Auditory Neurons with Transitory Axons to Visual Areas Form Short Permanent Projections.

The kitten's auditory cortex (including the first and second auditory fields AI and AII) is known to send transient axons to either ipsi- or contralateral visual areas 17 and 18. By the end of the first postnatal month the transitory axons, but not their neurons of origin, are eliminated. Here we investigated where these neurons project after the elimination of the transitory axon. Eighteen kittens received early (postnatal day (pd) 2 - 5) injections of long lasting retrograde fluorescent traces in visual areas 17 and 18 and late (pd 35 - 64) injections of other retrograde fluorescent tracers in either hemisphere, mostly in areas known to receive projections from AI and AII in the adult cat. The middle ectosylvian gyrus was analysed for double-labelled neurons in the region corresponding approximately to AI and AII. Late injections in the contralateral (to the analysed AI, AII) hemisphere including all of the known auditory areas, as well as some visual and 'association' areas, did not relabel neurons which had had transient projections to either ipsi- or contralateral visual areas 17 - 18. Thus, AI and AII neurons after eliminating their transient juvenile projections to visual areas 17 and 18 do not project to the other hemisphere. In contrast, relabelling was obtained with late injections in several locations in the ipsilateral hemisphere; it was expressed as per cent of the population labelled by the early injections. Few neurons (0 - 2.5%) were relabelled by large injections in the caudal part of the posterior ectosylvian gyrus and the adjacent posterior suprasylvian sulcus (areas DP, P, VP). Multiple injections in the middle ectosylvian gyrus relabelled a considerably larger percentage of neurons (13%). Single small injections in the middle ectosylvian gyrus (areas AI, AII), the caudal part of the anterior ectosylvian gyrus and the rostral part of the posterior ectosylvian gyrus relabelled 3.1 - 7.0% of neurons. These neurons were generally near (<2.0 mm) the outer border of the late injection sites. Neurons with transient projections to ipsi- or contralateral visual areas 17 and 18 were relabelled in similar proportions by late injections at any given location. Thus, AI or AII neurons which send a transitory axon to ipsi- or contralateral visual areas 17 and 18 are most likely to form short permanent cortical connections. In that respect, they are similar to medial area 17 neurons that form transitory callosal axons and short permanent axons to ipsilateral visual areas 17 and 18.

Neuronal expression of the ubiquitin ligase Nedd4-2 in rat dorsal root ganglia: Modulation in the spared nerve injury model of neuropathic pain.

Neuronal hyperexcitability following peripheral nerve lesions may stem from altered activity of voltage-gated sodium channels (VGSCs), which gives rise to allodynia or hyperalgesia. In vitro, the ubiquitin ligase Nedd4-2 is a negative regulator of VGSC α-subunits (Na(v)), in particular Na(v)1.7, a key actor in nociceptor excitability. We therefore studied Nedd4-2 in rat nociceptors, its co-expression with Na(v)1.7 and Na(v)1.8, and its regulation in pathology. Adult rats were submitted to the spared nerve injury (SNI) model of neuropathic pain or injected with complete Freund's adjuvant (CFA), a model of inflammatory pain. L4 dorsal root ganglia (DRG) were analyzed in sham-operated animals, seven days after SNI and 48h after CFA with immunofluorescence and Western blot. We observed Nedd4-2 expression in almost 50% of DRG neurons, mostly small and medium-sized. A preponderant localization is found in the non-peptidergic sub-population. Additionally, 55.7±2.7% and 55.0±3.6% of Nedd4-2-positive cells are co-labeled with Na(v)1.7 and Na(v)1.8 respectively. SNI significantly decreases the proportion of Nedd4-2-positive neurons from 45.9±1.9% to 33.5±0.7% (p<0.01) and the total Nedd4-2 protein to 44%±0.13% of its basal level (p<0.01, n=4 animals in each group, mean±SEM). In contrast, no change in Nedd4-2 was found after peripheral inflammation induced by CFA. These results indicate that Nedd4-2 is present in nociceptive neurons, is downregulated after peripheral nerve injury, and might therefore contribute to the dysregulation of Na(v)s involved in the hyperexcitability associated with peripheral nerve injuries.

Subarachnoid haemorrhage of unknown cause: Clinical, neuroradiological and evolutive aspects.

The clinical and radiological data of 52 patients with subarachnoid haemorrhage (SAH) and a negative panangiography were analysed with an average follow-up period of 3.8 years. Of these 52 patients, only one (1.9%) was subsequently found to have an aneurysm. Second angiography proved to be inconclusive in all 24 cases where it was performed. Of the 51 'true' non-aneurysmal SAH, 80% were in a good clinical grade on admission and 12% developed cerebral ischaemia. The mortality rate following SAH was 4%. There was one rebleeding. At follow-up examination, 87% of the patients had made a good recovery and 6% were left disabled due to SAH. Four patients with an aneurysmal pattern of SAH required a permanent shunt. All of the 22 patients with a perimesencephalic SAH were in a good neurological condition upon admission; one of them developed an angiography-induced transient cerebral ischaemia and another one suffered from a fatal rebleeding. None of the 21 survivors was disabled at follow-up examination. The clinical course of patients with SAH of unknown cause, especially those with a perimesencephalic pattern of haemorrhage, is good. Repeated angiography in this latter group is not useful. In the aneurysmal pattern SAH group, repeat angiography is advised only if there is strong computed tomographic (CT) scan suspicion of an aneurysm.

Bilateral olfactory sensory input enhances chemotaxis behavior.

Neural comparisons of bilateral sensory inputs are essential for visual depth perception and accurate localization of sounds in space. All animals, from single-cell prokaryotes to humans, orient themselves in response to environmental chemical stimuli, but the contribution of spatial integration of neural activity in olfaction remains unclear. We investigated this problem in Drosophila melanogaster larvae. Using high-resolution behavioral analysis, we studied the chemotaxis behavior of larvae with a single functional olfactory neuron on either the left or right side of the head, allowing us to examine unilateral or bilateral olfactory input. We developed new spectroscopic methods to create stable odorant gradients in which odor concentrations were experimentally measured. In these controlled environments, we observed that a single functional neuron provided sufficient information to permit larval chemotaxis. We found additional evidence that the overall accuracy of navigation is enhanced by the increase in the signal-to-noise ratio conferred by bilateral sensory input.

Activation of c-Jun in the nuclei of neurons of the CA-1 in thrombin preconditioning occurs via PAR-1.

Recently it has been shown that the c-Jun N-terminal kinase (JNK) plays a role in thrombin preconditioning (TPC) in vivo and in vitro. To investigate further the pathways involved in TPC, we performed an immunohistochemical study in hippocampal slice cultures. Here we show that the major target of JNK, the AP-1 transcription factor c-Jun, is activated by phosphorylation in the nuclei of neurons of the CA1 region by using phospho-specific antibodies against the two JNK phosphorylation sites. The activation is early and transient, peaking at 90 min and not present by 3 hr after low-dose thrombin administration. Treatment of cultures with a synthetic thrombin receptor agonist results in the same c-Jun activation profile and protection against subsequent OGD, both of which are prevented by specific JNK inhibitors, showing that thrombin signals through PAR-1 to JNK. By using an antibody against the Ser 73 phosphorylation site of c-Jun, we identify possible additional TPC substrates.

Dexamethasone stimulates the biochemical differentiation of fetal forebrain cells in reaggregating cultures.

The influence of dexamethasone on the development of neurons and oligodendrocytes was studied in serum-free, aggregating rat brain cell cultures. Synaptogenesis and myelination occur in this culture system. The concentration of myelin basic protein and the activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase were used as oligodendroglia and myelin markers. Choline acetyltransferase and acetylcholinesterase served as neuronal markers, glutamine synthetase reflected astrocyte differentiation, while ornithine decarboxylase served as a general marker for cell growth and maturation. This study showed that dexamethasone stimulated the differentiation of cholinergic neurons and astrocytes. The effect of dexamethasone on oligodendroglial differentiation and myelination depended on the stage of development: during the early phase of myelination dexamethasone had a stimulatory effect, whereas at a later stage it showed a significant inhibition.

Interaction between neuropeptide Y (NPY) and brain-derived neurotrophic factor in NPY-mediated neuroprotection against excitotoxicity : a role for microglia

The neuroprotective effect of neuropeptide Y (NPY) receptor activation was investigated in organotypic mouse hippocampal slice cultures exposed to the glutamate receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Exposure of 2-week-old slice cultures, derived from 7-day-old C57BL/6 mice, to 8 microm AMPA, for 24 h, induced degeneration of CA1 and CA3 pyramidal cells, as measured by cellular uptake of propidium iodide (PI). A significant neuroprotection, with a reduction of PI uptake in CA1 and CA3 pyramidal cell layers, was observed after incubation with a Y(2) receptor agonist [NPY(13-36), 300 nm]. This effect was sensitive to the presence of the selective Y(2) receptor antagonist (BIIE0246, 1 microm), but was not affected by addition of TrkB-Fc or by a neutralizing antibody against brain-derived neurotrophic factor (BDNF). Moreover, addition of a Y(1) receptor antagonist (BIBP3226, 1 microm) or a NPY-neutralizing antibody helped to disclose a neuroprotective role of endogenous NPY in CA1 region. Cultures exposed to 8 microm AMPA for 24 h, displayed, as measured by an enzyme-linked immunosorbent assay, a significant increase in BDNF. In such cultures there was an up-regulation of neuronal TrkB immunoreactivity, as well as the presence of BDNF-immunoreactive microglial cells at sites of injury. Thus, an increase of AMPA-receptor mediated neurodegeneration, in the mouse hippocampus, was prevented by neuroprotective pathways activated by NPY receptors (Y(1) and Y(2)), which can be affected by BDNF released by microglia and neurons.

Invasion of lesion territory by regenerating fibers after spinal cord injury in adult macaque monkeys.

In adult macaque monkeys subjected to an incomplete spinal cord injury (SCI), corticospinal (CS) fibers are rarely observed to grow in the lesion territory. This situation is little affected by the application of an anti-Nogo-A antibody which otherwise fosters the growth of CS fibers rostrally and caudally to the lesion. However, when using the Sternberger monoclonal-incorporated antibody 32 (SMI-32), a marker detecting a non-phosphorylated neurofilament epitope, numerous SMI-32-positive (+) fibers were observed in the spinal lesion territory of 18 adult macaque monkeys; eight of these animals had received a control antibody infusion intrathecally for 1month after the injury, five animals an anti-Nogo-A antibody, and five animals received an anti-Nogo-A antibody together with brain-derived neurotrophic factor (BDNF). These fibers occupied the whole dorso-ventral axis of the lesion site with a tendency to accumulate on the ventral side, and their trajectories were erratic. Most of these fibers (about 87%) were larger than 1.3μm and densely SMI-32 (+) stained. In the undamaged spinal tissue, motoneurons form the only large population of SMI-32 (+) neurons which are densely stained and have large diameter axons. These data therefore suggest that a sizeable proportion of the fibers seen in the lesion territory originate from motoneurons, although fibers of other origins could also contribute. Neither the presence of the antibody neutralizing Nogo-A alone, nor the presence of the antibody neutralizing Nogo-A combined with BDNF influenced the number or the length of the SMI-32 (+) fibers in the spinal lesion area. In summary, our data show that after a spinal cord lesion in adult monkeys, the lesion site is colonized by fibers, a large portion of which presumably originate from motoneurons.

c-Jun N-terminal kinase binding domain-dependent phosphorylation of mitogen-activated protein kinase kinase 4 and mitogen-activated protein kinase kinase 7 and balancing cross-talk between c-Jun N-terminal kinase and extracellular signal-regulated kinase pathways in cortical neurons

The c-Jun N-terminal kinase (JNK) is a mitogen-activated protein kinase (MAPK) activated by stress-signals and involved in many different diseases. Previous results proved the powerful effect of the cell permeable peptide inhibitor d-JNKI1 (d-retro-inverso form of c-Jun N-terminal kinase-inhibitor) against neuronal death in CNS diseases, but the precise features of this neuroprotection remain unclear. We here performed cell-free and in vitro experiments for a deeper characterization of d-JNKI1 features in physiological conditions. This peptide works by preventing JNK interaction with its c-Jun N-terminal kinase-binding domain (JBD) dependent targets. We here focused on the two JNK upstream MAPKKs, mitogen-activated protein kinase kinase 4 (MKK4) and mitogen-activated protein kinase kinase 7 (MKK7), because they contain a JBD homology domain. We proved that d-JNKI1 prevents MKK4 and MKK7 activity in cell-free and in vitro experiments: these MAPKK could be considered not only activators but also substrates of JNK. This means that d-JNKI1 can interrupt downstream but also upstream events along the JNK cascade, highlighting a new remarkable feature of this peptide. We also showed the lack of any direct effect of the peptide on p38, MEK1, and extracellular signal-regulated kinase (ERK) in cell free, while in rat primary cortical neurons JNK inhibition activates the MEK1-ERK-Ets1/c-Fos cascade. JNK inhibition induces a compensatory effect and leads to ERK activation via MEK1, resulting in an activation of the survival pathway-(MEK1/ERK) as a consequence of the death pathway-(JNK) inhibition. This study should hold as an important step to clarify the strong neuroprotective effect of d-JNKI1.

Spatial, temporal and subcellular localization of islet-brain 1 (IB1), a homologue of JIP-1, in mouse brain.

Islet-brain 1 (IB1) was recently identified as a DNA-binding protein of the GLUT2 gene promoter. The mouse IB1 is the rat and human homologue of the Jun-interacting protein 1 (JIP-1) which has been recognized as a key player in the regulation of c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways. JIP-1 is involved in the control of apoptosis and may play a role in brain development and aging. Here, IB1 was studied in adult and developing mouse brain tissue by in situ hybridization, Northern and Western blot analysis at cellular and subcellular levels, as well as by immunocytochemistry in brain sections and cell cultures. IB1 expression was localized in the synaptic regions of the olfactory bulb, retina, cerebral and cerebellar cortex and hippocampus in the adult mouse brain. IB1 was also detected in a restricted number of axons, as in the mossy fibres from dentate gyrus in the hippocampus, and was found in soma, dendrites and axons of cerebellar Purkinje cells. After birth, IB1 expression peaks at postnatal day 15. IB1 was located in axonal and dendritic growth cones in primary telencephalon cells. By biochemical and subcellular fractionation of neuronal cells, IB1 was detected both in the cytosolic and membrane fractions. Taken together with previous data, the restricted neuronal expression of IB1 in developing and adult brain and its prominent localization in synapses suggest that the protein may be critical for cell signalling in developing and mature nerve terminals.

Glutamine Synthetase is Expressed by Primary Sensory Neurons from Chick Embryos In Vitro but not In Vivo: Influence of Skeletal Muscle Extract.

Glutamine synthetase (GS) catalyses the ATP-dependent formation of glutamine from glutamate and ammonia. To determine whether dorsal root ganglion (DRG) cells from chick embryos express the enzyme in vivo or in vitro, GS was detected by immunocytochemical reaction either in vibratome sections of DRG or in dissociated DRG cell cultures. The immunocytochemical detection of GS showed that in vivo the DRG taken from chick embryos at day 10 (E10), E14, E18 or from chickens after hatching were free of any GS-positive ganglion cells; in contrast, in neuron-enriched cultures of DRG cells grown in vitro at E10, virtually all the neuronal cells (98.6 +/- 1.0%) express GS at 3, 5 or 7 days of culture. In mixed DRG cell cultures, only 83.6+/-4.6% of the neurons displayed a GS-immunoreactivity. In both culture conditions, neither the presence of horse serum nor the age of the culture appeared to affect the percentage of neurons which displayed a GS-immunoreactivity. After [3H]glutamine uptake, radioautographs revealed that only 80% of the neurons were labelled in neuron-enriched DRG cell cultures while 96% of the neurons were radioactive in mixed DRG cell cultures. Furthermore the most heavily [3H]glutamine-labelled neurons were exclusively found in mixed DRG cell cultures. Combination of both immunocytochemical detection of GS and radioautography after [3H]glutamine uptake showed that strongly GS-immunostained neurons corresponded to poorly radioactive ones and vice versa. When skeletal muscle extract (ME) was added to DRG cell cultures, the number of GS-positive neurons was reduced to 77.5 +/- 2.5% in neuron-enriched cultures or to 43.6 +/- 3.8% in mixed DRG cell cultures; in both types of culture, the intensity of the neuronal immunostaining was depressed. Furthermore, combined action of ME and non-neuronal cells potentiates the enzyme repression exerted separately by ME or non-neuronal cells. Since GS-immunoreactivity is expressed in DRG cells grown in vitro, but not in vivo, it is suggested that microenvironmental factors influence the expression of GS. More specifically, the repression of GS by primary sensory neurons grown in vitro may be strongly induced by soluble factors present in skeletal muscle, and to a lesser extent in brain, and potentiated by non-neuronal cells.

Neurochemical changes within human early blind occipital cortex.

Early blindness results in occipital cortex neurons responding to a wide range of auditory and tactile stimuli. These changes in tuning properties are accompanied by an extensive reorganization of the occipital cortex that includes alterations in anatomical structure, neurochemical and metabolic pathways. Although it has been established in animal models that neurochemical pathways are heavily affected by early visual deprivation, the effects of blindness on these pathways in humans is still not well characterized. Here, using (1)H magnetic resonance spectroscopy in nine early blind and normally sighted subjects, we find that early blindness is associated with higher levels of creatine, choline and myo-Inositol and indications of lower levels of GABA within the occipital cortex. These results suggest that the cross-modal responses associated with early blindness may, at least in part, be driven by changes within occipital biochemical pathways.

Patterns of calcium-binding proteins in human inferior colliculus: identification of subdivisions and evidence for putative parallel systems.

The subdivisions of human inferior colliculus are currently based on Golgi and Nissl-stained preparations. We have investigated the distribution of calcium-binding protein immunoreactivity in the human inferior colliculus and found complementary or mutually exclusive localisations of parvalbumin versus calbindin D-28k and calretinin staining. The central nucleus of the inferior colliculus but not the surrounding regions contained parvalbumin-positive neuronal somata and fibres. Calbindin-positive neurons and fibres were concentrated in the dorsal aspect of the central nucleus and in structures surrounding it: the dorsal cortex, the lateral lemniscus, the ventrolateral nucleus, and the intercollicular region. In the dorsal cortex, labelling of calbindin and calretinin revealed four distinct layers.Thus, calcium-binding protein reactivity reveals in the human inferior colliculus distinct neuronal populations that are anatomically segregated. The different calcium-binding protein-defined subdivisions may belong to parallel auditory pathways that were previously demonstrated in non-human primates, and they may constitute a first indication of parallel processing in human subcortical auditory structures.