Expression of substance P, neurokinin 1 receptors (NK1) and neurokinin 3 receptors in the developing mouse retina and in the retina of NK1 knockout mice.

To complete a series of studies on the expression of substance P and neurokinin receptors in mammalian retinas, we investigated the occurrence of these molecules in developing mouse retinas and in retinas of mice with genetic deletion of the neurokinin 1 receptor, the preferred substance P receptor. Using semi-quantitative reverse transcription-polymerase chain reaction, we measured detectable levels of the gamma isoform of preprotachykinin A (a substance P precursor) mRNA at postnatal day 4. Neurokinin 1 receptor and neurokinin 3 receptor mRNAs were also detected at postnatal day 4. While gamma preprotachykinin A and neurokinin 1 receptor mRNA levels significantly increased up to eye opening (postnatal day 11), neurokinin 3 receptor mRNA levels remained constant throughout development. Substance P, neurokinin 1 receptor and neurokinin 3 receptor immunoreactivities were present at postnatal day 5. Substance P was in amacrine cells, neurokinin 1 receptor in developing amacrine and bipolar cells and neurokinin 3 receptor in OFF-type cone bipolar cells. Interestingly, a transient increase in the density of neurokinin 1 receptor immunoreactive processes was observed at eye opening in lamina 3 of the inner plexiform layer, suggesting a role of substance P and neurokinin 1 receptor in this developmental phase. However, in neurokinin 1 receptor knockout retinas, besides a significant increase of the gamma preprotachykinin A mRNA levels, no major changes were detected: neurokinin 3 receptor mRNA levels as well as substance P and neurokinin 3 receptor immunostainings were similar to wild types. Together with previous studies, these observations indicate that there are major differences in neurokinin 1 receptor expression patterns among developing mammalian retinas. The observations in neurokinin 1 receptor knockout mice may not be applicable to rats or rabbits, and substance P and neurokinin 1 receptor may play different developmental roles in different species.

Expression of the neurokinin 1 receptor in the mouse retina.

Previous studies have revealed that the expression pattern of the neurokinin 1 receptor (the preferred receptor for substance P, SP) varies in different mammalian retinas. We investigated NK1 receptor expression in the mouse retina to provide background information for future studies in transgenic mice on SP functional roles in the retina. Mouse retinal sections were treated for single and double-label immunofluorescence. NK1 receptor immunoreactivity was in bipolar cells and in numerous amacrine cells. Double-label studies showed that NK1 receptor-expressing bipolar cells constituted a population of ON-type cone bipolar cells, since they were distinct from rod bipolar cells and contained glycine. They were nonrandomly distributed with highest density in central retina. These cells were similar and may correspond to the population of NK1 receptor-expressing bipolar cells of the rabbit retina. Different subsets of NK1 receptor-expressing amacrine cells were identified on the basis of the expression of selected neurotransmitter substances: i) about 23% of NK1 receptor-expressing amacrine cells also contained glycine; ii) the remaining 77% were likely to be GABAergic, although some inconsistency was observed in the GABA immunostaining obtained with two different GABA antibodies; iii) all dopaminergic amacrine cells also expressed NK1 receptors; iv) about one third of SP-containing amacrine cells also expressed NK1 receptors. These findings confirm and expand previous observations in rat and rabbit retinas. In particular, common to all three species is the expression of NK1 receptors in dopaminergic amacrine cells, indicating that SP neurotransmission may be a universal feature of the circuitry of the dopaminergic amacrine cell. Peculiar to the mouse retina is the presence of putative NK1 autoreceptors expressed by SP-containing amacrine cells.

Localization of S-100 protein in Müller cells of the retina--2. Electron microscopical immunocytochemistry.

The cellular and subcellular distribution of S-100 protein was investigated at the ultrastructural level in the rat retina by the immunocytochemical PAP method. S-100 appeared to be localized in the cytoplasm and nucleus of Müller cells, offering conclusive evidence that in the mammalian retina, the protein is confined to glial cells. S-100, as a marker for Müller cells, may be a useful tool in order to study the cytoarchitecture of the retina in normal as well as in pathologic conditions. In addition, the retina may represent a suitable model for further investigation on the biologic role of S-100.

Appearance of new alpha-bungarotoxin-acetylcholine receptors in cultured sympathetic ganglia of chick embryos.

alpha-Bungarotoxin (alpha-BuTX) has been used as a marker for studying the production of alpha-bungarotoxin-acetylcholine receptors (alpha-BuTX-AChRs) in explants of chick embryo sympathetic ganglia cultured in vitro. New alpha-BuTX-AChRs appear rapidly in the explants after blocking of the pre-existent ones with the toxin (40% of the total receptors at 3 h). There is a portion of alpha-BuTX-AChRs in the explants which for a short time is not accessible to the toxin. This portion constitutes the precursor pool of receptors and represents 18% of the total. The precursor pool of receptors supplies the neurons with new receptors for 1-2 h in the absence of protein synthesis. The appearance of new receptors from the precursor pool is an energy-dependent process.

Developmental distributive pattern of acetylcholinesterase in chick embryo ciliary ganglion.

AChE cytochemistry was performed in the chick ciliary ganglion (CG) during various embryonic stages. AChE first appeared in the RER of the neurons at 5 days of incubation (d.i.). Synaptic AChE appeared only later, parallely to the appearance of the calyciform synapses, i.e. at 9 d.i. At first AChE was mainly localized at the calyx side facing the satellite cell, thereafter extending to the neuronal side and especially labeling synaptic contacts occurring at points along the presynaptic membrane. Finally, at 15 d.i., i.e. when the calyx is morphologically mature, AChE reaction labels the whole contour of the calyciform nerve terminal. At 10 d.i., limited AChE-positive extrasynaptic areas of the ciliary neurons surface first appeared, thereafter extending up to affect at 15 d.i. large neuronal surfaces. Some hypotheses can be drawn from our results: (i) the earliest appearance of cytoplasmic AChE seems somehow independent of the establishing of functional synaptic contacts; (ii) the pattern of development of neuronal AChE suggests the existence of a sort of transynaptic control by presynaptic nerve terminals. However, it is possible that concomitant retrograde iris-dependent influences on ganglionic AChE concur in modulating neuronal AChE; (iii) the functional role possibly played by AChE localized at extra-synaptic level still remains to be clarified.

Studies on embryonic transplants to the transected spinal cord of adult rats.

Spinal cord tissue was obtained from 13- and 14-day embryonic rats and homologously grafted to the completely transected spinal cord of adult rats. Eight and 12 weeks after grafting, clinical, electrophysiological, histological, and neuroanatomical studies were performed. Motor performance of the hosts was assessed by the inclined-plane test. The conduction of nerve impulses across the lesion-transplantation site was evaluated by recording the spinal corticomotor and somatosensory evoked potentials. The survival, growth, differentiation, and parenchymal integration of the graft were documented histologically on semi-thin sections. The axonal interactions between the host spinal cord and the graft as well as the posttraumatic retrograde degeneration of corticospinal axons were investigated using the horseradish peroxidase (HRP) technique. Clinical and electrophysiological assessments did not demonstrate any functional activity of the graft. On histological examination, grafted neurons showed a survival rate of 55%. Such neurons exhibited a limited degree of growth and differentiation. The extent of parenchymal integration between the host spinal cord and the graft varied considerably among different specimens and in the various regions of every specimen. The HRP investigations demonstrated that some axonal interactions between the host spinal cord and the graft had occurred. Regenerated axons arising from both the spinal cord and the dorsal root ganglia of the host entered the graft and elongated in it. Also, axons from the grafted neurons were able to grow for some distance in the host spinal cord. The phenomenon of the posttraumatic retrograde degeneration of corticospinal axons was not affected by this embryonic tissue grafting.

Experimental repair of the oculomotor nerve: the anatomical paradigms of functional regeneration.

In adult guinea pigs, the oculomotor nerve was sectioned proximally (at the tentorial edge) or more distally (at the orbital fissure) and immediately repaired by reapproximation. During a 24-week postoperative period, extrinsic eye motility was assessed by analyzing the vestibulo-ocular reflexes. The regenerated oculomotor nerve was studied morphometrically on semi-thin histological sections at 16 and 24 weeks postinjury. The selectivity of muscle reinnervation was investigated by injection of both single (horseradish peroxidase) and double (fluorescent dyes) retrograde axonal tracers into the eye muscles. Following proximal repair of the oculomotor nerve, the degree of recovery of extraocular motility varied among different animals and remained stable over long-term observations. In animals with poor recovery, aberrant eye movements were always found, and the somatotopic map of the reinnervated eye muscles was greatly altered. Distortions of the central representation were also seen in those animals in which a good level of functional recovery was seen. However, in animals with good recovery, a topographic bias was re-established by about 65% of the original neuronal population, as opposed to 26% in the animals with poor recovery. Neurons located contralateral to the axotomized nucleus sprouted intra-axially and projected their axons to denervated eye muscles. The number and diameter of the regenerated axons, the number and soma diameter of the axotomized neurons, and the ratio of distal axonal branches to proximal supporting neurons were all related to the degree of functional recovery. Following repair of the oculomotor nerve at the orbital fissure, extraocular motility had recovered in all of the animals at 16 weeks without aberrant phenomena. Functional regeneration of the distally transected oculomotor nerve is thought to be the result of selective muscle reinnervation.

Superior cervical ganglion regenerating axons through peripheral nerve grafts and reversal of behavioral deficits in hemiparkinsonian rats.

The superior cervical ganglion (SCG) has been grafted to the brain of adult rats in an attempt to reverse the parkinsonian syndrome that follows destruction of central dopamine systems. However, the main limitation to this approach is the massive cell death that occurs in the grafted SCG after direct transplantation into the brain. In adult rats, 6-hydroxydopamine (6-OHDA) was stereotactically injected into the right substantia nigra (SN). One month later, dopamine denervation was assessed using the apomorphine-induced rotational test. In rats with a positive test, an autologous peripheral nerve (PN) graft was tunneled from the right cervical region to the ipsilateral parietal cortex. One end of PN graft was sutured to the transected postganglionic branch of the SCG and the other end was inserted into a surgically created cortical cavity. The apomorphine test was repeated at 3 days and again at 1, 3, and 5 months after surgery. The brain, SCG, and PN graft were studied under light and electron microscopy and with the tyrosine hydroxylase immunohistochemical and horseradish peroxidase tracing methods. Three days after grafting, there were no significant differences on the apomorphine test as compared to the preoperative test. Conversely, 1,3, and 5 months after grafting, the number of rotations was reduced by 69% (+/-20.2), 66.6% (+/-17.1), and 72.5% (+/-11.3), respectively. Control rats that received a free PN graft to the brain and underwent section of the postganglionic branch of the SCG did not show significant changes on the apomorphine test after surgery. Histological examination revealed that the PN graft was mostly reinnervated by amyelinic axons of small caliber. Approximately 40% of the SCG neuronal population that normally projects to the postganglionic branch survived axotomy and regenerated the transected axons into the PN graft. Axons arising from the SCG elongated the whole length of the graft, crossed the graft-brain interface and extended into brain regions adjacent to the denervated striatum up to 2037 micrometer from the graft insertion site. This work shows that the ingrowth of catecholamine-regenerating axons from the SCG to dopamine-depleted brain parenchyma significantly reduces behavioral abnormalities in hemiparkinsonian rats. This effect cannot be ascribed either to the brain cavitation or to the PN tissue placement in the brain.

Oculomotor nerve regeneration in rats. Functional, histological, and neuroanatomical studies.

To study oculomotor nerve regeneration in rats, the oculomotor nerve was approached microsurgically and was sectioned at the base of the skull. The nerve stumps were reapproximated and affixed with a plasma clot in Group I animals and were separated by a gap in Group II animals. Visceral eye motility was evaluated weekly between 1 day and 40 weeks after surgery by recording the pupillary diameter under standardized photic stimulation. Somatic eye motility was assessed after 26 weeks by measuring the ocular displacement evoked by vestibular stimulation in the horizontal and vertical planes. Nerve regeneration was documented histologically and morphometrically at 8, 16, and 40 weeks after section. The selectivity of axonal regeneration to the extraocular muscles was investigated after 26 weeks by mapping (with injection of retrograde horseradish peroxidase) the motoneurons that supplied each reinnervated muscle. Between 6 and 20 weeks after section, the pupil diameter showed a progressive reduction in Group I rats, and no changes were observed in Group II rats. Compared with normal rats, the amplitude of horizontal and vertical ocular displacements was decreased, respectively, by 30% and 45% in Group I and by 65% and 80% in Group II. In Group I rats, the vestibular stimulation in the horizontal plane evoked anomalous eye movements with vertical components. On histological examination, regenerated nerves showed a progressive increase of axonal diameter and myelin-sheath thickness. Reinnervated muscles were associated with a less specific, bilateral representation in the midbrain compared with normal muscles, which have unilateral representation. The changes of the somatotopic organization were interpreted as being the result of the misdirected regrowth of axons in the postlesional nerve stump and of the collateral sprouting in the midbrain.

The third nerve transection and regeneration in rats with preliminary results on the sixth nerve transection and regeneration in guinea pigs.

The relationship between the phenomenon of the nonselective reinnervation and the functional recovery after section and repair of the highly organized third cranial nerve motor system in rats was studied. The same relationship after section and repair of the more simply organized sixth cranial nerve motor system in guinea pigs is presented as preliminary results. Anatomical demonstration of nonselective reinnervation was obtained by injecting horseradish peroxidase (HRP) into the extraocular muscles. A bilateral reinnervation of previously ipsilateral innervated muscles both in the third and the sixth nerve was interpreted as a plastic response of the brain stem neurons to the nerve injury. Functional recovery, evaluated by measuring with an infrared light technique the horizontal and vertical vestibulo-ocular reflexes, was excellent for the rectus lateralis muscle while it was relatively poor i.e. partial for the muscles depending on the third nerve. These data suggest that one of the most important factors influencing the functional recovery after section and repair of a peripheral nerve is the complexity of the nerve motor system organization.

Effects of L-carnitine, L-acetylcarnitine and gangliosides on the regeneration of the transected sciatic nerve in rats.

Three pharmacological agents, L-carnitine, L-acetylcarnitine and gangliosides, were tested for their ability to enhance the regeneration of the rat sciatic nerve following transection and microsurgical repair. The drugs were administered intraperitoneally at the dose of 50 mg/kg/d for 28 and 56 d postoperatively. At the end of treatment, the motor function recovery of the peroneal component of the sciatic nerve was assessed and the regenerated nerves were analysed morphometrically on histological semi-thin sections. Also, the reinnervated extensor digitorum longus (EDL) muscles were studied histochemically using the adenosine-triphosphatase (ATP-ase) technique 56 d after surgery. Motor function assessment at 56 d after nerve repair revealed that L-acetylcarnitine-treated animals recovered a clinical grade significantly higher (p less than 0.05) than the control animals. Twenty-eight days after nerve repair, the number of myelinated fibres was significantly higher (p less than 0.05) in L-acetylcarnitine and ganglioside-treated animals than in control animals. However, 56 d after nerve repair the number of regenerated fibres in all the drug-treated groups was not significantly different from that of the control group. The EDL muscles of the drug-treated animals did not show significant differences from those of control animals with respect to fibre composition and fibre diameter although the L-acetylcarnitine-treated animals exhibited a significantly lower (p less than 0.05) degree of muscle atrophy than did the control animals. The results of the present work seem to indicate that L-acetylcarnitine and to a lesser extent gangliosides exert some favourable effect on the regeneration of the transected sciatic nerve in rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Levocarnitine acetyl prevents cell death following long-term section of the vagus nerve in rats.

Levocarnitine acetyl has previously been found to significantly prevent axotomy-induced cell death in the spinal cord motor nucleus 9 and 12 months after section of the sciatic nerve in rats. In the present paper, the effects of levocarnitine acetyl on axotomy-induced cell death in the brain stem motor nuclei 90 days after section of the vagus nerve were studied. The right vagus nerve was cut at the neck. To prevent regeneration, a 5 mm-long segment of the vagus nerve was excised and the distal stump was displaced caudally. After surgery, a group of rats (n = 6) was treated with levocarnitine acetyl dissolved in the drinking water (75 mg/kg/day) (Group I). A second group of operated rats (n = 4) received drinking water alone. (Group II). Ninety days postoperatively, in the rats of both groups the proximal nerve stump of the vagus nerve was injected with horseradish peroxidase to label retrogradely the brain stem motoneurons of the dorsal motor vagal and the ambiguus nuclei. The brain stem nuclei were also labelled by horseradish peroxidase in three unoperated control rats (Group III). In the Group II rats, the number of horseradish peroxidase-labelled motoneurons of the dorsal motor vagal nucleus was found to be significantly smaller than in either the Group I (p < 0.01) or the Group III (p < 0.02) animals. In the Group I rats, the number of motoneurons of the dorsal motor vagal nucleus was not significantly smaller compared to the Group III rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the degenerative and regenerative phenomena occurring after transection and repair of the sciatic nerve in rats: effects of acetyl-L-carnitine.

The effects of acetyl-L-carnitine on some degenerative and regenerative phenomena following sciatic nerve transection in rats, were studied. In Experiment 1, acetyl-L-carnitine was administered intraperitoneally at the dose of 50 mg/kg/day for 28 and 56 days following transection and microsurgical repair of the sciatic nerve. On day 56, the acetyl-L-carnitine-treated rats showed a significantly (p less than 0.05) better motor recovery ("clinical assessment") of the peroneal component of the sciatic nerve than the control rats. Twenty-eight days after nerve repair, the acetyl-L-carnitine-treated rats showed a significantly higher (p less than 0.05) number of myelinated axons in the postlesional nerve stump than control rats. Finally, the treated rats had a significantly lower (p less than 0.05) presence of atrophic fibres in the extensor digitorum longus muscle. In Experiment 2 the sciatic nerve was cut. To prevent spontaneous regeneration, a metallic clip was applied to the distal nerve stump and then the nerve stumps were positioned in different anatomical compartments. After surgery, a group of rats was treated with acetyl-L-carnitine dissolved in the drinking water (75 mg/kg/day). Another group of rats received normal water and served as the control group. Three, 6, 9, 12 and 18 months postoperatively, in the rats of both groups, the proximal sciatic nerve stump was injected with horseradish peroxidase to label the spinal cord neurons of the sciatic nerve nucleus. While in untreated rats the number of horseradish peroxidase-labelled neurons decreased with the increase in denervation time, in acetyl-L-carnitine-treated rats the number of horseradish peroxidase-labelled neurons remained stable for as long as 12 months of denervation and decreased only after 18 months of denervation. Furthermore, acetyl-L-carnitine-treated rats showed a significantly higher (p less than 0.05) number of horseradish peroxidase-labelled neurons with respect to untreated rats both after 9 and 12 months of denervation. In Experiment 3, the sciatic nerve was cut and then repaired after periods of 3, 6, 9, 12, and 18 months. Four months after nerve repair, the sciatic nerve was again cut and the proximal nerve stump was injected with horseradish peroxidase to label the spinal cord neurons of the sciatic nerve nucleus. Both acetyl-L-carnitine-treated and untreated rats showed a tendency to have an increased number of horseradish peroxidase-labelled neurons with respect to intact rats of correspondent ages.(ABSTRACT TRUNCATED AT 400 WORDS)

Increased expression of Aquaporin 4 in the rat hippocampus and cortex during trimethyltin-induced neurodegeneration.

Trimethyltin chloride (TMT) is a neurotoxicant producing neuronal degeneration and reactive astrogliosis in the mammalian central nervous system, especially the hippocampus. A previous magnetic resonance imaging investigation in TMT-treated rats evidenced dilation of lateral ventricles, also suggesting alterations in blood-brain barrier permeability and brain edema. Aquaporin 4 (AQP4), a glial water channel protein expressed mainly in the nervous system, is considered a specific marker of vascular permeability and thought to play an important role in brain edema (conditions). We studied AQP4 expression in the hippocampus and cerebral cortex of TMT-treated rats in order to explore the molecular mechanisms involved in brain edema occurring in these experimental conditions. Real-time PCR and western blotting data showed significant up-regulation of both AQP4 mRNA and protein levels starting 14 days after TMT treatment in the hippocampus and cortex. Parallel immunofluorescence studies indicated intense astrogliosis and AQP4 immunoreactivity diffusely pronounced in the hippocampal and cortex areas starting 14 days after TMT intoxication. In order to study the effects of TMT on vascular integrity, double-label immunofluorescence experiments for rat immunoglobulin G (IgG) and rat endothelial cell antigen-1 (RECA-1) or neuronal nuclei (NeuN) (endothelial and neuronal markers respectively) were performed. The results indicated, at 21 and 35 days after treatment, the presence of rat IgG in paravasal parenchyma and in some neuronal cells of the hippocampus and cortex. The extravasated IgG staining was temporally correlated with over-expression of neuronal vascular endothelial growth factor (VEGF) and the active phosphorylated form of its neuronal receptor (VEGFR-2P), suggesting that these factors may cooperate in mediating vascular leakage.

Cathepsin D plays a crucial role in the trimethyltin-induced hippocampal neurodegeneration process.

Trimethyltin chloride (TMT) is known to produce neuronal damage in the rat hippocampus, especially in the CA(1)/CA(3) subfields, together with reactive astrogliosis. Previous studies indicate that in cultured rat hippocampal neurons the Ca(2+) cytosolic increase induced by TMT is correlated with apoptotic cell death, although some molecular aspects of the hippocampal neurodegeneration induced by this neurotoxicant still remain to be clarified. Cathepsin D (Cat D) is a lysosomal aspartic protease involved in some neurodegenerative processes and also seems to play an important role in the processes that regulate apoptosis. We investigated the specific activity and cellular expression of Cat D in the rat hippocampus in vivo and in cultured organotypic rat hippocampal slices. The role of Cat D in cell death processes and the mechanisms controlling Cat D were also investigated. Cat D activity was assayed in hippocampus homogenates of control and TMT-treated rats. In order to visualize the distribution of Cat D immunoreactivity in the hippocampus, double-label immunofluorescence for Cat D and Neu N, GFAP, OX42 was performed. In addition, in order to clarify the possible relationship between Cat D activity, neuronal calcium overload and neuronal death processes, organotypic hippocampal cultures were also treated with a Cat D inhibitor (Pepstatin A) or Calpain inhibitor (Calpeptin) or an intracellular Ca(2+) chelator (BAPTA-AM) in the presence of TMT. TMT treatment in rat hippocampus induced high levels of Cat D activity both in vivo and in vitro, in glial cells and in CA(3) neurons, where a marked TMT-induced neuronal loss also occurred. Cat D is actively involved in CA3 neuronal death and the protease increase is a calcium-Calpain dependent phenomenon.

[Preliminary observations on ciliary ganglia in organ culture]. / Osservazioni preliminari sul ganglio ciliare in coltura d'organo.

Preliminary observations have been made concerning some ultrastructural aspects of the choroid neurons in organ-cultured ciliary ganglia. Reactive nuclear as well as cytoplasmic modifications have been described. They consist mainly of nuclear dense bodies, arrays of smooth endoplasmic reticulum cisternae, clusters of condensed mitochondria. These modifications would confirm the existence of an interaction occurring between the neurons of the ciliary ganglion and their target organs.

Acetylcholinesterase localization at synapses in chick embryo ciliary ganglion.

The cytochemical localization of acetylcholinesterase (AChE) at calyciform synapses of the chick ciliary ganglion during embryonic development has been investigated. AChE activity is present at the surface membrane of newly formed calyciform synapses and closely follows the progressive enlargement of the synaptic area. The occurrence of a retrograde iris-dependent influence on ganglionic AChE is considered. AChE seems to be a suitable marker for synaptic maturation.

[Cholinergic enzymes in the ciliary ganglia of the chicken and pigeon]. / Enzimi colinergici nel ganglio ciliare di pollo e di piccione.

In the present paper we have comparatively analyzed acetylcholinesterase (AChE) and cholinacetyltransferase (ChAT) activity in chick and pigeon ciliary ganglion. AChE specific activity in the pigeon ciliary ganglion is remarkably higher than the one occurring in the chick; conversely the ChAT specific activity is similar in the chick as well as in the pigeon. Higher AChE activity found in the pigeon ciliary ganglion can be partially attributed to a selective accumulation of the enzyme in already described membrane-limited formations typical of the choroid neurons. After post-ganglionic axotomy such formations undergo a progressive disappearance which parallels the decrease of AChE activity. The present data suggest the hypothesis that the structures under investigation as well as ganglionic AChE are possibly controlled through a retrograde mechanism by their target organ.