bluuebunny/crossref_metadata_2025 huggingface dataset:
Acetylcholinesterase Adaptation to Voluntary Wheel Running is Proportional to the Volume of Activity in Fast, but not Slow, Rat Hindlimb Muscles
Authors: Gisiger Victor , Bélisle Manon , Gardiner Phillip F.
Published: April 2006

Chronic enhancement of neuromuscular activity by forced exercise training programmes results in selective adaptation of the G4 acetylcholinesterase (AChE) molecular form in hindlimb fast muscles of the rat, with only minor and non‐selective AChE changes in the soleus. In order to shed further light on the physiological significance of this G4 adaptation to training, we turned to a voluntary exercise model. The impact of 5 days and 4 weeks of voluntary wheel cage running on AChE molecular forms was examined in four hindlimb fast muscles and the slow‐twitch soleus from two rat strains. Inbred Fisher and Sprague– Dawley rats, placed in live‐in wheel cages, exercised spontaneously for distances which progressively increased up to an average of ∼3 and 18 km/day, respectively, by the end of week 4. Fast muscles responded to this voluntary activity by massive G4 increases (up to 420%) with almost no changes in A12, so that by week 4 the tetramer became the main AChE component of these muscles. The additional G4 was composed primarily of amphiphilic molecules, suggesting a membrane‐bound state. The G4 content of fast muscles was highly correlated with the distance covered by the rats during the 5 days before they were killed (r= 0.850‐0.879, P < 0.001 in three muscles). The soleus muscle, in turn, responded to wheel cage activity by a marked selective reduction of its asymmetric forms—up to 45% for A12. This A12 decline, already maximal by day 5 of wheel cage running, showed no relationship with the distance covered. The present results constitute strong new evidence suggesting that the role of AChE in neuromuscular transmission is not limited solely to the rapid inactivation of just‐released acetylcholine.
Nerve Growth Factor and Basic Fibroblast Growth Factor Protect Cholinergic Neurons Against Quinolinic Acid Excitotoxicity in Rat Neostriatum
Authors: Pérez‐Navarro Esther , Alberch Jordi , Arenas Ernest , Calvo Nancy , Marsal Jordi
Published: April 2006

In the present work we have characterized a possible mechanism leading to the early survival of neostriatal cholinergic neurons after quinolinic acid injection. Different doses of quinolinic acid were injected in rat neostriatum and two different parameters were analysed 7 days after the lesion: choline acetyltransferase (ChAT) activity and nerve growth factor (NGF) levels. We have observed that ChAT activity decreased (until 68 nmol quinolinic acid) and NGF levels increased (until 34 nmol quinolinic acid) in a dose‐dependent manner. In order to characterize the time‐course of the lesion on NGF levels and ChAT activity, and the possible protective effect of NGF and basic fibroblast growth factor (bFGF) on cholinergic neurons, we have used the quinolinic acid dose (68 nmol) at which the first decrease of ChAT activity was observed. ChAT activity and NGF levels showed different patterns of response to quinolinic acid injection, since the maximal effect was reached at 1 day for ChAT activity and at 2 days for NGF levels. NGF or bFGF simultaneously injected with quinolinic acid (68 nmol) completely prevented the decrease in ChAT activity in a dose‐dependent manner but NGF was more effective than bFGF. Furthermore, differences observed in ChAT activity after NGF but not bFGF treatment were correlated with changes in the number of ChAT immunoreactive cells. Finally, we have also observed that, although bFGF alone was not able to modify NGF levels, bFGF simultaneously injected with quinolinic acid produced an increase of NGF levels higher than that observed after quinolinic acid injection alone. Our results show that NGF and bFGF protect striatal cholinergic neurons against quinolinic acid injury, and bFGF is able to potentiate the increase of NGF after the lesion, suggesting a cooperative action between different trophic factors in neuronal protection after excitotoxic injury. Thus, administration of trophic factors may be relevant in the prevention and treatment of neurodegenerative disorders, such as Huntington's disease.
Secondary Cell Death and the Inflammatory Reaction After Dorsal Hemisection of the Rat Spinal Cord
Authors: Dusart I. , Schwab M. E.
Published: April 2006

Local spinal cord lesions are often greatly enlarged by secondary damage, a process which leads to massive additional cell death. This process is poorly understood. In order to investigate which types of cells could play a role in increasing the size of the lesion, we have analysed the events occurring at rat spinal cord lesion sites from 1 h to 3 months after partial transection using cell type‐specific markers. One hour after transection, the lesion site was small and corresponded to the zone of primary mechanical damage. Extravasation of blood and an opening of the blood – brain barrier occurred. Rapidly thereafter, at 3 and 6 h, an area of secondary cell death developed around the zone of the primary lesion. This secondary cell death, which was probably largely of the necrotic type, affected neurons, macroglia and microglial cells indiscriminately. It was virtually complete at 12 h. Recruitment of inflammatory cells followed a time course which lagged behind that of secondary cell death. Adhesion of neutrophils to the inside of blood vessels was observed at 3 h. They appeared in large numbers at 6 h at the site of the primary lesion, but not yet in the area of secondary cell death. They were numerous throughout the lesion site at 24 h and then disappeared rapidly. Proliferation and recruitment of macrophages and microglial cells became predominant 2 days after injury. Their density was highest within the lesion site between 4 and 8 days. Very few astrocytes were present in the lesion site during the first week. In contrast, the surrounding area contained numerous activated astrocytes, which began to delineate the lesion site. After 2 weeks, the microglial cells and macrophages progressively disappeared from the lesion site, and a cavity formed. A glial scar surrounded this cavity and consisted of reactive astrocytes and activated microglial cells. The time course of the cellular reactions observed here suggests that secondary damage is not primarily due to destructive effects of neutrophils and macrophages. The inflammatory process after spinal cord transection is qualitatively similar to that observed outside the CNS. Inflammatory cells, which can release cytokines and growth factors, could play important roles in protective reactions of the tissue and glial scar formation.
Life‐spanning Behavioural and Adrenal Dysfunction Induced by Prenatal Hypoxia in the Rat is Prevented by the Calcium Antagonist Nimodipine
Authors: Nyakas Csaba , Buwalda Bauke , Markel Éva , Korte S. Mechiel , Luiten Paul G. M.
Published: April 2006

The long‐term behavioural effects of prenatal chronic anaemic hypoxia were investigated in young (5 months old), late adult (19 months) and aged Wistar rats (23–26 months). Sodium nitrite (2 g/l) offered in the drinking water during the second half of pregnancy served to evoke prenatal hypoxia. In parallel to nitrite treatment the Ca2+ channel blocker nimodipine (10 mg/kg) or vehicle alone was administered intragastrically once daily. Open‐field activity, intermale social behaviour, learning ability in a black–white discrimination paradigm and fear‐induced emotionality were assessed at different ages. Plasma corticosterone response to novelty stress was measured by blood sampling through chronic venous canulas at the age of 28 months. The nitrite‐exposed 5‐month‐old offspring started exploration in a novel open‐field with considerable delay. This delayed start‐latency was augmented in 19‐ and 23‐month‐old rats, pointing to exaggerated suppression of behavioural arousal. Nitrite‐induced hypoxia decreased the duration of social interactions during ageing. Aged rats exposed to nitrite were unable to learn a black–white discrimination but showed a normal generalized conditioned fear response (immobility) to the test situation as a whole. The conditioned fear‐induced vocalization was more frequent among hypoxic aged animals. The aged hypoxic rats displayed a prolonged plasma corticosterone stress response and had higher adrenal weight than their controls. The abnormal open‐field, social, learning and emotional behaviours, as well as the altered plasma corticosterone response, were prevented by prenatal nimodipine treatment.
Alterations in GAP‐43 and Synapsin Immunoreactivity Provide Evidence for Synaptic Reorganization in Adult Cat Dorsal Lateral Geniculate Nucleus following Retinal Lesions
Authors: Baekelandt V. , Arckens L. , Annaert W. , Eysel U. T. , Orban G. A. , Vandesande F.
Published: April 2006

Growth‐associated protein‐43 (GAP‐43) and synapsin were used as molecular markers for synaptic reorganization in the adult cat visual system following sensory deprivation. Small binocular retinal lesions (central 10°) were made with a xenon light photocoagulator in adult cats. One, 3, 5 and 7 weeks after induction of the lesion, the neuropil levels of synapsin and GAP‐43 in the dorsal lateral geniculate nucleus (dLGN) and area 17 were determined by immunocytochemistry. GAP‐43 displayed a moderately low basal level in the dLGN of normal adult cats. The parvocellular C layers and the interlaminar plexi were characterized by higher immunoreactivity for GAP‐43. Lesion‐induced alterations were observed in all layers: GAP‐43 immunoreactivity increased in the part of the dLGN representing central vision. This increase was maximal 3 weeks after the lesion. Under our experimental conditions, sensory deprivation did not significantly alter GAP‐43 levels in the visual cortex. The changes in synapsin immunoreactivity were also restricted to the dLGN. In this nucleus, synapsin immunoreactivity decreased in all layers in the part subserving central vision 1 week after lesion. By 3 weeks after lesion, the level of synapsin had already returned to normal. This study provides evidence for a capacity for structural remodelling in primary sensory brain areas such as the dLGN throughout adult life. The observed changes in GAP‐43 and synapsin in the dLGN suggest that synaptic reorganization is induced by retinal lesions. Normalization of synaptic density and activity could be important for the survival of the partially deafferented geniculate neurons.
The Topography of Tangential Inhibitory Connections in the Postnatally Developing and Mature Striate Cortex of the Cat
Authors: Albus K. , Wahle P.
Published: April 2006

Clustered intrinsic connections in the striate cortex of kittens originate from an unclustered, diffusely organized pattern prevailing during the first postnatal week. In order to study the participation of inhibitory neurons in this reorganization of the connections, we determined the topography of the inhibitory tangential connections in the striate cortex of cats ranging in age between 7 and 330 days by combining retrograde transport of fluorescent microspheres with GABA immunohistochemistry. After small intracortical injections of tracer, neurons containing either microspheres only (non‐GABAergic neurons) or GABA‐like immunoreactivity in addition to microspheres (GABAergic neurons) are labelled at various horizontal distances from the injection. At the end of the first postnatal week, both GABAergic and non‐GABAergic neurons are distributed in the horizontal plane in an unclustered fashion. During the second postnatal week, the tangential connections rearrange rapidly to form clusters. The tendency of the cells to form clusters is much weaker, however, in GABAergic than in non‐GABAergic neurons. In regions >500 μm distant from the centre of injection ∼90% of the non‐GABAergic neurons (range 87.5‐92.6%) but only 63% (range 57.1 – 72.3%) of the GABAergic neurons reside within the clusters formed by the non‐GABAergic neurons. These proportions do not change systematically with age. In the regions outside the non‐GABAergic clusters, GABAergic neurons appear to be evenly distributed and not to aggregate in clusters. From postnatal day 7 forward GABAergic neurons largely retain their overall distribution and density in the horizontal plane. When considering all cortical layers (including the superficial white matter) the lateral spread of the GABAergic neurons is more restricted than that of the non‐GABAergic neurons. Systematic changes in the lateral spread of inhibitory connections according to postnatal age were not observed. We conclude that, like the non‐GABAergic neurons, the GABAergic neurons have attained an adult‐like topography in the horizontal plane by about the end of the second postnatal week. From that time until adulthood they display much weaker clustering, a higher relative occurrence of short axon collaterals and a more restricted lateral distribution than do the excitatory neurons.
Developmental Changes in Spontaneous GABAA‐mediated Synaptic Events in Rat Hippocampal CA3 Neurons
Authors: Hosokawa Y. , Sciancalepore M. , Stratta F. , Martina M. , Cherubini E.
Published: April 2006

Ongoing spontaneous postsynaptic potentials (SPSPs) were intracellularly recorded at 34‐36°C from hippocampal CA3 neurons in slices obtained from postnatal days (P) 0 – 6 and 7 – 31. SPSPs occurred randomly, and their frequency distribution was fitted by a single exponential function. They were little affected by kynurenic acid, but were reversibly blocked by bicuculline, implying that they were mediated by GABAA receptors. The mean amplitude was 4.53 ± 0.89 mV in control conditions and 4.07 ± 0.79 mV in kynurenic acid. In kynurenic acid (with CsCl‐filled microelectrodes), SPSPs reversed polarity at 2.4 ± 2 mV. When tetrodotoxin (1 μM) was added to kynurenic acid solution, GABAA‐mediated miniature postsynaptic potentials (MPSPs) were recorded. Under these conditions large events disappeared. The mean amplitude of MPSPs was 2.51 ± 0.43 mV. The mean frequency decreased from 2.96 ± 1.04 Hz in kynurenic acid to 0.4 ± 0.15 Hz in kynurenic acid plus tetrodotoxin. In contrast with P0 – P6, at P7 – P31 SPSPs were significantly affected by kynurenic acid. The mean amplitude of SPSPs shifted from 4.71 ± 0.82 mV in control conditions to 3.79 ± 0.76 mV in kynurenic acid. At this developmental stage, the reversal potential of GABAA‐mediated SPSPs shifted towards more negative values (‐23.7 ± 1.3 mV). Addition of tetrodotoxin to kynurenic acid solution abolished larger events and revealed GABAergic MPSPs. The mean amplitude of MPSPs was 2.72 ± 0.5 mV, a value very close to that observed at P0 – P6. Synaptic currents were recorded at 22 – 24°C from voltage‐clamped CA3 pyramidal neurons (at P6) using the tight‐seal whole‐cell recording technique. Cells were dialysed with CsCl solution and held at ‐70 mV. Spontaneous GABAA‐mediated miniature postsynaptic currents (MPSCs) were recorded in the presence of kynurenic acid and tetrodotoxin. The decay time of MPSCs was fitted with a single exponential and was 29 ± 3 ms. No correlation was found between the peak amplitude of individual events and their rise or decay time constant. The mean amplitude distribution of MPSCs was 12 ± 4.3 pA. In outside‐out patches from acutely dissociated CA3 hippocampal neurons, GABA (50 μM) activated single‐channel events of 24 and 35 pS conductance. Therefore one quantal current represents the simultaneous opening of five to seven GABAA receptor channels on the postsynaptic cell. These data show also that in the immediate postnatal period spontaneous glutamatergic potentials are poorly developed and start appearing towards the end of the first postnatal week concomitant with the shift of GABA from the depolarizing to the hyperpolarizing direction.
Transplanted Transgenically Marked Oligodendrocytes Survive, Migrate and Myelinate in the Normal Mouse Brain as They Do in the Shiverer Mouse Brain
Authors: Lachapelle F. , Duhamel‐Clerin E. , Gansmüller A. , Evercooren A. Baron‐Van , Villarroya H. , Gumpel M.
Published: April 2006

The dye Hoechst 33342 was combined with an immunodetectable transgene product (chloramphenicol acetyltransferase, CAT) expressed in differentiated oligodendrocytes to trace their fate after transplantation in the normal and the shiverer mouse brain. In the shiverer brain, the technique allowed us to visualize grafted cells inside myelin basic protein‐positive myelin patches. Most of these cells were CAT‐positive/Hoechst 33342‐negative, reinforcing our hypothesis that cell division probably follows migration of grafted oligodendrocytes. Correlation of their morphology and distribution with their location in the host CNS suggested a local effect on the cell division and morphogenesis of the grafted material. When compared with transplantation of fragments of normal newborn donor tissue into the newborn shiverer brain, no difference could be seen between the behaviour of normal and transgenic oligodendrocytes. In the normal brain, transgenic oligodendrocytes survived at least 150 days and successfully myelinated the host axons. The timing of differentiation of grafted cells was similar in both types of recipient brains. Migration occurred rostrally and caudally. Although migrating cells could be observed along the meninges and the blood vessels, migration occurred preferentially along white matter tracts. The extent of migration was influenced by the site of implantation, and grafted cells could be found up to 6 mm from the grafting point. No differences in the timing of differentiation or the pattern or extent of migration could thus be demonstrated when transgenic oligodendrocytes were transplanted in the normal or the shiverer brain. This validates our previous studies using the newborn shiverer mouse as recipient.
The Antennal Motor System of Crickets: Proctolin in Slow and Fast Motoneurons as Revealed by Double Labelling
Authors: Bartos Marlene , Allgäuer C. , Eckert M. , Honegger H. W.
Published: April 2006

This study decribes proctolin‐like immunoreactivity (PLI) of identified antennal motoneurons in the brain of adult crickets (Gryllus bimaculatus). The motoneurons were first backfilled with the fluorescent dye Lucifer Yellow and then immunohistochemically labelled with an antibody against proctolin. Altogether 14 of the 17 excitatory antennal motoneurons, including physiologically fast and slow types, showed PLI. The only common inhibitor consistently demonstrated a weak positive PLI. PLI was also present in the dendritic arborizations and varicosities of motor axons in the intrinsic antennal muscles. Densitometric measurements of motoneuron somata showed significant differences in the intensity of PLI in different types of antennal motoneurons, suggesting that antennal motoneurons produce different amounts of proctolin. Identical motoneuron somata display a large variance of PLI intensities in different brains. This observation may indicate up‐and down‐regulation of proctolin in individual crickets.
Human and Rat Primary C‐Fibre Afferents Store and Release Secretoneurin, a Novel Neuropeptide
Authors: Kirchmair Rudolf , Marksteiner Josef , Troger Josef , Mahata Sushil K. , Mahata Manjula , Donnerer Josef , Amann Rainer , Fischer‐Colbrie Reiner , Winkler Hans , Saria Alois
Published: April 2006

Secretoneurin is a recently discovered neuropeptide derived from secretogranin II (SgII). Since this peptide could be detected in the dorsal horn of the spinal cord we studied whether it is localized in and released from primary afferent neurons. Secretoneurin was investigated with immunocytochemistry and radioimmunoassay in spinal cord, dorsal root ganglia and peripheral organs. SgII mRNA was determined in dorsal root ganglia. Normal rats and rats pre‐treated neonatally with capsaicin to destroy selectively polymodal nociceptive (C‐) fibres were used. Slices of dorsal spinal cord were perfused in vitro for release experiments. Immunocytochemistry showed a distinct distribution of secretoneurin‐immunoreactivity (IR) in the spinal cord and lower brainstem. A particularly high density of fibres was found in lamina I and outer lamina II of the caudal trigeminal nucleus and of the spinal cord. This distribution was qualitatively identical in rat and human post‐mortem tissue. Numerous small diameter and some large dorsal root ganglia neurons were found to contain SgII mRNA. Capsaicin treatment led to a marked depletion of secretoneurin‐IR in the substantia gelatinosa, but not in other immunopositive areas of the spinal cord and to a substantial loss of small (<25 μm) SgII‐mRNA‐containing dorsal root ganglia neurons. Radioimmunoassay revealed a significant decrease of secretoneurin‐IR in the dorsal spinal cord, the trachea, heart and urinary bladder of capsaicin‐treated rats. Perfusion of spinal cord slices with capsaicin as well as with 60 mM potassium led to a release of secretoneurin‐IR. In conclusion, secretoneurin is a neuropeptide which is stored in and released from capsaicin‐sensitive, primary afferent (C‐fibre) neurons. It may, therefore, be a novel peptidergic modulator of pain transmission or of C‐fibre mediated non‐nociceptive information.
Physiological Properties of Neuronal Nicotinic Receptors Reconstituted from the Vertebrate β2 Subunit and Drosophilaα Subunits
Authors: Bertrand D. , Ballivet M. , Gomez M. , Bertrand S. , Phannavong B. , Gundelfinger E. D.
Published: April 2006

Three cDNAs (ALS, Dα2 and ARD) isolated from the nervous system of Drosophila and encoding putative nicotinic acetylcholine receptor subunits were expressed in Xenopus oocytes in order to study their functional properties. Functional receptors could not be reconstituted from any of these subunits taken singly or in twos and threes. In contrast, large evoked currents (in the μA range) were consistently observed upon agonist application on oocytes co‐injected with ALS or Dα2 in combination with the chick β2 structural subunit. The ALS/β2 and Dα2/β2 receptors are highly sensitive to acetylcholine and nicotine, and their physiological properties resemble those of native or reconstituted receptors from vertebrates. Although the physiological properties of ALS/β2 and Dα2/β2 receptors are quite similar, clear differences appear in their pharmacological profiles. The ALS/β2 receptor is highly sensitive to α‐bungarotoxin while the Dα2/β2 receptor is totally insensitive to this agent. These results demonstrate that the Drosophila ALS and Dα2 cDNAs encode neuronal nicotinic subunits responding to physiological concentrations of the agonists acetylcholine and nicotine.
Inhibition of c‐Fos Protein Expression in Rat Spinal Cord by Antisense Oligodeoxynucleotide Superfusion
Authors: Gillardon F. , Beck H. , Uhlmann E. , Herdegen T. , Sandkühler J. , Peyman A. , Zimmermann M.
Published: April 2006

Peripheral noxious stimulation leads to a rapid and transient expression of c‐fos, c‐jun and other immediate‐early genes (IEGs) in the spinal cord. However, the role of IEG encoded transcription factors in plasticity of spinal neurons remains speculative. In the present study we have shown that superfusion of rat spinal cord with antisense oligodeoxynucleotides complementary to c‐fos mRNA suppresses heat‐induced c‐Fos protein expression without affecting other members of the Fos and Jun family, thus providing a technique to determine the function of IEGs in vivo.
Neuroanatomical Characterization of a New Mutant Rat with Dopamine Depletion in the Substantia Nigra
Authors: Clarke D. J. , Payne A. P.
Published: April 2006

A new mutant rat strain with movement disorders has arisen spontaneously in a closed colony of Albino–Swiss rats at the Department of Anatomy, Glasgow University. The animals possess a progressive locomotor disorder which is first apparent at around post‐natal day 10. We here report that tyrosine hydroxylase immunocytochemistry demonstrates that these mutants have profound depletions in dopaminergic systems in the substantia nigra and corresponding areas of neostriatum.
Voltage‐gated Currents in Rabbit Retinal Astrocytes
Authors: Clark Beverley A. , Mobbs Peter
Published: April 2006

The voltage‐gated currents of the astrocytes associated with the retinal capillaries of the rabbit retina were studied using whole‐cell patch clamp recording. The resting potential of these cells was −70 ± 4.8 mV (mean ± SEM; n= 54), and the input resistance and cell capacitance were 558 ± 3.6 MΩ and 19.5 ± 1.8 pF respectively. Depolarization to potentials positive to −50 mV evoked rapidly activating inward and outward currents. The inward current was transient, eliminated by substitution of choline for Na+ in the bathing solution, and reduced by 50% in the presence of 1 μM tetrodotoxin. The time‐to‐peak of the Na+ current was more than twice that for the Na+ current found in retinal neurons. The glial Na+ current was half‐inactivated at −55 mV. A transient component of the outward K+ current was blocked by external 4‐aminopyridine while a more sustained component was blocked by external tetraethylammonium. At potentials between −150 and −50 mV the membrane behaved Ohmically. Voltage‐gated currents in retinal astrocytes recorded in situ appear qualitatively similar to those described for some glial cells in vitro.
A Neurotoxic Prion Protein Fragment Induces Rat Astroglial Proliferation and Hypertrophy
Authors: Forloni Gianluigi , Del Bo Roberto , Angeretti Nadia , Chiesa Roberto , Smiroldo Simona , Doni Rinaldo , Ghibaudi Elena , Salmona Mario , Porro Monica , Verga Laura , Giaccone Giorgio , Bugiani Orso , Tagliavini Fabrizio
Published: April 2006

Prion‐related encephalopathies are characterized by the accumulation of an abnormal prion protein isoform (PrPSc) and the deposition of PrP amyloid in the brain. This process is accompanied by neuronal loss and astrogliosis. We recently showed that a synthetic peptide corresponding to residues 106–126 of human PrP is amyloidogenic and causes neuronal death by apoptosis in vitro. In the present study we investigated the effects of 1‐ and 14‐day exposures of rat astroglial cultures to mtcromolar concentrations of this peptide as well as peptides homologous to other portions of PrP, a peptide corresponding to residues 25–35 of amyloid‐β protein, and a scrambled sequence of PrP 106–126. No significant changes were observed after 1‐day exposure of cultures to any peptide. Conversely, 14‐day treatment with PrP 106–126 (50 μM) resulted in a 5‐fold increase in glial fibrillary acidic protein (GFAP) expression, as evaluated by Northern and Western blot analyses, and a 1.5‐fold increment in cell number. Light and electron microscopy immunohistochemistry showed an enlargement in size and density of astroglial processes, and an increase in GFAP‐immunoreactive intermediate filaments. These changes were not observed after 14‐day treatment of cultures with the other peptides, including PrP 106–126 scrambled. The increase in GFAP expression of astroglial cultures exposed to PrP 106–126 was quantitatively similar to that found in scrapie‐infected hamster brains. These results suggest that the PrP region corresponding to residues 106–126 is biologically active, and that cerebral accumulation of peptides including this sequence might be responsible for both the neuronal degeneration and the astrogliosis that occur in prion‐related encephalopathies.
The Presynaptic Cell Determines the Number of Synapses in the Drosophila Optic Ganglia
Authors: Canal I. , Fariñas I. , Gho M. , Ferrús A.
Published: April 2006

The role of the pre‐ and postsynaptic cells in determining the number of synapses has been investigated in retina mosaics of the gigas (gig) mutant of Drosophila. Mutant photoreceptors are two to three times larger than those of the wild type, while adjacent cells in the mosaic retina and the lamina are normal in size. Serial electron microscope reconstructions of mosaic lamina cartridges show that gig photoreceptors establish more synapses upon lamina neurons than the normal photoreceptors do. By contrast, the number of feedback synapses that lamina neurons make onto gig photoreceptors does not increase. The increment in the number of synapses correlates positively with the increment of presynaptic cell membrane, resulting in constancy of synapse density. The phototactic response of flies bearing a gig eye is abnormal, indicating that the extra synapses are functional.
Acetylcholinesterase‐containing Intrinsic Neurons in the Rat Main Olfactory Bulb: Cytological and Neurochemical Features
Authors: Le Jeune H. , Jourdan F.
Published: April 2006

Acetylcholinesterase (AChE) histochemistry in light and electron microscopy was used to identify cholinoceptive neurons in the olfactory bulb of adult and 15‐day‐old rats. Double‐labelling experiments using AChE histochemistry and either tyrosine hydroxylase or GABA immunocytochemistry with light microscopy were also performed in order to specify the chemical nature of cholinoceptive neurons. Superficial short‐axon cells and several morphological subtypes of deep short‐axon cells (second‐order interneurons) are the most numerous AChE‐containing intrinsic neurons in the olfactory bulb. Short‐axon interneurons seem to be the only neurons expressing AChE in the deep olfactory bulb since the numerous granule cells (first‐order interneurons) were never found to be AChE‐positive, even in electron microscopy. In the superficial olfactory bulb, cholinoceptive cells belong to several neuronal categories. In addition to the intensely labelled superficial short‐axon cells, a few periglomerular cells (first‐order interneurons) display weak but significant AChE expression, clearly visible in electron microscopy. Both ultrastructural and double‐labelling observations support the hypothesis that a subset of superficial tufted cells is also cholinoceptive. The coexistence of AChE and tyrosine hydroxylase in large neurons located in the glomerular and superficial external plexiform layers indicates that some, if not all, cholinoceptive tufted cells belong to the dopaminergic population previously observed in this area. These observations indicate that several types of intrinsic neurons express AChE and can be tentatively considered as cholinoceptive. Our results provide an anatomical substrate for hypotheses concerning the complex effects of acetylcholine in the processing of sensory information in the olfactory bulb.
Polarized Distribution of the Trans‐Golgi Network Marker TGN38 During the In Vitro Development of Neocortical Neurons: Effects of Nocodazole and Brefeldin A
Authors: Lowenstein P. R. , Morrison E. E. , Bain D. , Shering A. F. , Banting G. , Douglas P. , Castro M. G.
Published: April 2006

Neurons are polarized secretory cells whose cytoplasm and plasma membrane are polarized to form two compartments: dendrites and axons. In mature, fully polarized neurons, the microtubule‐associated protein Map2 is targeted to dendrites, while tau is mainly restricted to axons. However, the intraneuronal distribution of secretory pathway organelles, such as the endoplasmic reticulum and the Golgi complex, which give rise to all constitutive, regulated and lysosome vesicles, is poorly understood. Thus, to investigate the distribution of the trans‐Golgi network during the development and maturation of rat neocortical neurons in vitro, we have utilized an antibody recognizing a 38 kDa trans‐Golgi network‐specific protein, TGN38, and immunofluorescence microscopy. Before neurons have established polarity, TGN38 immunoreactivity outlines several vesicles dispersed throughout the cell body cytoplasm; these converge close to a major Map2‐immunopositive process during the establishment of neuronal polarity, and later merge into a single structure located at the base of a thick Map2‐immunopositive process, ∼18 h after plating. At this stage TGN38 immunoreactivity is located within 45° of the major Map2‐immunoreactive process in 54% of neurons, while in only 6% of cells it is located at the opposite pole. After 3 days in vitro, during the segregation of microtubule‐associated proteins to either dendrites or axons, TGN38 immunoreactivity clusters continue to be located close to a major dendrite, and in some neurons these clusters begin to enter a major Map2‐immunoreactive process. At 10 days in vitro TGN38 immunoreactivity extends into a major dendrite for 5–30 μm in many neurons. Thus, the distribution of TGN38 immunoreactivity becomes polarized, being localized within a single, usually the major, neocortical dendrite. Our results also show that the morphological appearance of TGN38‐immunoreactive structures is microtubule‐dependent, since nocodazole treatment of polarized neurons induces scattering of TGN38‐immunoreactive vesicles throughout the cell body's cytoplasm. Treatment with brefeldin A induces scattering of small TGN38‐immunoreactive vesicles throughout the neuronal cytoplasm and processes, a different response to that observed in non‐neuronat cells.
Cytosolic Ca2+ Binding Proteins during Rat Brain Ageing: Loss of Calbindin and Calretinin in the Hippocampus, with no Change in the Cerebellum
Authors: Villa Antonello , Podini Paola , Panzeri Maria Carla , Racchetti Gabriella , Meldolesi Jacopo
Published: April 2006

The expression of two cytosolic, high affinity Ca2+‐binding proteins, calbindin‐28 and calretinin, has been investigated in the cerebellum and hippocampus of young and old rats (from 12 days to 30 months) by combining immunofluorescence and Western blotting. Three markers, calreticulin (the major Ca2+ binding protein within the lumen of the endoplasmic reticulum), MAP‐2 (a microtubule binding protein concentrated in neuronal dendrites) and synaptophysin (an integral protein of synaptic vesicles), were studied in parallel. In the cerebellar cortex a rise from 12 to 60 days was observed with calbindin‐28 and, especially, calretinin, concentrated in the Purkinje and granule neurons, respectively. The level of expression of the two proteins subsequently remained high and the distribution was unchanged, even in the cerebellum of old animals. A completely different pattern was observed in the hippocampus. Here calretinin, present especially in fibres and interneurons, was abundant in the young, decreased in the adult and reached low values in the old rats. Calbindin‐28 accumulated during growth, especially in a subpopulation of CA1 pyramidal cells and in the mossy fibres of CA3, then declined, although irregularily, during ageing. These changes of the two proteins were more marked in the dorsal and central parts than in the ventral part of the hippocampus. In the same brain areas the levels of expression of the three additional markers and their distribution within neurons and synapses were unchanged by ageing. These last results demonstrate that ageing is not accompanied by a marked loss of hippocampal neurons, yet most of the latter cells appear to modify their general Ca2+ homeostasis as a consequence of the decreased level of cytosolic Ca2+ binding proteins. These events could contribute to the changes in neuronal functioning that have been reported in the aged hippocampus, whereas those of the cerebellum appear to be sustained by other mechanisms.
Defined Neuronal Arborizations by Guided Outgrowth of Leech Neurons in Culture
Authors: Fromherz Peter , Schaden Herbert
Published: April 2006

Identified neurons of Hirudo medicinalis were cultivated on a protein extract of the extracellular matrix (ECM) of the leech. Microscopic patterns of active ECM protein were prepared by UV photolithography using aluminium masks. The shape of the patterns was visualized by a colour pattern formed in a dye–polymer substrate. The neurons were explanted on the root of branched ECM patterns. The patterns guided the outgrowth of neurites along linear lanes and they induced a bifurcation of the neurites under certain conditions. Neurons with a reproducible, regular shape of arborization were obtained within 1–2 days.
The Effects of Dopamine on the Subthreshold Electrophysiological Responses of Rat Prefrontal Cortex Neurons In Vitro
Authors: Geijo‐Barrientos Emilio , Pastore Carlos
Published: April 2006

The rat prefrontal cortex is densely innervated by dopaminergic fibres originating in the mesencephalic ventral tegmental area, and dopamine application in vivo has an inhibitory effect. We have studied the effects of dopamine on the persistent sodium current that is present in prefrontal cortex neurons and on the subthreshold electrophysiological responses generated by that current: a slow depolarization and a fast oscillatory activity. Experiments were made in coronal slices of rat frontal cortex (300–400 pm thickness) and intracellular recordings from regularly spiking cells were obtained with 3 M potassium acetate‐filled glass microelectrodes (80–150 MΩ). Dopamine was applied dissolved in the extracellular medium and, in current‐clamp recordings, reversibly inhibited the slow subthreshold depolarization. Dopamine was ineffective when applied after tetrodotoxin (1 μM) had blocked the action potentials. This inhibition was dose‐dependent in the range of 0.1–10 μM. Dopamine, applied at 10 μM, decreased the steady‐state firing frequency and also inhibited the subthreshold fast oscillatory activity. The currents activated in the subthreshold range were recorded with the single‐electrode voltage‐clamp technique and a clear persistent, tetrodotoxin‐sensitive component was isolated. This component was inhibited by 50% in a reversible way by 20 μM dopamine. These results show that dopamine increases the threshold for spike firing and suggest a mechanism for the inhibitory action of this neurotransmitter in the prefrontal cortex.
Regulation of Rat Sympathetic Nerve Density by Target Tissues and NGF in Maturity and Old Age
Authors: Thrasivoulou C. , Cowen T.
Published: April 2006

Previous studies in our laboratory using a transplantation model have shown that target tissues of some autonomic neurons, including cerebral blood vessels, exert a controlling influence on nerve fibre loss in old age. The present study was undertaken in order to discover whether the influence of targets extends to controlling age changes in specific populations of nerves. In old rats, we have demonstrated a significant decrease of ‐50% in the sympathetic innervation of middle cerebral arteries, using tyrosine hydroxylase‐like immunoreactivity. Following transplantation, tyrosine hydroxylase‐like immunoreactive nerve density on both young and old implanted middle cerebral arteries mirrored the nerve densities seen in normal, non‐transplanted vessels. Furthermore, implanted tissue from old donors became reinnervated with a nerve density ‐50% less than that of young implanted vessels. Treatment of transplants with nerve growth factor, however, was able to reverse these age changes and restore the sympathetic innervation of aged middle cerebral arteries to levels above those seen in young middle cerebral arteries. These results suggest that the pattern and density of sympathetic innervation that the middle cerebral artery receives is determined by the target rather than by the neurons supplying the tissue. The ability of nerve growth factor to induce regrowth in sympathetic neurons innervating ageing target tissues implies that age‐related neuronal atrophy may be due to reduced synthesis or availability of target‐derived neurotrophic factors.
Block of c‐Fos and JunB Expression by Antisense Oligonucleotides Inhibits Light‐induced‐Phase Shifts of the Mammalian Circadian Clock
Authors: Wollnik F. , Brysch W. , Uhlmann E. , Gillardon F. , Bravo R. , Zimmermann M. , Schlingensiepen K. H. , Herdegen T.
Published: April 2006

Light‐induced phase shifts of circadian rhythmic locomotor activity are associated with the expression of c‐Jun, JunB, c‐Fos and FosB transcription factors in the rat suprachiasmatic nucleus, as shown in the present study. In order to explore the importance of c‐Fos and JunB, the predominantly expressed AP‐1 proteins for the phase‐shifting effects of light, we blocked the expression of c‐Fos and JunB in the suprachiasmatic nucleus of male rats, housed under constant darkness, by intracerebroventricular application of 2 μ1 of 1 mM antisense phosphorothioate oligodeoxynucleotides (ASO) specifically directed against c‐fos and JunB mRNA. A light pulse (300 lux for 1 h) at circadian time 15 induced a significant phase shift (by 125 ± 15 min) of the circadian locomotor activity rhythm, whereas application of AS0 6 h before the light pulse completely prevented this phase shift. Application of control nonsense oligodeoxynucleotides had no effect. ASO strongly reduced the light‐induced expression of c‐Fos and JunB proteins. In contrast, light pulses with or without the control nonsense oligodeoxynucleotides evoked strong nuclear c‐Fos and JunB immunoreactivity in the rat suprachiasmatic nucleus. These results demonstrate for the first time that inducible transcription factors such as c‐Fos and JunB are an essential part of fundamental biological processes in the adult mammalian nervous system, e.g. of light‐induced phase shifts of the circadian pacemaker.
A Minimal CGRP Gene Promoter is Inducible by Nerve Growth Factor in Adult Rat Dorsal Root Ganglion Neurons But Not in PC12 Phaeochromocytoma Cells
Authors: Watson Andrea , Ensor Elizabeth , Symes Aviva , Winter Janet , Kendall Giles , Latchman David
Published: April 2006

The calcitonin/CGRP gene is transcribed in thyroid C cells and some neuronal cells but not in other cell types. Although the promoter sequences mediating gene activity in thyroid C cells have been extensively studied, the elements responsible for promoter activity in neuronal cells and its stimulation by nerve growth factor (NGF) have not previously been defined. We report the first use of the calcium phosphate procedure to successfully transfect adult rat dorsal root ganglion neurons, which naturally express the calcitonin/calcitonin gene‐related peptide (CGRP) in an NGF‐inducible manner. This method was used to characterize the elements in the calcitonin/CGRP promoter which are responsible for its basal activity and NGF inducibility in DRG neurons and in PC12 cells, a neuronally derived cell line which does not naturally express the calcitonin/CGRP gene. Although the sequences required for basal activity are similar in each cell type, we show that a minimal calcitonin/CGRP promoter is NGF‐responsive in dorsal root ganglion cells, but that upstream sequences are required for such inducibility in PC12 cells.
The Classification and Properties of Nociceptive Afferent Units from the Skin of the Anaesthetized Pig
Authors: Lynn B. , Faulstroh K. , Pierau F.‐K.
Published: April 2006

The afferent properties of nerve fibres innervating the hairy skin of the pig hind limb were investigated by recording from 142 single units from the saphenous nerve. Identified single units were isolated using maximal electrical stimulation of the nerve trunk. Afferent units were classified on the basis of their responses to a range of stimuli, both thermal (heating to 60°C and cooling to 10°C) and mechanical (air jet, von Frey type filaments with forces of 0.1–250 mN, and strong pressure with a blunt needle). A‐fibre units (conduction velocity 6.3–64 m/s, n= 60) fell into categories that have been described in hairy skin in other mammalian species. Most were mechanoreceptors, although seven typical A‐fibre mechanical nociceptors with large, multipoint fields were also isolated. No cutaneous receptive field could be found for 15% of A‐fibre units. Out of 62 C‐fibre units (conduction velocity 0.49–2 m/s) 40% had no cutaneous field for pressure, heat or cold. Of the C‐fibre units with cutaneous fields, 42% were polymodal nociceptors, 38% were mechanoreceptors with a variety of properties, including some excited by noxious heat, and 19% were heat‐only nociceptors. C‐polymodal nociceptors had large receptive fields up to 12.5 mm across and did not sensitize following strong heating. Twenty units conducted at 2–6.3 m/s, between the main C‐ and A‐fibre bands, and were varied in their responses. Some had properties identical to C‐fibre mechanoreceptors whilst four were sensitive cold thermoreceptors and one was a polymodal nociceptor. Two units were mechanical nociceptors with small receptive fields. The innervation of pig skin thus has some features like that of primates, such as the presence of C‐heat nociceptors and the large receptive fields of C‐polymodal nociceptors. However, other features were like non‐primates (e.g. the minimal heat‐sensitivity of A‐mechanical nociceptors) or were unique (the heat‐sensitive mechanoreceptors and the lack of heat sensitization in C‐polymodal nociceptors from hairy skin).
Expression of the Highly Polysialylated Neural Cell Adhesion Molecule During Postnatal Myelination and Following Chemically Induced Demyelination of the Adult Mouse Spinal Cord
Authors: Oumesmar B. Nait , Vignais L. , Duhamel‐Clerin E. , Avellana‐Adalid V. , Rougon G. , Evercooren A. Baron‐Van
Published: April 2006

We have investigated the expression of the highly polysialylated neural cell adhesion molecule in the mouse spinal cord during postnatal myelination and in the adult after chemically induced demyelination. By double immunohistochemistry, using a monoclonal antibody (anti‐Men B) which specifically recognizes polysialic acid (PSA) units on neural cell adhesion molecule (N‐CAM), and an anti‐myelin basic protein, a caudorostral gradient of expression of PSA‐NCAM was observed at postnatal day 1 (P1), which was inversely related to the gradient of myelination. At P7, PSA‐NCAM labelling decreased relative to P1. In white matter, this decrease was correlated with the progression of myelination. PSA‐NCAM immunoreactivity persisted in as yet unmyelinated structures, i.e. the corticospinal tract, the dorsomedial part of the ventral funiculus and the lateral funiculi, and decreased with the onset of myelination of these structures at P15. In the adult, PSA‐NCAM expression remained in discrete structures, i.e. Iaminae I and II of the dorsal horn and lamina X around the central canal. The ependymal cells and the astrocyte endfeet under the meninges were also labelled. In addition, PSA‐NCAM expression was reinduced on various cells and structures after lysolecithin‐induced demyelination of the adult mouse spinal cord. At early times after demyelination, PSA‐NCAM was expressed on glial cells around the lesion but also at a distance from this zone. Seven days after injection, cellular PSA‐NCAM expression was found around but also within the lesion. This expression was totally abolished 15 days after injection. Double immunohistochemistry for PSA and cell‐specific markers showed that the cells which expressed PSA‐NCAM after demyelination were oligodendrocyte precursors, reactive astrocytes and Schwann cells. PSA‐NCAM re‐expression on all cell types was transient and ceased when myelin repair was accomplished. The spatial and temporal regulation of PSA‐NCAM expression during development and after demyelination suggests a role for PSA‐NCAM in glial plasticity during the myelination and remyelination processes.
Receptor Expression in Olfactory Neurons During Rat Development: In Situ Hybridization Studies
Authors: Strotmann J. , Wanner I. , Helfrich T. , Breer H.
Published: April 2006

In situ hybridization approaches have been employed to explore the onset and time course of odorant receptor expression during the prenatal development of rats. The critical phase for the maturation of the olfactory system from embryonic day (E) 12 to E18 was analysed. The onset of expression of four receptor genes (OR5, OR14, OR37 and OR124) was found between day E12 and E14. In the early phase of development (E14) a distinct receptor subtype was expressed only in a few hundred neurons; the number increased about two‐ to three‐fold within a 2 day interval. From the very beginning, spatial segregation of receptor subtypes in distinct expression zones was observed.