Edinger-Westphal Neurons Research Articles

Edinger-Westphal peptidergic neurons enable maternal preparatory nesting.

SummaryOptimizing reproductive fitness in mammalians requires behavioral adaptations during pregnancy. Maternal preparatory nesting is an essential behavior for the survival of the upcoming litter. Brain-wide immediate early gene mapping in mice evoked by nesting sequences revealed that phases of nest construction strongly activate peptidergic neurons of the Edinger-Westphal nucleus in pregnant mice. Genetic ablation, bidirectional neuromodulation, and in vitro and in vivo activity recordings demonstrated that these neurons are essential to modulate arousal before sleep to promote nesting specifically. We show that these neurons enable the behavioral effects of progesterone on preparatory nesting by modulating a broad network of downstream targets. Our study deciphers the role of midbrain CART+ neurons in behavioral adaptations during pregnancy vital for reproductive fitness.

CRF family peptides are differently altered by acute restraint stress and chronic unpredictable stress

Corticotropin-releasing factor (CRF) acts to promote stress-like physiological and behavioral responses and is mainly expressed in the paraventricular hypothalamic nucleus (PVN). Urocortin 1 (Ucn1) is also a ligand to CRF type 1 and 2 receptors that has been associated with the stress response. Ucn1 neurons are primarily found in the Edinger–Westphal (EW) nucleus. It has been previously proposed that CRF and Ucn1 differently modulate stress responses to distinct types of stressors. The present study used male Wistar rats to compare the effects of acute restraint stress and unpredictable chronic stress (UCS) through Fos-immunoreactivity (Fos-ir) on CRF-containing neurons of PVN and Ucn1-containing EW centrally projecting neurons. Results showed that PVN neurons responded to both acute restraint and UCS. Also for the PVN, unspecific variables, dependent on the time animals remained in the laboratory, do not seem to alter Fos-ir, since no significant differences between acute and chronic control groups were found. On the other hand, EW neurons were only activated in response to acute restraint stress. Also, for this nucleus a significant difference was found between acute and chronic control groups, suggesting that unspecific variables, dependent on the time animals remain in the laboratory, interfere with the nucleus activation. These results suggest that CRF/Ucn1 neuronal circuits encompass two interconnected systems, which are coordinated to respond to acute stressors, but are differentially activated during chronic unpredictable stress.

Distinct glutaminyl cyclase expression in Edinger–Westphal nucleus, locus coeruleus and nucleus basalis Meynert contributes to pGlu-Aβ pathology in Alzheimer’s disease

Glutaminyl cyclase (QC) was discovered recently as the enzyme catalyzing the pyroglutamate (pGlu or pE) modification of N-terminally truncated Alzheimer’s disease (AD) Aβ peptides in vivo. This modification confers resistance to proteolysis, rapid aggregation and neurotoxicity and can be prevented by QC inhibitors in vitro and in vivo, as shown in transgenic animal models. However, in mouse brain QC is only expressed by a relatively low proportion of neurons in most neocortical and hippocampal subregions. Here, we demonstrate that QC is highly abundant in subcortical brain nuclei severely affected in AD. In particular, QC is expressed by virtually all urocortin-1-positive, but not by cholinergic neurons of the Edinger–Westphal nucleus, by noradrenergic locus coeruleus and by cholinergic nucleus basalis magnocellularis neurons in mouse brain. In human brain, QC is expressed by both, urocortin-1 and cholinergic Edinger–Westphal neurons and by locus coeruleus and nucleus basalis Meynert neurons. In brains from AD patients, these neuronal populations displayed intraneuronal pE-Aβ immunoreactivity and morphological signs of degeneration as well as extracellular pE-Aβ deposits. Adjacent AD brain structures lacking QC expression and brains from control subjects were devoid of such aggregates. This is the first demonstration of QC expression and pE-Aβ formation in subcortical brain regions affected in AD. Our results may explain the high vulnerability of defined subcortical neuronal populations and their central target areas in AD as a consequence of QC expression and pE-Aβ formation.

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 ID-like potassium channels since low concentrations (200 μM) of the potassium channel blocker 4-aminopyridine elicited repetitive firing. In all neurons, low threshold Ca2+ spikes were seen and these were blocked by nickel(II) chloride hexahydrate, suggesting that they are mediated via low voltage–activated Ca2+ 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.

Alcohol-induced c-Fos expression in the Edinger-Westphal nucleus: pharmacological and signal transduction mechanisms.

Mapping inducible transcription factors has shown that the Edinger-Westphal nucleus is preferentially sensitive to alcohol intoxication. Herein, we characterize the pharmacological and signal transduction mechanisms related to alcohol-induced c-Fos expression in Edinger-Westphal neurons. Using immunohistochemistry, we show that pretreatment with gamma-aminobutyric acid (GABA)-ergic antagonists (4 mg/kg bicuculline and 45 mg/kg pentylenetetrazole) attenuates induction of c-Fos expression by alcohol (2.4 g/kg, intraperitoneal). In addition, 10 mg/kg 2-(2,3-dihydro-2-methoxy-1,4-benzodioxin-2-yl)4,5-dihydro-1H-imidazole (RX 821002), an alpha(2A/D)-adrenoceptor antagonist, and 20 mg/kg haloperidol, a dopamine antagonist, also block alcohol-induced c-Fos expression in Edinger-Westphal neurons. No effects were seen in alcohol-induced c-Fos after the pretreatment of 20 mg/kg propranolol (beta-adrenoceptor antagonist), 10 mg/kg 2-(2-(4-(2-methoxyphenyl)piperazin-1-yl) ethy)-4,4-dimethyl-1,3-(2H,4H)-isoquinolindione dihydrochloride (ARC 239) (alpha(2B/C)-adrenoceptor antagonist), or 30 mg/kg naltrexone (opioid antagonist). Although positive modulators for the GABA(A) receptor (20 mg/kg 3alpha-hydroxy-5alpha-pregnan-20-one and 10-30 mg/kg chlordiazepoxide) and opioid receptor (10 mg/kg morphine) produced significant elevations, agonists for alpha(2)-adrenoceptors (clonidine) and dopamine receptors (apomorphine) had no effect on Edinger-Westphal c-Fos expression. These findings suggest that alcohol-induced c-Fos expression in Edinger-Westphal results from direct interactions with GABA(A) receptors, which are modified by alpha(2A/D)-adrenoceptors and dopamine receptors. Also using immunohistochemistry to identify potential intracellular mechanisms associated with alcohol-induced c-Fos expression in Edinger-Westphal, we show time-dependent increases in serine 727 phospho-signal transducer and activator of transcription 3 (Stat3) but no changes in phospho-cAMP response element-binding protein and phospho-Elk1. Time-dependent increases in phospho-extracellular signal-regulated kinase (ERK) 1/2 were found to occur simultaneously with increases in serine 727 phospho-Stat3. Finally, blockade of ERK 1/2 phosphorylation with the mitogen-activated protein kinase (MEK) 1/2 inhibitor SL327 blocked alcohol-induced c-Fos expression, suggesting that alcohol induces c-Fos in Edinger-Westphal neurons through activation of the MEK1/2-ERK1/2-Stat3 pathway.

Subcortical neural circuits for ocular accommodation and vergence in primates.

Our current knowledge of the neural bases of vergence and accommodation has increased significantly over the past few years. The behavior of medial rectus motoneurons during vergence, which has been reported by a number of investigators, is described. The behavior of Edinger-Westphal neurons during accommodation is also described, as are the characteristics of midbrain near-response neurons in the supraoculomotor area. Evidence that some of these near-response neurons provide the vergence input to medial rectus motoneurons and possibly the accommodation input to Edinger-Westphal neurons is reviewed. Anatomical studies have shown that the midbrain near-response region receives input from two deep cerebellar nuclei, the posterior interposed and the fastigial nucleus. Single-unit recording in the posterior interposed nucleus has revealed cells that increase their activity during the far-response, and the behavior of these neurons is reviewed. In addition, studies of a precerebellar nucleus, the nucleus reticularis tegmenti pontis, have revealed some cells that increase their activity during the near-response and others that do so during the far-response. The behavior of these neurons is reviewed. This review documents the great strides that are occurring in our understanding of the anatomy and physiology of the neural pathways controlling vergence and accommodation in the primate.

Transneuronal retinal input to the primate Edinger-Westphal nucleus.

The Edinger-Westphal nucleus of the oculomotor nuclear complex provides preganglionic parasympathetic innervation to the pupil. We labelled its retinal input by transneuronal autoradiography after an eye injection of [3H]proline in the macaque monkey. The primary retinal projection to the pretectum terminated in the ipsilateral and contralateral olivary nuclei. These nuclei were intensely labelled and sharply delimited, with a mean diameter of 590 microm and a rostrocaudal length of 2.52 mm. The caudal half of the olivary nucleus on each side broke into multiple clumps of label. Fragments of label also surrounded each olivary nucleus. The exact pattern of pretectal labelling varied considerably among animals and even from side to side in the same animal. In 5 of 6 monkeys, label from the olivary nucleus reached the Edinger-Westphal nucleus transneuronally. In transverse sections, the Edinger-Westphal label appeared as a circular patch located on either side of the midbrain ventral to the cerebral aqueduct in the central gray matter. It averaged 230 microm in diameter and 610 microm in length. In Nissl-stained sections, the autoradiographic label corresponded to a distinct nucleus comprised of neurons that were smaller than neurons in nearby somatic subdivisions of the oculomotor complex. The mean area of Edinger-Westphal neurons was 295 microm2. Transneuronal retinal input to the Edinger-Westphal nucleus mediating pupillary constriction terminates in a single, well-defined cell group in the midbrain.

Plasma membrane calcium ATPase in synaptic terminals of chick Edinger-Westphal neurons

The plasma membrane calcium ATPase pump (PMCA) is one of two major mechanisms known to be involved in extruding calcium from cells. The monoclonal antibody 5F10 was used to examine the distribution of PMCA in chick Edinger-Westphal neurons, a population of cholinergic preganglionic neurons whose cells bodies reside in the Edinger-Westphal nucleus in the brainstem and whose axons form synaptic terminals on parasympathetic neurons in the ciliary ganglion. Definitive PMCA immunoreactivity was undetectable in Edinger-Westphal cell bodies in the brainstem. In contrast, immunoreactivity for PMCA was robust in ciliary ganglia and resembled patterns of immunoreactivity for the synaptic vesicle antigen SV-2, suggesting that PMCA is expressed in Edinger-Westphal synaptic terminals. Moreover, PMCA immunoreactivity co-localized with immunoreactivity for enkephalin and substance P, two neuropeptides known to be expressed in Edinger-Westphal synaptic terminals. Fine structure studies revealed that PMCA immunoreactivity is associated with synaptic vesicles rather than the plasma membrane in Edinger-Westphal terminals. In immunodot assays, synaptic vesicles purified fromTorpedo electric organ are also immunoreactive for PMCA as well as SV-2.Torpedo vesicles are negative for the sarcoplasmic/endoplasmic reticulum ATPase, suggesting that the observed PMCA immunoreactivity is not associated with smooth endoplasmic reticulum. Immunoblot analysis confirmed that 51710 recognizes a protein with the correct molecular mass for PMCA in tissue homogenates of chick cerebellum, chick ciliary ganglia, andTorpedo synaptic vesicles. These findings describe a previously unrecognized location for PMCA in the membranes of cholinergic synaptic vesicles. Relevance to previous data and possible functions are discussed.

Nicotinic acetylcholine receptors in separate brain regions exhibit different affinities for methyllycaconitine.

The family of nicotinic acetylcholine receptors contains numerous subtypes. Since the subunit compositions of most native neuronal nicotinic receptors are unknown, an important method for distinguishing subtypes of functional neuronal receptors is based on pharmacological criteria, such as affinity for snake toxins. We have now examined the affinities of native chick nicotinic receptors for methyllycaconitine, a toxin purified from Delphinium. We find that methyllycaconitine is a potent antagonist at central nicotinic receptors located on Edinger-Westphal neurons, producing nearly complete functional blockade of nicotinic responses at 10 nM. In marked contrast, methyllycaconitine is 1000-fold less potent at blocking nicotinic responses in the lateral spiriform nucleus. Methyllycaconitine inhibits kappa-bungarotoxin-sensitive nicotinic receptors in ciliary ganglia at 0.5-1.0 microM. Radioligand binding studies also reveal heterogeneity in the affinity of the toxin for nicotinic receptors. Methyllycaconitine binds most avidly to [125I] alpha-bungarotoxin sites in brain (Ki = 5.4 nM), and is 200-fold less potent at muscle nicotinic receptors (IC50 = 1.1 microM). The least potent binding of the toxin is to [3H]nicotine sites in brain (Ki = 3.7 microM). Methyllycaconitine is thus a useful pharmacological tool for distinguishing certain subtypes of native nicotinic receptors. The relatively low affinity of the toxin for nicotinic receptors in the lateral spiriform nucleus is consistent with the known properties of these receptors, which include a high affinity for [3H]nicotine and a lack of sensitivity to alpha- and kappa-bungarotoxin. On the basis of high affinity for methyllycaconitine and insensitivity to alpha-bungarotoxin, the nicotinic receptors in the Edinger-Westphal nucleus are unlike any previously described nicotinic receptor subtype.

Agrin mRNA expression in the developing chick Edinger-Westphal nucleus.

Agrin is a large extracellular matrix protein that directs the accumulation of acetylcholine receptors at the neuromuscular junction. Recent evidence suggests that agrin may be involved in organizing synapses in the visual system as well. Focussing on the pathway that controls accommodation and pupilloconstriction, this study examined the temporal pattern of agrin expression with reference to the organization of cholinergic synapses between embryonic chick Edinger-Westphal and ciliary ganglion neurons. In situ hybridization with an S35-labeled agrin cRNA probe was used to characterize agrin expression in the Edinger-Westphal nucleus during development. Agrin mRNA was detected in the Edinger-Westphal nucleus at all time points studied, from embryonic day 7 (E7, Hamburger and Hamilton stage 31) through newly hatched chicks. Throughout this period, agrin mRNA expression in Edinger-Westphal neurons was lower than in nearby oculomotor and trochlear neurons, suggesting that cells projecting to neuronal targets may require less agrin than those projecting to muscle. Agrin mRNA expression in the Edinger-Westphal nucleus at E7, E8, E9, and E10 was significantly higher than at E12. The early appearance of agrin mRNA coincides with the period during which acetylcholine receptors are being organized on ciliary ganglion neurons, consistent with the possibility that agrin contributes to neuron-neuron synapse formation in this pathway.

Behavior of identified Edinger-Westphal neurons during ocular accommodation

1. The present study used single-unit recording and antidromic activation techniques in alert rhesus monkeys to examine the static and dynamic behavior of 21 parasympathetic, preganglionic neurons of the Edinger-Westphal nucleus (EW) during ocular accommodation. 2. All identified EW neurons were active when viewing at optical infinity with an average firing rate of 11.6 spikes/s. During near viewing, there was a linear relationship between firing rate and accommodation with an overall gain for the population of preganglionic EW neurons of 3.3 (spikes/s)/diopter. 3. The activity of eight identified EW neurons was studied during viewing of targets with conflicting vergence and accommodative demands to dissociate their vergence and accommodation responses. With normal viewing these responses are so closely matched that it cannot be determined if the activity of a cell is related to vergence or to accommodation, but with dissociated viewing these relationships can be determined. Under this viewing condition, six preganglionic EW neurons showed the same relationship to accommodation as they did during normal viewing. However, the activity of two cells could not be explained solely by accommodation, and they showed some activity related to vergence. 4. Microstimulation at the sites of identified EW neurons produced accommodation in the ipsilateral eye. Repeated measures of the effect of microstimulation yielded a value of 75 ms for the latency of the response. This latency was essentially the same in both animals tested. 5. The activity of identified EW neurons is related to the velocity of accommodation as well as to static accommodation. The relationship between accommodation velocity and firing rate was studied for 15 identified EW neurons during sine-wave tracking of targets moving in depth. All of these cells showed a clear relationship between firing rate and accommodation velocity. Overall, this group of identified EW neurons showed a velocity sensitivity of 1.2 (spikes/s)/(diopter/s) and an estimated neural time constant of 380 ms. 6. Eleven neurons encountered near to preganglionic EW neurons could not be antidromically activated by stimulation of the oculomotor nerve. These neurons had statistically higher gains with respect to the near response; indeed, there was no overlap between the gains of these neurons and the gains of preganglionic EW neurons. Upon dissociation of vergence from accommodation, they were found to be related to either vergence or to vergence and accommodation but not solely to accommodation.

Multiple subtypes of voltage-gated calcium currents in the Edinger-Westphal nucleus

Avian Edinger-Westphal (EW) neurons provide a unique opportunity to compare electrophysiologically the membranes of cell bodies and terminals in the same population of neurons. Axons that originate from neurons in the lateral region of the EW nucleus form a morphologically distinct presynaptic terminal, known as a calyx, on ciliary ganglion neurons [32]. Several studies have shown that calyciform terminals in the ciliary ganglion exhibit predominantly N-type, high-voltage-activated (HVA) calcium channels [35–37,45]. The goal of this study was to characterize and compare the calcium currents expressed in EW cell somas with those reported in the terminals. Whole-cell patch-clamp techniques were used to record from cell bodies in the lateral EW nucleus in slice preparations. Slices were obtained from embryonic day 16 chicks, matching the age of the embryos in which calyces were studied. Recordings were localized to the lateral region of the EW nucleus using Lucifer yellow fills. Voltage-step commands from −70 to 0 mV produced calcium currents with both a sustained and an inactivating component. Depolarization steps to 0 mV from a holding potential of −40 mV eliminated the inactivating component. These recordings suggested the presence of both LVA and HVA calcium currents.Application of 0.1 mM NiCl2 produced a reversible decrease in the amplitude of the whole-cell calcium current, preferentially affecting the inactivating component. The Ni2+ -sensitive current activated and inactivated rapidly in a voltage-dependent manner. Treatment with 0.1 mM cadmium chloride caused a reversible reduction in the amplitude of the calcium current. ω-Conotoxin (CTX) (0.002 mM) produced an irreversible decrease in the amplitude of the calcium current. An unidentified component of the calcium current, which was Ni2+- and CTX-insenive, constituted ≈10–15% of the current. Nifedipine (0.010 mM) had no detectable effects on the calcium currents. These results suggest that, in contrast to their terminals, cell bodies in the lateral region of the EW nucleus express T-type, LVA as well as N-type, HVA calcium channels.

Synaptic Vesicle Proteins and Target-Dependent Morphogenesis of Calyx-like Contacts in Vitro

Chick Edinger-Westphal neurons form large, calyciform synaptic terminals, also known as calyces, on a subpopulation of ciliary ganglion neurons in vivo. This distinctive morphology is mimicked by contacts formed between Edinger-Westphal and ciliary ganglion neurons in culture. The work reported here finds that calyx-like contacts in culture are immunoreactive for the two vesicle proteins SV-2 and synaptotagmin, supporting the hypothesis that these contacts are synaptic terminals developing in vitro. When Edinger-Westphal neurons are cultured with ciliary ganglion neurons, calyx-like contacts are three times more frequent than when Edinger-Westphal neurons are cultured inappropriately with sympathetic neurons. Furthermore, calyx-like contacts are rare in cultures of ciliary ganglion neurons alone, even though ciliary ganglion neurons are known to functionally innervate themselves in culture (11). These results suggest that calyx morphogenesis is favored when Edinger-Westphal neurons interact specifically with ciliary ganglion neurons and discouraged when they interact with inappropriate neurons. That this specificity is expressed in culture, in the absence of normal tissue topography and patterned activity, adds to the evidence that direct surface interactions between target cells and growing neurites are involved in the morphogenesis of synaptic terminals in vivo.

The peripheral and central projections of the Edinger-Westphal nucleus in the rat. A light and electron microscopic tracing study

The peripheral and central efferent projections of the rostral part of the Edinger-Westphal nucleus in the rat were investigated at the light and electron microscopic level by means of iontophoretic injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin and retrograde tracer injections of Fast blue and Nuclear yellow into the facial nucleus and into the principal olive. Two pathways leaving the rostral part of the Edinger-Westphal nucleus were studied, a peripheral and a central descending pathway. Fluorescent experiments demonstrated that the central pathway fibers originated from distinct individual Edinger-Westphal neurons. These neurons were mainly distributed throughout the rostral part of the Edinger-Westphal nucleus and had fusiform cell bodies. The neurons rarely form collateral projections. The central descending pathway left the Edinger-Westphal nucleus medially and terminated bilaterally in the principal olive, in the subnuclei A, B and C of the inferior olive and ipsilaterally in the medial accessory olive. The central pathway also terminated contralaterally in the lateral parabrachial nucleus, the facial nucleus, the trigeminal brainstem nuclear complex, the lateral reticular nucleus and the rostroventral reticular nucleus. The projection to the facial nucleus provides evidence for the existence of a polysynaptic loop forming the central part of the corneal blink reflex. Projections from the Edinger-Westphal nucleus to the cerebellar cortex or the deep nuclei, as described in cat and primate, could not be confirmed. The peripheral pathway left the Edinger-Westphal nucleus ventrally and terminated on dendrites of ciliary ganglion cells, along smooth muscle cells of ciliary ganglion associated arterioles and in the proximity of ciliary ganglion associated venules. The central and peripheral terminals that originate in the Edinger-Westphal nucleus all had similar ultrastructural features: clear, round vesicles and electron dense mitochondria. The terminals originating from the central descending pathway were often found to be arranged in glomerular-like structures. The central and peripheral terminals made asymmetric synaptic membrane specializations (Gray type one), except terminals innervating the ciliary ganglion associated vessels, which showed no synaptic contacts.

Different responses to opioids measured in terminals and somas of Edinger-Westphal neurons

Neurotransmitter receptors on the axon terminals of a neuron can be located a considerable distance away from comparable receptors on the cell body or dendrites of the same neuron. We examined the effects of activating either nerve terminal receptors or those located on or near the somas of chick Edinger-Westphal neurons. Cell body responses were measured via intracellular recording in a brain slice preparation. To measure nerve terminal responses, intracellular recordings were obtained from the large, calyciform nerve endings in intact ciliary ganglia, which emanate from neurons of the lateral Edinger-Westphal nucleus. Cell bodies of Edinger-Westphal neurons responded to leucine-enkephalin with a dose-dependent hyperpolarization that was associated with a decrease in input resistance. In spontaneously active Edinger-Westphal somas, leucine-enkephalin caused marked inhibition of suprathreshold and subthreshold activity, indicating that, as with a number of other central neurons, the major effect of opioids was to reduce excitability. The response to opioids was sensitive to naloxone (1 μM) and was a direct effect, since it was not blocked by either 0.5 μM tetrodotoxin or 100 μM cadmium. More selective mu ([ d-Ala 2, N-Me-Phe 4, Gly 5-ol]-enkephalin) and delta ([ d-Ser 2]-leucine-enkephalin-Thr and [ d-Pen 2,5]-enkephalin) opioid agonists produced effects similar to those of leucine-enkephalin. Opioids produced strikingly different effects in the nerve terminals of Edinger-Westphal neurons, where the major effect was a depolarization associated with a decrease in input resistance. The effects of opioids in the terminals were reduced in a low sodium buffer, indicating that they were dependent on the presence of extracellular sodium. In contrast, altering extracellular potassium or calcium had little effect on the response. The actions of the tachykinin peptide substance P were also examined in both preparations. Substance P elicited similar responses in both terminals and cell bodies of Edinger-Westphal neurons. These responses consisted of a membrane depolarization associated with little or no change in input resistance and increased cell excitability. The present results indicate that in at least one type of vertebrate neuron, opioid receptors exert different actions depending upon their localization within the neuron. Since opioid receptors are dependent on coupling to G-proteins to elicit effects on membrane-spanning ion channels, it thus appears that neurotransmitter receptors can be functionally coupled to different ion channels within the same neuron.

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.

Preganglionic neurons from the Edinger-Westphal nucleus: Growth and histochemical characterization in cell culture

The Edinger-Westphal (EW) nucleus, also known as the accessory oculomotor nucleus in the chick, provides the cholinergic preganglionic input to the parasympathetic ciliary ganglion. In addition to acetylcholine, many EW neurons have been shown to contain enkephalin-like and/or substance P-like immunoreactivity. Establishment of EW neurons in culture would make possible study of their interactions with ciliary ganglion neurons in vitro and in addition would provide a valuable system for studying cholinergic/peptidergic neurons of the vertebrate central nervous system. We describe here dissociated cell cultures established from midbrain tissue containing the EW nucleus. In these cultures, 86% of the cells with neuronal morphology were positive for intracellular acetylcholinesterase activity, 54% were positive for enkephalin-like immunoreactivity, and 4% were positive for substance P-like immunoreactivity. The proportions of neurons that scored as labeled were even higher if the number of positive cells was compared to the number of cells in sister cultures immunoreactive for the large neurofilament protein polypeptide. When the cultures were stained simultaneously for acetylcholinesterase activity and enkephalin-like immunoreactivity, 34% of the cells with neuronal morphology were positive for both. In cultures derived from adjacent tissue regions very few cells expressed both activities. These results suggest that the cells expressing both acetylcholinesterase activity and enkephalin-like immunoreactivity in culture are EW neurons. The putative EW neurons survive for weeks in vitro in the absence of their normal target, the ciliary ganglion.

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.

Edinger-Westphal nucleus: Projections to the brain stem and spinal cord in the cat

The efferent connections of the Edinger-Westphal (EW) nucleus of the cat have been examined using the autoradiographic anterograde axonal transport technique. Following injections of [ 3H]amino acids into the EW nucleus, fibers could be traced from this region to a number of sites in the caudal brain stem and spinal cord heretofore not known to receive an afferent input from this nucleus. Two descending pathways have been identified. One pathway travels in the medialmost aspect to the medial longitudinal bundle and terminates in the dorsal accessory olive. The other pathway leaves the nucleus laterally, coursing through the medial tegmentum, and then shifts to a ventrolateral position in the rostral rhombencephalon. Some fibers of this lateral pathway curve dorsally and terminate in the medial parabrachial nucleus. The remainder of this fiber system lies ventral to the spinal trigeminal complex, with some axons terminating in the subtrigeminal nucleus, while other fibers continue ventral to the caudal part of the spinal trigeminal nucleus and appear to terminate in the marginal layer and ventromedial part of this nucleus. Another component of this system terminates between the gracile and medial cuneate nuclei. The main pathways from the EW nucleus to the spinal cord include (1) some fibers which course through the dorsal column nuclei into the ventromedial part of the dorsal columns, and (2) other fibers which continue caudally immediately along the ventrolateral aspect of the spinal trigeminal nucleus and then proceed to the spinal cord in the region between the dorsal horn and the lateral cervical nucleus. These EW fibers appear to terminate mainly in Rexed's lamina I (marginal layer); other fibers, from both tracts appear to terminate in lamina V. There was no evidence for any ascending projections from the EW nucleus. Confirmatory data were obtained from a series of horseradish peroxidase (HRP) experiments, in which EW neurons were retrogradely labeled following injections into the dorsal column nuclei, spinal trigeminal nucleus, inferior olivary nucleus, or spinal cord. These results clearly indicate that the traditional view of the EW nucleus as merely a parasympathetic preganglionic nucleus should be seriously questioned.