Acute pain increases phosphorylation of DCLK-long in the Edinger-Westphal nucleus but not in the hypothalamic paraventricular nucleus of the rat.

UNLABELLED: The doublecortin-like kinase (DCLK) gene is crucially involved in neuronal plasticity and microtubule-guided retrograde transport of signaling molecules. We have explored the possibility that DCLK is involved in pain-induced signaling events in adult male Wistar rats. Our results show that both DCLK-short and DCLK-long splice variants are present in the cell body and proximal dendrites of neurons in stress-related nuclei, ie, the paraventricular nucleus of the hypothalamus (PVN) and the non-preganglionic Edinger-Westphal nucleus (npEW) in the rostroventral periaqueductal grey. We found that DCLK-long but not DCLK-short is phosphorylated in its serine/proline-rich domain. Furthermore, we demonstrate that phosphorylation of DCLK-long in the npEW is increased by acute pain, whereas DCLK-long phosphorylation in the PVN remains unaffected. This is the first report revealing that DCLK isoforms in the PVN and npEW occur in the adult mammalian brain and that pain differentially affects DCLK-long-mediated neuronal plasticity in these 2 stress-sensitive brain centers. PERSPECTIVE: Pain is a burden for society and the individual, and although the mechanisms underlying pain are relatively well known, its treatment remains difficult and incomplete. Pain stress can lead to diseases like chronic pain and depression. The differential DCLK-phosphorylation in stress-sensitive brain areas is a potential novel therapeutic target in pain research.

Immunohistochemical determination of the sympathetic pathway in the orbit via the cranial nerves in humans.

OBJECT: The present study was undertaken to elucidate the extent and precise distribution of the postganglionic sympathetic fibers in the cranial nerves projecting to the orbit and to reconstruct sympathetic routes in the orbit in humans. For this purpose, the authors made an immunohistochemical determination of the sympathetic fibers by using an antibody against norepinephrine-synthetic enzyme, tyrosine hydroxylase (TH). METHODS: Specimens containing the orbit and the cavernous sinus were obtained from formalin-fixed human cadavers. First, it was confirmed that the superior cervical ganglion contained strongly immunostained TH-positive neuronal cell bodies and fibers. After careful dissection of the cranial nerves projecting to the orbit, different segments of each cranial nerve were processed for immunohistochemical analysis for TH. All of the intraorbital cranial nerves contained TH-positive sympathetic fibers, although the amounts were very different in each cranial nerve. At the proximal site of the common tendinous ring, TH-positive fibers were found mainly in the abducent and trochlear nerves. At the distal site of this ring, TH-positive fibers were lost or markedly reduced in number in the abducent and trochlear nerves and were distributed mostly in the ophthalmic and oculomotor nerves. Among the cranial nerves projecting to the orbit, the ophthalmic nerve and its bifurcated nerves--frontal, lacrimal, and nasociliary--contained numerous TH-positive fibers. CONCLUSIONS: The authors conclude that the postganglionic sympathetic fibers are distributed to all cranial nerves projecting to the orbit and that the ophthalmic nerve provides a major sympathetic route in the orbital cavity in humans.

Preganglionic endings from nucleus of Edinger-Westphal in pigeon ciliary ganglion contain neuronal nitric oxide synthase.

The avian ciliary ganglion (CG) controls choroidal blood flow by its choroidal neurons, and pupil constriction and accommodation by its ciliary neurons. It was previously reported that both choroidal and ciliary neurons label positively for NADPH diaphorase (NADPHd), a marker for nitric oxide synthase (NOS). To assess if this labeling is preganglionic or postganglionic and to determine if it is attributable to neuronal NOS (nNOS), we studied pigeon CG using NADPHd histochemistry and nNOS immunohistochemistry (IHC). Short-duration staining times by NADPHd histochemistry yielded intense labeling of structures that appeared to be the cap-like endings on ciliary neurons and the boutonal endings on choroidal neurons that arise from the nucleus of Edinger-Westphal (EW), and light or no postganglionic perikaryal staining. The light postganglionic staining that was observed tended to be localized to ciliary neurons. Consistent with this, NADPHd+ nerve fibers were observed in the postganglionic ciliary nerves but rarely in the postganglionic choroidal nerves. These same staining times yielded robust staining of neurons in the orbital pterygopalatine microganglia network, which are known to be nNOS+. Diffuse staining of CG perikarya was observed with longer staining durations, and this staining tended to mask the preganglionic labeling. Preganglionic NADPHd+ staining in CG with short staining times was blocked by the NOS inhibitors iodonium diphenyl (IDP) and dichlorophenol-indophenol (DPIP), but the diffuse postganglionic staining observed with the longer staining times was not completely blocked. Labeling of CG sections for substance P (SP) by IHC (which labels EW-originating preganglionic endings in CG) and subsequently for NADPHd confirmed that NADPHd was localized to preganglionic endings on CG neurons. Immunohistochemical double labeling for nNOS and SP or enkephalin further confirmed that nNOS is found in boutonal and cap-like endings in the CG. Two studies were then carried out to demonstrate that the nNOS+ preganglionic endings in CG arise from EW. First, NADPHd+ and nNOS+ neurons were observed in EW in pigeons treated with colchicine to enhance perikaryal labeling. Second, NADPHd+ and nNOS+ preganglionic endings were eliminated from CG ipsilateral to an EW lesion. These various results indicate that NOS is present in EW-arising preganglionic endings on choroidal and ciliary neurons in avian CG. NOS also appears to be found in some ciliary neurons, but its presence in choroidal neurons is currently uncertain.

Expression of a serine protease (motopsin PRSS12) mRNA in the mouse brain: in situ hybridization histochemical study.

Serine proteases are considered to play several important roles in the brain. In an attempt to find novel brain-specific serine proteases (BSSPs), motopsin (PRSS-12) was cloned from a mouse brain cDNA library by polymerase chain reaction (PCR). Northern blot analysis demonstrated that the postnatal 10-day mouse brain contained the most amount of motopsin mRNA. At this developmental stage, in situ hybridization histochemistry showed that motopsin mRNA was specifically expressed in the following regions: cerebral cortical layers II/III, V and VIb, endopiriform cortex and the limbic system, particularly in the CA1 region of the hippocampal formation. In addition, in the brainstem, the oculomotor nucleus, trochlear nucleus, mecencephalic and motor nuclei of trigeminal nerve (N), abducens nucleus, facial nucleus, nucleus of the raphe pontis, dorsoral motor nucleus of vagal N, hypoglossal nucleus and ambiguus nucleus showed motopsin mRNA expression. Expression was also found in the anterior horn of the spinal cord. The above findings strongly suggest that neurons in almost all motor nuclei, particularly in the brainstem and spinal cord, express motopsin mRNA, and that motopsin seems to have a close relation to the functional role of efferent neurons.

Organization of choline acetyltransferase-containing structures in the cranial nerve motor nuclei and spinal cord of the monkey.

Cholinergic structures in the cranial nerve motor nuclei and ventral and lateral horns of the spinal cord of the monkey, Macaca fuscata, were investigated immunohistochemically with a monoclonal antibody against monkey choline acetyltransferase (ChAT). ChAT-immunoreactive perikarya and dendrites were present in the oculomotor, trochlear, abducent, trigeminal motor, facial and hypoglossal nuclei, nucleus of Edinger-Westphal, nucleus ambiguus, dorsal nucleus of the vagus, lamina IX of the cervical, thoracic and lumbar spinal cords, and intermediolateral nucleus of the thoracic spinal cord. The neuropil of the trigeminal motor, facial and hypoglossal nuclei, nucleus ambiguus and lamina IX of the cervical, thoracic and lumbar spinal cords contained many ChAT-positive bouton-like structures and they were seemingly in contact with perikarya and dendrites of motoneurons, suggesting that motoneurons in these nuclei are cholinoceptive as well as cholinergic. The oculomotor, trochlear and abducent nuclei, nucleus of Edinger-Westphal, dorsal nucleus of the vagus and intermediolateral nucleus of the thoracic spinal cord contained a small number of ChAT-immunoreactive bouton-like structures, but they did not contact with perikarya and dendrites of ChAT-positive neurons. These observations suggest that the organization of the motor nuclei is complex, at least regarding the cholinoceptivity.

An immunohistochemical study on manganese superoxide dismutase in Parkinson's disease.

We report an immunohistochemical study on manganese superoxide dismutase (Mn SOD) in Parkinson's disease (PD) patients and age-matched control subjects. Overall appearance of immunostaining intensity of nigral neurons did not differ significantly between the PD patients and the control subjects. However, when the immunostaining intensity of each neuron was semiquantitatively analyzed, both very intensely stained (more than normal) neurons as well as neurons stained only weakly were more frequently detected in the lateral part than in the medial and the central parts of the substantia nigra in PD patients. As a result, the proportion of normally stained neurons was significantly smaller in the lateral part of the substantia nigra in PD patients; however, the overall distribution of the neurons among the three rating grades for immunostaining did not differ significantly. The immunostaining intensity of the neuropils in the medial and the central part of the substantia nigra tended to be more intense in PD patients than in the control subjects. Our results suggest up-regulation of Mn SOD mainly in the dendritic processes of the less involved nigral neurons.

Altered gravity affects succinate dehydrogenase reactivity in specific nuclei of fish brain.

The effect of long-term (10 days) altered gravitational conditions upon succinate dehydrogenase (SDH) reactivity in total brain as well as in individual brain nuclei of developing cichlid fish larvae has been investigated by means of semiquantitative histochemical methods (densitometric grey value analysis). Increasing acceleration from near weightlessness (spaceflight) via 1 g controls to 3 g hypergravity (centrifuge) resulted in slightly increased total brain SDH reactivity. When focusing on distinct neuronal integration centres within the same brains in order to find the anatomical substratum of the gross histochemical data, significant effects of altered gravity on vestibulum-related brain parts were obtained. The total brain results may therefore represent the sum of such particular indirect effects but may also comprise in addition a non vestibular-related general and therefore direct influence of altered gravitational conditions, possibly on all cells.

Influence of visual deprivation on the visual and oculomotor system: acetylcholinesterase activity in oculomotor neurons.

The effects of visual deprivation (dark-rearing) on neurons in both the visual and oculomotor systems of black mice were studied using morphologic and histochemical techniques. In the neurons of the dorsal nucleus of the lateral geniculate body, the cytoplasm of dark-reared mice was less developed and the cytoplasm/nucleus ratio was significantly smaller in the dark-reared mice than in the controls. In contrast, large motoneurons in the oculomotor nucleus did not show any ultrastructural changes and the cytoplasm/nucleus ratio was normal. However, in large motoneurons of dark-reared mice, acetylcholinesterase (AChE)-positive areas were scattered in the cytoplasm, and the ratio of the AChE-positive areas to the cytoplasm area was significantly smaller than that in the controls.

Hypertrophy of motor neurons in the oculomotor nucleus of the rat following removal of the contralateral extraocular muscles.

Motor neurons of the oculomotor nucleus of the rat were identified immunohistochemically using a monoclonal antibody against choline acetyltransferase (ChAT). The number and size of the cell bodies were examined following removal of the extraocular muscles on one side. 35 days postoperatively, motor neurons of the oculomotor nucleus ipsilateral to the muscle removal are undiminished in number and are of normal size when compared with littermate control animals. Cholinergic cells in the contralateral nucleus are significantly larger than normal (+23%). This hypertrophy appears to persist at least until 300 days after operation, the longest survival time examined.

Co-localization of acetylcholinesterase and choline acetyltransferase in the rat cerebrum.

Acetylcholinesterase-histochemistry has been widely used for localizing cholinergic neurons despite specificity problems. The distribution of cells stained with this method has never been directly compared on a histochemical level with the specific cholinergic marker, choline acetyltransferase. We recently reported the immunohistochemical localization of choline acetyltransferase using monoclonal antibodies [Levey A. I., Armstrong D., Atweh S. F., Terry R. D. & Wainer B. H. (1983) J. Neurosci 3, 1-9]. Here we report the development of a combined histochemical and immunohistochemical method for the co-localization of the 2 cholinergic markers, and their comparison in the rat cerebrum. Although the precise relationship between the markers was complex, the important results were: (1) all neurons which contained choline acetyltransferase also contained some acetylcholinesterase; (2) many acetylcholinesterase-containing neurons did not contain any demonstrable choline acetyltransferase; (3) all neurons which stained intensely for acetylcholinesterase in the neostriatum and basal forebrain also contained choline acetyltransferase; and (4) many choline acetyltransferase-containing neurons did not stain intensely for acetylcholinesterase. The results corroborate the assumption that choline acetyltransferase is a more specific marker for cholinergic neurons than acetylcholinesterase. Intense staining for acetylcholinesterase can be reliably used in some regions of the cerebrum for identifying cholinergic neurons, however, it should be recognized that this criterion s not essential for all cholinergic neurons.

[Enzyme histochemical investigations of the nucleus nervi oculomotorii of Salmo irideus (Gibbons 1855) during biomorphosis. I. Transmitter enzymes]. / Enzymhistochemische Untersuchungen am Nucleus nervi oculomotorii von Salmo irideus (Gibbons 1855) in der Biomorphose.

1. The activities of the transmitter enzymes acetylcholine esterase (AChE), monoamine oxidase (MAO) and gamma-aminobutyrate transaminase (GABA-TR) were investigated in the nucleus nervi oculomotorii of Salmo irideus (Teleostei) during biomorphosis. 2. AChE as well as MAO-activity could be demonstrated already before the time of hatching, whereas GABA-TR occured from the 10th day post hatching. 3. In earliest stages the AChE activity was found in the neuronal pericarya of the nucleus n. III, but later on a strong increase occured in vestibular presynaptic terminals establishing synaptic contacts with the lateral surface of the large pericarya, especially in the medial part of the dorsal subnucleus and the ventral subnucleus. In adults the enzyme activity was localized in vestibular nerve fiber terminals exclusively due to the loss of enzyme activity in the pericarya. 4. The activity of MAO was found surrounding the neurons of the nucleus nervi oculomotorii with stronger deposits of reaction product at the medial surface that is not occupied by cholinergic terminals. 5. Besides non reacting neurons in all parts of the nucleus some cells in the transition zone between the medial and lateral area of the dorsal subnucleus, exhibited high cytoplasmatic GABA-TR activity. 6. The different ways of chemical transmission between nerve terminals and the neurons of the nucleus nervi oculomotorii are discussed.

Topographical distribution of acetylcholinesterase and butyrylcholinesterase in the diencephalon and mesencephalon of the garden lizard (Calotes versicolor).

The present report incorporates the histochemical mapping of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) among the various nuclei and fiber tracts of the diencephalon and mesencephalon of Calotes veriscolor. The various nuclei, for both enzymes, present varying degrees of staining, ranging from negative nuclei, on the one hand, to mild and intense staining on the other hand. Almost all of the fiber tracts reveal intense activity in BChE preparations, while they demonstrate mild and moderate activities for AChE. The nature of the various nuclei in relation to enzymatic patterns is discussed.

Trigeminal representations of the masticatory and extraocular proprioceptors as revealed by horseradish peroxidase retrograde transport.

Horseradish peroxidase has been injected in the masticatory and extraocular muscles in newborn and adult cats to identify the cells of origin for the muscle endings. Labeled motoneurons in the nuclei of the III, IV, V and VI nerves have been observed. They are the parent cells of the motor terminals taking up the enzyme in the muscle injected. Labeled ganglionic cells have been found scattered all along the ipsilateral mesencephalic nucleus of the V nerve after injection of both the jaw closing and the jaw opening muscles. Labeled cells have also been found in the ipsilateral caudal part of the same nucleus after injection of the extraocular muscles. These results are interpreted as due to enzyme uptake by the sensory endings of the muscle studied. Moreover cell bodies in the semilunar ganglion were found marked for both groups of muscles injected showing a second ganglionic representation for the sensory endings.