotropin-releasing factor (CRF) and expresses CRF receptors, suggesting that endogenous CRF impacts on this system. The present study characterized interactions between CRF and the dorsal raphe serotonin (5-HT) system. The effects of intracerebroventricularly (i.c.v.) administered CRF on microdialysate concentrations of 5-HT in the lateral striatum of freely moving rats were determined. CRF had biphasic effects, with 0.1 and 0.3 microgram decreasing, and 3.0 micrograms increasing 5-HT dialysate concentrations. i.c.v. administration of CRF inhibited neuronal activity of the majority of dorsal raphe neurons at both low (0.3 microgram) and high (3 micrograms) doses. Likewise, intraraphe administration of CRF (0.3 and 1.0 ng) had predominantly inhibitory effects on discharge rate. Together, these results suggest that CRF is positioned to regulate the function of the dorsal raphe serotonergic system via actions within the cell body region. This regulation may play a role in stress-related psychiatric disorders in which 5-HT has been implicated.

Activation of locus coeruleus neurons in the rat by a benzazocine derivative (UM 1046) that mimics opiate withdrawal.

The drug UM 1046 (N-cyclopropylmethyl-1,2,3,4,5,6-hexahydro-8-hydroxy-6-methyl-3-benzazoc ine), administered to opiate-naive monkeys, produces certain behavioral responses resembling antagonist-precipitated morphine withdrawal (Swain and Seevers, 1975). In the present study, UM 1046 (5.6 mg/kg, i.v.), administered to naive rats, produced a syndrome that consisted primarily of retching, chewing, teeth chattering, shaking and abnormal postures. It was of interest to determine whether UM 1046 had an effect on noradrenergic neurons of the locus coeruleus (NE-LC) since increased activity of these cells has been reported to occur during antagonist-precipitated opiate withdrawal (Aghajanian, 1978) and after the administration of drugs that mimic this syndrome in normal animals. (i.e. methylxanthine phosphodiesterase inhibitors, Grant and Redmond, 1981). In the present study, UM 1046 (1.0-5.6 mg/kg, i.v.) caused a dose-dependent increase (of up to 200%) in the spontaneous discharge rate of noradrenergic neurons of the locus coeruleus in rats anesthetized with halothane. The time-course of this effect was similar to the time-course of the behavioral syndrome described above. Stimulation of central muscarinic receptors is integrally involved in the response to this drug since the effects of UM 1046 (5.6 mg/kg) were antagonized by scopolamine (0.5 mg/kg, i.v.), but not by methylscopolamine (1.0 mg/kg, i.v.). Unlike systemic administration, iontophoretic application of UM 1046 did not consistently increase the spontaneous discharge rate of these cells, indicating that the site of action of the drug may be outside the nucleus locus coeruleus. This study complements previous findings of increased activity in noradrenergic neurons of the locus coeruleus during withdrawal-like behavior. In addition, the results are compatible with others which suggest that a cholinergic link is essential for the withdrawal-like actions of the benzazocine.

Cocaine effects on brain noradrenergic neurons of anesthetized and unanesthetized rats.

The present study characterized and quantified the effects of systemically administered cocaine on spontaneous, sensory-evoked and stress-elicited activity of noradrenergic locus coeruleus (LC) neurons of anesthetized and unanesthetized rats. Cocaine (0.1-3.0 mg/kg, i.v.) decreased LC spontaneous discharge rate and discharge evoked by repeated sciatic nerve stimulation in halothane-anesthetized rats. In unanesthetized rats cocaine (0.3-10.0 mg/kg, i.v.) also decreased LC spontaneous discharge rate and LC discharge evoked by repeated auditory stimulation. However, analysis of variance revealed a statistically significant shift to the right in the cocaine dose-response curves for effects on tonic and evoked LC discharge in unanesthetized compared to anesthetized rats. Thus, cocaine was somewhat less potent in inhibiting tonic and evoked discharge of unanesthetized rats compared to anesthetized rats. In anesthetized rats cocaine (1.0 mg/kg) did not affect LC activation by intracerebroventricularly (i.c.v.) administered corticotropin-releasing factor (3.0 micrograms in 3.0 microliters) or by hemodynamic stress elicited by i.v. nitroprusside infusion. The present findings demonstrate that cocaine has similar effects on LC neurons of anesthetized and unanesthetized rats but that it is less potent in unanesthetized rats. These effects of cocaine at noradrenergic cell bodies acting in concert with its effects at noradrenergic terminals in LC target regions may be important in the overall action of cocaine on arousal and cortical information processing.

Dissociation of locus coeruleus activity and blood pressure. Effects of clonidine and corticotropin-releasing factor.

In order to determine whether pharmacologically-induced alterations in the spontaneous activity of neurons in the locus coeruleus are associated with changes in blood pressure, the activity of the locus coeruleus and blood pressure were recorded simultaneously in anesthetized rats after the administration of agents known to affect both of these parameters. Spontaneous activity of the locus coeruleus was decreased by intracerebroventricular (i.c.v.) administration of both clonidine and St 91, [2,(2,6-diethyl-phenylimino)imidazolidine chloride], a charged analogue of clonidine. However, only clonidine decreased the mean blood pressure after intracerebroventricular administration suggesting that either the receptors mediating decreases in the activity of the locus coeruleus are different to those mediating hypotension, or that St 91 does not distribute to sites involved in the control of blood pressure even after intracerebroventricular administration. Intravenous administration of clonidine, but not of St 91, decreased the activity of the locus coeruleus and produced a prolonged hypotension, thus suggesting a central mechanism for these effects. Both clonidine and St 91 administered intravenously, produced a brief initial period of hypertension which was not associated with consistent changes in the spontaneous activity of the locus coeruleus. Thus, noradrenergic agonists can decrease the activity of the locus coeruleus without affecting blood pressure, and increase blood pressure without affecting the activity of the locus coeruleus. The spontaneous activity of cells in the locus coeruleus was increased by 100% after the intracerebroventricular administration of corticotropin-releasing factor (CRF; 3.0 micrograms).(ABSTRACT TRUNCATED AT 250 WORDS)

Distinguishing characteristics of serotonin and non-serotonin-containing cells in the dorsal raphe nucleus: electrophysiological and immunohistochemical studies.

The membrane properties and receptor-mediated responses of rat dorsal raphe nucleus neurons were measured using intracellular recording techniques in a slice preparation. After each experiment, the recorded neuron was filled with neurobiotin and immunohistochemically identified as 5-hydroxytryptamine (5-HT)-immunopositive or 5-HT-immunonegative. The cellular characteristics of all recorded neurons conformed to previously determined classic properties of serotonergic dorsal raphe nucleus neurons: slow, rhythmic activity in spontaneously active cells, broad action potential and large afterhyperpolarization potential. Two electrophysiological characteristics were identified that distinguished 5-HT from non-5-HT-containing cells in this study. In 5-HT-immunopositive cells, the initial phase of the afterhyperpolarization potential was gradual (tau=7.3+/-1.9) and in 5-HT-immunonegative cells it was abrupt (tau=1.8+/-0.6). In addition, 5-HT-immunopositive cells had a shorter membrane time constant (tau=21.4+/-4.4) than 5-HT-immunonegative cells (tau=33.5+/-4.2). Interestingly, almost all recorded neurons were hyperpolarized in response to stimulation of the inhibitory 5-HT(1A) receptor. These results suggested that 5-HT(1A) receptors are present on non-5-HT as well as 5-HT neurons. This was confirmed by immunohistochemistry showing that although the majority of 5-HT-immunopositive cells in the dorsal raphe nucleus were double-labeled for 5-HT(1A) receptor-IR, a small but significant population of 5-HT-immunonegative cells expressed the 5-HT(1A) receptor. These results underscore the heterogeneous nature of the dorsal raphe nucleus and highlight two membrane properties that may better distinguish 5-HT from non-5-HT cells than those typically reported in the literature. In addition, these results present electrophysiological and anatomical evidence for the presence of 5-HT(1A) receptors on non-5-HT neurons in the dorsal raphe nucleus.

Fos immunoreactivity in hypocretin-synthesizing and hypocretin-1 receptor-expressing neurons: effects of diurnal and nocturnal spontaneous waking, stress and hypocretin-1 administration.

Hypocretin/orexin modulates sleep-wake state via actions across multiple terminal fields. Within waking, hypocretin may also participate in high-arousal processes, including those associated with stress. The current studies examined the extent to which alterations in neuronal activity, as measured by Fos immunoreactivity, occur within both hypocretin-synthesizing and hypocretin-1 receptor-expressing neurons across varying behavioral state/environmental conditions associated with varying levels of waking and arousal. Double-label immunohistochemistry was used to visualize Fos and either prepro-hypocretin in the lateral hypothalamus or hypocretin-1 receptors in the locus coeruleus and select basal forebrain regions involved in the regulation of behavioral state/arousal. Animals were tested under the following conditions: 1). diurnal sleeping; 2). diurnal spontaneous waking; 3). nocturnal spontaneous waking; and 4). high-arousal waking (diurnal novelty-stress). Additionally, the effects of hypocretin-1 administration (0.07 and 0.7 nmol) on levels of Fos were examined within these two neuronal populations. Time spent awake, scored for the 90-min preceding perfusion, was largely comparable in diurnal spontaneous waking, nocturnal spontaneous waking and high-arousal waking. Nocturnal spontaneous waking and high-arousal waking, but not diurnal spontaneous waking, were associated with increased levels of Fos within hypocretin-synthesizing neurons, relative to diurnal sleeping. Within hypocretin-1 receptor-expressing neurons, only high-arousal waking was associated with increased levels of Fos. Hypocretin-1 administration dose-dependently increased levels of Fos within hypocretin-1 receptor-expressing neurons to levels comparable to, or exceeding, levels observed in high-arousal waking. Combined, these observations support the hypothesis that hypocretin neuronal activity varies across the circadian cycle. Additionally, these data suggest that waking per se may not be associated with increased hypocretin neurotransmission. In contrast, high-arousal states, including stress, appear to be associated with substantially higher rates of hypocretin neurotransmission. Finally, these studies provide further evidence indicating coordinated actions of hypocretin across a variety of arousal-related basal forebrain and brainstem regions in the behavioral state modulatory actions of this peptide system.

Corticotropin-releasing factor innervation of the locus coeruleus region: distribution of fibers and sources of input.

Electrophysiologic studies support the hypothesis that corticotropin-releasing factor, the neurohormone that initiates adrenocorticotropin release during stress, also serves as a neurotransmitter in the pontine noradrenergic nucleus, the locus coeruleus. To elucidate the circuitry underlying proposed corticotropin-releasing factor neurotransmission in the locus coeruleus, the present study utilized immunohistochemical techniques to characterize corticotropin-releasing factor innervation of rat locus coeruleus and pericoerulear regions. Corticotropin-releasing factor-like immunoreactive fibers were identified in the locus coeruleus of colchicine- and non-colchicine-treated rats. However, corticotropin-releasing factor innervation of pericoerulear regions rostral and lateral to the locus coeruleus was more dense than that of the locus coeruleus proper. Double-labeling studies utilizing antisera directed against corticotropin-releasing factor and tyrosine hydroxylase indicated that corticotropin-releasing factor-like immunoreactive fibers overlap with tyrosine hydroxylase-like immunoreactive processes of locus coeruleus neurons, particularly in rostral medial and lateral regions. A group of corticotropin-releasing factor-like immunoreactive neurons was localized just lateral to the locus coeruleus and numerous corticotropin-releasing factor-like immunoreactive neurons were visualized just ventral to the rostral pole of the locus coeruleus in a region corresponding to Barrington's nucleus. None of these corticotropin-releasing factor-like immunoreactive neurons were tyrosine hydroxylase-positive. To determine the source of corticotropin-releasing factor-like immunoreactive fibers in the locus coeruleus, injections of the retrograde tracer [wheat germ agglutinin conjugated to inactivated (apo) horseradish peroxidase coupled to gold particles] were made into the locus coeruleus and sections were processed for corticotropin-releasing factor-like immunoreactivity.(ABSTRACT TRUNCATED AT 250 WORDS)