Urocortin expression in the Edinger-Westphal nucleus is down-regulated in transgenic mice over-expressing neuronal corticotropin-releasing factor

In recent years a large body of evidence has emerged linking chronic stress with increased vulnerability for depression and anxiety disorders. As corticotropin-releasing factor (CRF) is hypersecreted under these psychological conditions, we used our CRF-overexpressing (CRF-OE) mouse line to study underlying brain mechanisms possibly causing these disorders. Urocortin (Ucn), a recently discovered member of the CRF peptide family may play a role in the pathophysiology of stress-induced disorders. Stressors recruit Ucn-immunoreactive neurons in the Edinger-Westphal nucleus (E-WN), which is the major site of Ucn expression. Furthermore, E-WN Ucn mRNA levels are upregulated in CRF-deficient mice. Based on these findings, we hypothesized the down-regulation of E-WN Ucn in CRF-OE mice and consequently, altered responsiveness to stressful stimuli. Our results support this hypothesis as we found weaker immunohistochemical labeling with anti-Ucn and a six times weaker Ucn mRNA signal in E-WN in CRF-OE mice. Moreover, E-WN Ucn-expressing neurons mounted a response to acute challenge in CRF-OE mice too. From these results it is concluded that the CRF and E-WN Ucn neuronal systems work in concert in response to acute challenges, but are inversely regulated in their activities during chronic hyperactivity of the hypothalamo-pituitary-adrenal axis.     

The Edinger-Westphal nucleus of the juvenile rat contains transient- and repetitive-firing neurons

Classically, the Edinger-Westphal nucleus is described as containing neurons controlling accommodation and pupillary constriction via projections to the ciliary ganglion. However, in several species including rat, some Edinger-Westphal neurons have ascending or descending CNS projections suggesting that the Edinger-Westphal nucleus might also have non-ocular functions. To further characterize the function of this nucleus we studied the electrophysiological properties of Edinger-Westphal neurons in a slice preparation from juvenile rats. The position of the Edinger-Westphal nucleus was determined using an immunohistochemical procedure directed at the peptide Urocortin, which is expressed in Edinger-Westphal neurons. Passive and active membrane responses were investigated and two different neuron types were identified. One type had a transient firing response to 400 ms depolarizing current pulses and one type had a repetitive firing response. Transient-firing neurons had an outward rectifying response inhibiting firing, possibly due to slowly inactivating I(D)-like potassium channels since low concentrations (200 microM) of the potassium channel blocker 4-aminopyridine elicited repetitive firing. In all neurons, low threshold Ca(2+) spikes were seen and these were blocked by nickel(II) chloride hexahydrate, suggesting that they are mediated via low voltage-activated Ca(2+) channels. Some biocytin-labeled neurons had axons or axonal collaterals projecting laterally or dorsally, suggesting possible non-ocular targets. In conclusion, the rat Edinger-Westphal nucleus contains two separate types of neurons with distinct electrophysiological properties.     

Cerebellar cortical and nuclear afferents from the Edinger-Westphal nucleus in the cat

Summary The cerebellar projection from the Edinger-Westphal nucleus was studied in the cat by means of retrograde transport of the wheat germ agglutinin-horseradish peroxidase complex. The present findings give evidence that the flocculus is the main terminal area. However, small tracer implants and injections into various parts of the cerebellar nuclei and cortex revealed projections also to the fastigial and interposed nuclei and to most parts of the anterior and posterior lobe cortices. The projecting neurons are small and located bilaterally throughout the Edinger-Westphal nucleus. No topical differences between the projections to different parts of the cerebellum were found.     

The reciprocal connections of endomorphin 1- and endomorphin 2-containing neurons between the hypothalamus and nucleus tractus solitarii in the rat

In the CNS, endomorphin 1- and endomorphin 2-immunoreactive neuronal cell bodies have been principally found both in the hypothalamus and nucleus tractus solitarii. Functionally, the hypothalamus and nucleus tractus solitarii are closely related in many aspects, especially in visceral functions. On the other hand, there are also many endomorphin-immunoreactive fibers and terminals in the two regions. In the present study, to investigate whether endomorphin 1-immunoreactive and endomorphin 2-immunoreactive neurons in the hypothalamus and nucleus tractus solitarii project reciprocally between these two regions, fluorescent retrograde labeling combined with immunofluorescence histochemical staining for endomorphin 1 and endomorphin 2 was used. After injection of Fluoro-Gold into the nucleus tractus solitarii of rats, endomorphin 1/Fluoro-Gold or endomorphin 2/Fluoro-Gold double-labeled neuronal cell bodies were predominantly observed in the arcuate nucleus of the hypothalamus, a few of which were also observed in the posterior hypothalamic area and periventricular hypothalamic nucleus. After injection of Fluoro-Gold into the medial zone of hypothalamic tuberal region and the lateral hypothalamic area, respectively, endomorphin 1/Fluoro-Gold or endomorphin 2/Fluoro-Gold double-labeled neuronal cell bodies were found chiefly in the medial, commissural, lateral and gelatinous parts of the nucleus tractus solitarii. These results provide morphological evidence that there exist reciprocal endomorphinergic connections between the hypothalamus and nucleus tractus solitarii.     

Edinger-Westphal neurons that project to spinal cord contain substance P

Combined retrograde transport and immunocytochemical methods were used to determine whether Edinger-Westphal neurons projecting to spinal cord also demonstrate substance P-like immunoreactivity (SPLI). Large injections of horseradish peroxidase (HRP) into cervical and lumbar enlargements retrogradely labeled cells throughout the length of the Edinger-Westphal complex (EW). Nearly all HRP-labeled EW neurons also stained for SPLI, evidence that EW is the origin of a direct substance P pathway linking rostral mesencephalon with spinal cord.     

Eye movement-related responses of neurons in human subthalamic nucleus

Intraoperative microelectrode single unit recordings are routinely made in the subthalamic nucleus (STN) of awake and alert Parkinsons disease (PD) patients during surgery for implantation of deep brain stimulation (DBS) electrodes. These recordings not only assist in determining the optimal target for electrode implantation, but also offer the unique opportunity to study movement-evoked responses from the basal ganglia. We report on the responses of human STN neurons to eye movements from eight PD patients (five men and three women). Twenty percent (18/89) of tested STN neurons showed responses to eye movements. Patients made pro-saccades, voluntary saccades or smooth pursuit eye movements in four directions: up, down, left, right. The majority of STN neurons (72% or 13/18), that responded to eye movements were found in the ventral half of the nucleus, while 58% (22/38) of STN neurons that had somatic responses were found in the dorsal half of the nucleus. The firing rate for STN oculomotor neurons was 33±15 Hz (n=18), which was not different from that reported previously for STN neurons. Most neurons only responded to eye movements in a single direction, but 17% (3/18) showed responses to more than one direction. The majority of responses (17/18) to eye movements were increases in firing rate although one neuron did show a pause in firing with eye movement onset. The phasic changes in firing rate in response to eye movement usually occurred up to 250 ms following eye movement onset. Neurons were found that showed task-specific responses to cued versus self-paced saccades, responded to both passive limb movement and voluntary eye movement, and appeared to show either visual or attentional responses. These human physiological data, in conjunction with previous anatomical studies, suggest that the STN might have an oculomotor role. Although there is no evidence that STN is responsible for driving eye movements, it may have a role in either sensory feedback, corollary discharge, or in focusing the substantia nigra pars reticulata to allow a saccade to occur through disinhibition of the superior colliculus.     

Immunohistochemical localization of AMPA-type glutamate receptor subunits in the nucleus of the Edinger-Westphal in embryonic chick

The ischemic damage in the hippocampal CA1 region following transient forebrain ischemia, delayed neuronal death, is a typical apoptotic response, but the underlying mechanisms are not fully understood. We have reported that mild hyperthermia (38 °C) accelerates DNA fragmentation of the gerbil CA1 pyramidal neurons following transient forebrain ischemia. Recently, we reported that galectin-3, a β-galactosidase-binding lectin, is spatio-temporally expressed only by activated microglial cells located within CA1 region following transient forebrain ischemia in gerbils. Furthermore, expression of galectin-3 and Iba-1 (a specific microglial cell marker) are strongly reduced by hypothermia during ischemic insult. To further elucidate the effect of hyperthermia on the expression of galectin-3 by micloglia in delayed neuronal death, we examined immunohistochemical expression of galectin-3 and Iba-1, in situ terminal dUTP-biotin nick end labeling of DNA fragmentation (for determination of cell death) and hematoxylin and eosin staining (for morphological observation). We observed that between 37 °C and 39 °C, there was a temperature-dependent enhancement of galectin-3 expression in microglial cells in the CA1 region following transient ischemia. Apoptotic DNA fragmentation, detected by TUNEL staining, was observed in CA1 region in normothermia. This TUNEL staining was enhanced by hyperthermia at 37.5 °C and 38 °C, but not at 39 °C. Ischemia-induced neuronal degeneration in CA1 region in gerbil hippocampus subjected to hyperthermia (37.5 °C, 38 °C and 39 °C) observed by HE staining is similar to that in normothermic gerbils. These findings imply that galectin-3 expression in microglia may influence the survival of CA1 pyramidal neurons in cases such as hyperthermia-related neuronal injury.     

Immunohistochemical localization of AMPA-type glutamate receptor subunits in the nucleus of the Edinger-Westphal in embryonic chick

The Edinger-Westphal nucleus (EW) in birds is responsible for the control of pupil constriction, accommodation, and choroidal blood flow. The activation of EW neurons is mediated by the neurotransmitter glutamate, in large part through AMPA-type glutamate receptors (GluRs), whose behavior varies according to the subunit composition. We investigated the developmental expression of the GluR subunits in EW of the chick (Gallus gallus) using immunohistochemistry on tissue from embryonic days 10 through 20 (E10–E20). Of the three antibodies used, one recognized the GluR1 subunit, another the GluR4 subunit, and the third recognized a sequence common to GluR2 and GluR3 subunits. No immunolabeling of EW neurons for any GluR subunits was observed prior to E12, although immunolabeling was seen in somatic oculomotor prior to E12. At E12, immunoreactivity for each of the three antibodies was in only approximately 2% of EW neurons. By E14, the abundance of GluR1+ perikarya in EW had increased to 13%, and for GluR2/3 had increased to 48%. The perikaryal abundance of the immunoreactivity for GluR1 and GluR2/3 declined to 3% and 23%, respectively, by E16. At E14, 33% of EW neurons immunolabeled for GluR4, and their frequency increased to 43% by E16, and remained at that approximate percentage through hatching. The increased expression of GluR1 and GluR4 in EW at E14 coincides with the reported onset of the expression of the calcium-binding protein parvalbumin, and the calcium currents associated with AMPA receptors formed by these two subunits may play a role in the occurrence of parvalbumin expression.     

Characterization of gabaergic neurons within the human medial mamillary nucleus

The morphology, distribution and relative frequency of GABAergic neurons in the medial mamillary nucleus (MMN) of normal human individuals was studied using a glutamic acid decarboxylase (GAD) antiserum. GAD-immunoreactive (GAD-IR) neurons were found sparsely distributed throughout the MMN and most displayed a simple bipolar morphology. A small population of large diameter GAD-IR neurons was found in the white matter capsule adjacent to the ventral border of the MMN. Results of double-labeling experiments revealed no evidence of calretinin, parvalbumin or calbindin immunoreactivities co-localizing with GAD-IR neurons. GAD-IR neurons of the MMN had an average somal area of 138+/-41 microm2, compared with the average somal area of 384+/-137 microm2 for the population of MMN neurons as a whole. GAD-IR neurons had a tendency to cluster in groups of two (and occasionally three) and showed a distribution gradient across the MMN with higher densities being found near the insertion of the fornix, the origin of the mamillo-thalamic tract and toward the medial MMN border. Quantitative estimates of GAD-IR neuron frequency revealed the GAD-IR phenotype to constitute an average of 1.7% percent of the total neuron population within the human MMN. These findings suggest that inhibitory activity within the human MMN is regulated in part by a small population of intrinsic GABAergic interneurons.     

Peptide 19-containing neurons in the medullary dorsal horn, subnuclei interpolaris and oralis, and nucleus principalis of the rat

Peptide 19 (PEP 19) is a 7.6 kDa polypeptide which can bind to calmodulin and inhibit calcium-calmodulin signaling. In this study, PEP 19-immunoreactivity (ir) was examined in the rat trigeminal sensory nuclei. Numerous PEP 19-immunoreactive (ir) neurons were detected in the medullary dorsal horn (MDH) and rostral parts of the trigeminal sensory nuclei (subnuclei interpolaris and oralis, and nucleus principalis). The mean numbers ± S.D. per section of PEP 19-ir neurons were 104.2 ± 30.4 in the MDH, 137.8 ± 39.5 in the subnucleus interpolaris, 129.2 ± 46.9 in the subnucleus oralis and 157.2 ± 34.1 in the nucleus principalis. In the MDH, small to medium-sized PEP 19-ir neurons were abundant within superficial laminae. PEP 19-ir neurons with various cell body sizes were also distributed in the rostral parts of the trigeminal sensory nuclei. A double immunofluorescence analysis also demonstrated that many PEP 19-ir neurons co-expressed parvalbumin (PV)-ir in the MDH (9.0%), subnucleus oralis (7.7%) and nucleus principalis (19.7%). In the subnucleus interpolaris, such neurons were relatively rare (1.7%). PEP 19-ir neurons were mostly devoid of calbindin D-28k. In addition, a retrograde tracing method revealed that a substantial number of PEP 19-ir neurons projected to the thalamus. PV-ir was common in thalamus-projecting PEP 19-ir neurons. These findings suggest that PEP 19-ir neurons in the MDH may have a function in modulation of nociceptive and thermo-receptive signaling. It is also likely that PEP 19-ir neurons in rostral parts of the trigeminal sensory nuclei are related to transduction of mechano-receptive information from facial regions to the thalamus.     

Edinger-Westphal nucleus: cells that project to spinal cord contain corticotropin-releasing factor

Combined retrograde transport and immunocytochemical methods were used to determine whether neurons in the Edinger-Westphal complex (EW) that project to the spinal cord also demonstrate corticotropin-releasing factor-like immunoreactivity (CRF-LI). Large injections of horseradish peroxidase (HRP) into cervical spinal cord retrogradely labeled cells throughout the extent of the EW complex. Most retrogradely labeled EW neurons also exhibited CRF-LI, evidence that EW is the origin of a direct CRF-containing pathway which links the rostral mesencephalon with spinal cord.     

A double-label study of efferent projections from the Edinger-Westphal nucleus in goldfish and kelp bass

The Edinger-Westphal nucleus in goldfish was identified by retrograde labeling from the ciliary ganglion. In the same animals a few neurons near this nucleus (perinuclear Edinger-Westphal neurons) were labeled by a different retrograde tracer injected into the cerebellum. No double-labeled cells were found. Similar results were obtained in kelp bass, except that in this species no cerebellar-projecting perinuclear neurons were observed. Cerebellar-projecting Edinger-Westphal neurons have previously been described in some mammals, but not in other vertebrates. Therefore the homology of cerebellar-projecting cells of the Edinger-Westphal region in mammals and teleost fishes is doubtful.     

Morphology of the neurons in the human lateral geniculate nucleus and their normal development

Summary Neurons in the adult human lateral geniculate nucleus have been classified using Golgi preparations. The neuronal classes correspond to those previously described in monkey (Saini and Garey 1981). The commonest are multipolar neurons with either radiate or tufted dendritic trees. Also seen frequently are bipolar neurons with two or three diametrically opposed dendrites. Rarer classes include neurons with beaded dendrites and those with axon-like dendritic processes, perhaps inter-neurons. Neurons are also found in the circumgenicule capsule. Most neurons have dendrites restricted to the laminae, but some dendrites cross the borders of both magno- and parvocellular laminae. Somata are also seen in interlaminar zones with dendrites reaching the adjacent laminae. No significant difference, apart from size, is found between neurons in magno- and parvocellular laminae.Most neuronal types are found at birth. They are, however, strikingly different from their adult forms in having growth cones and filopodia and an abundance of dendritic and somatic spines and hair-like processes. Morphological maturity is reached by about nine months postnatally. Similar maturational changes occur in monkey in the first two months of life (Garey and Saini 1981) and in both monkey and man this period of maturation of the lateral geniculate nucleus corresponds to increasing visual acuity and a time when each species is most likely to be affected by visual deprivation.Supported by the Swiss National Science Foundation (3.612.80)The work described in this paper forms part of a study for a doctoral dissertation in the University of Lausanne by C. de Courten     

Urocortin 1 distribution in mouse brain is strain-dependent

Urocortin 1 has been implicated in a number of specific behaviors, which include energy balance, stress reactivity and ethanol consumption. To elucidate genetically influenced differences in the mouse urocortin 1 system, we performed immunohistochemical characterization of urocortin 1 distribution in C57BL/6J and DBA/2J mouse brain. Urocortin 1 analysis reveals strain-dependent differences in distribution of urocortin 1 immunoreactive neurons and neuronal fibers. In both strains, the highest number of urocortin 1-positive neurons was observed in the Edinger-Westphal nucleus and lateral superior olive. Urocortin 1-positive neurons were detected in the dorsal nucleus of the lateral lemniscus of DBA/2J mice, but were absent in the C57BL/6J strain. Differences in urocortin 1 fibers were detected in many areas throughout the brain, and were most apparent in the septal areas, thalamic areas, several midbrain regions, and medulla. Strain-dependent distribution of urocortin 1-containing cells and fibers suggests that differences in this neuropeptide system may underlie differences in behavior and physiological responses between these strains. Further, we found that in both mouse strains, urocortin 1 in the Edinger-Westphal nucleus and choline acetyltransferase are not coexpressed. We show that the urocortin 1-positive neurons of this brain area form a separate population of cells that we propose to be called the non-preganglionic Edinger-Westphal nucleus.     

Neurotensin expressing neurons developed earlier than vasoactive intestinal polypeptide and vasopressin expressing neurons in the human suprachiasmatic nucleus

Development of neurotensin (NT), vasoactive intestinal polypeptide (VIP), vasopressin (AVP) and neuropeptide-Y (NPY) expressing neurons was investigated in the human fetal suprachiasmatic nucleus of nine subjects ranging from 20-40 weeks of gestation using immunocytochemistry and morphometry. Results obtained showed that NT expressing neurons developed earlier than VIP, AVP and NPY expressing neurons. Consistent with results obtained from animal studies, we also found VIP expressing neurons were born earlier than AVP expressing neurons. Whether the NT expressing neurons play a role in generating circadian rhythms in the early life of humans needs to be further investigated.     

Urocortin and the brain

Urocortin is a member of the corticotropin-releasing hormone (CRH) family of peptides. In the brain, its potent suppression of food intake is mediated by CRH receptors (CRHR). Urocortin also participates in the regulation of anxiety, learning, memory, and body temperature, and it shows neuroprotection. This review will summarize the location of urocortin-producing neurons and their projections, the pharmacological evidence of its actions in the CNS, and information acquired from knockout mice. Urocortin interacts with leptin, neuropeptide Y, orexin, and corticotropin in the brain. Also produced by the GI tract, heart, and immune cells, urocortin has blood concentrations ranging from 13 to 152 pg/ml. Blood-borne urocortin stimulates the cerebral endothelial cells composing the blood-brain barrier and crosses the blood-brain barrier by a unique transport system. Overall, urocortin acts on a broad neuronal substrate as a neuromodulator important for basic survival.     

Influence of aging on the seasonal rhythm of the vasopressin-expressing neurons in the human suprachiasmatic nucleus

The mammalian suprachiasmatic nucleus (SCN) is considered to be a major component of the biological clock implicated in the temporal organization of a variety of physiological endocrine and behavioral processes. There is now a great deal of evidence indicating that many of these rhythms are progressively disturbed during senescence. The present study was aimed at investigating the influence of aging on the seasonal rhythm of the vasopressin (VP)-expressing neurons in the human SCN. To that end brains obtained at autopsy of 48 human subjects ranging in age from 6 to 91 years were studied. Subjects were divided into two age groups viz. “young subjects” (up to 50 years) and “elderly subjects” (over 50 years). It is shown that the number of VP-immunoreactive neurons in the human SCN exhibits a marked annual oscillation in young but not in elderly people. Whereas in young subjects low VP-immunoreactive neuron numbers were found during the summer (May-July) and peak values in autumn (September-November) the SCN of elderly people showed a disrupted annual cycle with a reduced amplitude. These data suggest that the biosynthesis of vasopressin in the human SCN exhibits a seasonal rhythm that becomes disturbed later in life.