bluuebunny/crossref_metadata_2025 huggingface dataset:
The dopamine D2 receptor subfamily in rat retina: ultrastructural immunogold and in situhybridization studies
Authors: Derouiche Amin , Asan Esther
Published: March 2003

Dopamine, a major neurotransmitter in the vertebrate retina, is released from interplexiform cells and a restricted subset of amacrine cells. Dopamine effects vary between different retinal cell types, most likely due to differences in cell‐specific receptor subtype expression. Identification of cells expressing receptors of the D2‐subfamily (D2R, D3R, D4R) on a light microscopical level has rendered equivocal results, and no information is as yet available concerning the subcellular distribution of receptor protein. In the present study, D2R and D2/3R subtype‐specific antisera, and D2R‐, D3R‐ and D4R‐specific oligonucleotide probes were used for ultrastructural and in situ hybridization analyses of the receptor subtype distribution in the rat retina.

Light and electron microscopy showed that in addition to the known localization of intense D2R‐immunoreactivity in all dopaminergic cells immunoreactive for tyrosine hydroxylase (TH), homogeneous, less intense D2R‐immunoreactivity was also seen throughout the inner plexiform layer (IPL). Ultrastructurally, many additional amacrine cell processes devoid of TH‐immunoreactivity at all levels of the inner plexiform layer were immunoreactive. D2R‐immunoreactivity was found mainly on intracellular vesicles, and immunoreactivity associated with the plasma membrane was always extrasynaptic. No D2R‐immunoreactivity was found in amacrine cell somata postsynaptic to the so‐called dopaminergic ‘ring endings’. Many D2R‐mRNA reactive cells were observed throughout the inner nuclear layer. Morphologically, labelled cells resemble amacrines and bipolars but not horizontal cells. Reactivity with splice variant‐specific oligonucleotide probes suggested that the D2LR variant is the predominant if not the only D2R isoform in the rat retina. D2R‐mRNA reactivity was not observed in other retinal layers, in particular not in photoreceptor inner segments, which displayed D4R‐mRNA reactivity. D3R‐mRNA reactivity was not detected.

The results indicate that D2‐like responses are mediated through the D2R subtype, by an autoreceptor mechanism in dopaminergic cells, and by volume transmission in non‐dopaminergic cells of the inner retina. D2‐like responses in photoreceptors probably represent D4R activation.
Conditioned enhancement and suppression in the developing auditory midbrain
Authors: Thornton Sally K. , Semple Malcolm N. , Sanes Dan H.
Published: March 2003

Neural responses in the adult central auditory system to binaural stimuli can be altered by preceding acoustic events, including auditory motion. To determine whether the juvenile auditory system also exhibits this feature, we have examined interaural level difference (ILD) processing in the developing gerbil. A long binaural stimulus was followed without interruption by modulation of the level difference (virtual acoustic motion), which in turn was followed smoothly by a new steady state ILD. Auditory responses of single neurons in the inferior colliculus (IC) were assessed for sensitivity to the final steady state ILD. The response of EI neurons (excited by contralateral stimulation and inhibited ipsilaterally) was examined at postnatal (P) days 17–18, P24–25, and in adult animals. In adult animals, a sudden reduction of the inhibitory stimulus level resulted in a long‐lasting (median = 4.3 s) enhanced discharge rate (conditioned enhancement). In P17–18 animals, conditioned enhancement only lasted for 1.2 s. When the inhibitory stimulus level was suddenly increased, adult neurons often displayed a conditioned suppression of discharge rate (median = 4.5 s), whereas P17–18 neurons remained suppressed for a much briefer period (median = 1.2 s). Moreover, the difference between conditioned responses and control discharge rates was three–four times greater in adult neurons compared to those recorded in P17–25 animals.

Because conditioned responses are sensitive to the relative balance of contralateral excitation and ipsilateral inhibition, we examined the relationship between excitatory and inhibitory thresholds. In adult animals, excitatory thresholds were an average of 12 dB lower than inhibitory thresholds, while at P17–25 excitatory and inhibitory thresholds were roughly the same. These results indicate that computational properties of juvenile and adult IC neurons differ quantitatively, and this may reflect an imbalance between excitation and inhibition. The developmental differences described herein may limit the ability of young animals to locate a sound source with the latency and accuracy of an adult.
Rhombomere origin plays a role in the specificity of cranial motor axon projections in the chick
Authors: Warrilow Joanne , Guthrie Sarah
Published: March 2003

Guidance of cranial motor axons to their targets conforms to a segmental plan in the chick embryo. Trigeminal motor neurons lie within rhombomeres 2 and 3 and project via an exit point in rhombomere 2 to innervate the first branchial arch. Facial motor neurons lie within rhombomeres 4 and 5 and grow out via an exit point in rhombomere 4 to innervate the second branchial arch. We have investigated the axial level‐specific matching of motor neurons and branchial arches using donor to host transplantation in avian embryos. Previous work has shown that rostrocaudal reversal of a single hindbrain segment (rhombomere 3) leads to misprojection of a contingent of trigeminal axons via the facial nerve exit point. Using the same experimental manipulation in chick embryos and quail–chick chimaeras, we have analysed the pathways of these aberrant projections. We have found that in the majority of embryos analysed from stage 19 to 31, trigeminal axons from the transplanted rhombomere projected towards second branchial arch muscles, in addition to their normal first arch muscle targets. However, from stage 32 to 36, aberrant projections to second arch‐derived muscles were detected only in a small minority of embryos. These experiments show that trigeminal motor neurons show a lack of specificity in their early projection into the periphery but that inappropriate projections may be later eliminated. This suggests that segmental mechanisms intrinsic to the hindbrain specify motor neurons with respect to their eventual innervation pattern.
Sprouting and regeneration after pyramidotomy and blockade of the myelin‐associated neurite growth inhibitors NI 35/250 in adult rats
Authors: Raineteau Olivier , Z'Graggen Werner J. , Thallmair Michaela , Schwab Martin E.
Published: March 2003

After a selective unilateral lesion of the corticospinal tract (CST) at the level of the brainstem (pyramidotomy) and neutralization of the myelin associated neurite growth inhibitors NI‐35/250 with the monoclonal antibody (mAb) IN‐1, we had previously observed a strong behavioural recovery in parallel with an enhanced structural plasticity of the lesioned as well as the unlesioned CST. The present study focuses on the regenerative response of the cut CST axons at the lesion site in these adult rats. The results show a strong enhancement of regenerative sprouting of CST fibres by treatment with the mAb IN‐1. Successful elongation of these sprouts through the pyramidal decussation and into the cervical spinal cord was also dependent on the presence of this antibody. In the spinal cord, regenerating fibres were rarely found in the position of the former CST; most of the fibres were distributed seemingly randomly over the entire lateral extent of the spinal cord.
Pre‐ or post‐training injection of buspirone impaired retention in the inhibitory avoidance task: involvement of amygdala 5‐HT1A receptors
Authors: Liang K. C.
Published: March 2003

The present study investigated the effect of buspirone on memory formation in an aversive learning task. Male Wistar rats were trained on the inhibitory avoidance task and tested for retention 1 day after training. They received peripheral or intra‐amygdala administration of buspirone or other 5‐HT1A drugs either before or after training. Results indicated that pretraining systemic injections of buspirone caused a dose‐dependent retention deficit; 5.0 mg/kg had a marked effect and 1.0 mg/kg had no effect. Post‐training injections of the drug caused a time‐dependent retention deficit, which was not due to a state‐dependent effect on retrieval. When training in the inhibitory avoidance task was divided into a context‐training phase and a shock‐training phase, buspirone impaired retention only when administered in the shock‐training phase, suggesting that the drug influenced memory processing of affective events. Further results indicated that post‐training intra‐amygdala infusion of buspirone or the 5‐HT1A agonist 8‐hydroxy‐di‐n‐propylaminotetralin (8‐OH‐DPAT) caused a time‐dependent and dose‐dependent retention deficit. Post‐training intra‐amygdala infusion of the 5‐HT1A antagonist WAY100635 (N‐(2‐(4‐(2‐methoxyphenyl)‐1‐piperazinyl)‐N‐(2‐pyridyl) cyclohexane carboxamine maleate) attenuated the memory‐impairing effects of buspirone. These findings suggest that buspirone may modulate memory storage processes in the inhibitory avoidance task through an action on amygdaloid 5‐HT1A receptors.
ARNT2, a transcription factor for brain neuron survival?
Authors: Drutel Guillaume , Héron Anne , Kathmann Markus , Gros Claude , Macé Séverine , Plotkine Michel , Schwartz Jean‐Charles , Arrang Jean‐Michel
Published: March 2003

The processes responsible for the limited ability to divide and long survival of neurons are not well understood but may involve aryl hydrocarbon receptor nuclear translocator 2 (ARNT2), a recently identified protein, apparently belonging to the basic helix–loop–helix superfamily of transcription factors, which is expressed almost exclusively in brain during the whole lifetime. In agreement, we show, in the rat, that ARNT2 immunoreactivity could be observed only within nuclei of brain neurons and of dividing and neuronal PC12 cells, a localization consistent with a role in transcription regulation. Cell death elicited either by focal ischaemia in brain or oxidative stress in PC12 cells was largely preceded by an almost complete suppression of ARNT2 expression. In contrast, when PC12 cell cycle progression was impaired, ARNT2 expression was enhanced. Finally, the downregulation of ARNT2 levels induced by antisense oligonucleotides prevented PC12 cell proliferation and induced apoptosis. These observations support the hypothesis that ARNT2 is a neuronal transcription factor, regulating cell cycle progression and preventing cell death, whose sustained expression might ensure brain neuron survival.
Presence of a reduced opioid response in interleukin‐6 knock out mice
Authors: Bianchi Mauro , Maggi Roberto , Pimpinelli Federica , Rubino Tiziana , Parolaro Daniela , Poli Valeria , Ciliberto Gennaro , Panerai Alberto E. , Sacerdote Paola
Published: March 2003

Cytokines are known to influence neuronal functions. The purpose of this study was to investigate the putative role of the cytokine interleukin‐6 (IL‐6) in the pathways involved in opioid‐mediated responses, by using IL‐6‐deficient mice. We reported that with a thermal stimulus IL‐6‐knock‐out (IL‐6KO) mice presented nociceptive thresholds similar to those measured in their controls. However, they showed a reduced analgesic response both to the restraint stress and to the administration of low doses of morphine. Hypothalamic levels of the opioid peptide β‐endorphin were significantly higher in IL‐6KO mice than they were in their controls. The development of tolerance to the analgesic effect of morphine was more rapid in IL‐6‐deficient mice than in wild‐type controls. Binding experiments showed that the number of opioid receptors in the midbrain, but not in the hypothalamus, decreased in IL‐6KO mice. Autoradiographic binding analysis revealed that the density of μ receptors diminished while the δ‐opioid receptors did not. These results suggest that IL‐6 is necessary for a correct development of neuronal mechanisms involved in the response to both endogenous and exogenous opiates.
Behavioural consequences of oligodendrocyte progenitor cell transplantation into experimental demyelinating lesions in the rat spinal cord
Authors: Jeffery N. D. , Crang A. J. , O'Leary M. T. , Hodge S. J. , Blakemore W. F.
Published: March 2003

Glial cell transplantation has the potential to be developed into a clinical treatment for human demyelinating diseases because of its demonstrated efficacy in remyelinating experimentally demyelinated axons. As a step towards clinical application it is necessary to demonstrate that the procedure is safe and efficacious in promoting behavioural recovery. In this study we transplanted glial cell progenitors into demyelinating lesions induced by intraspinal injection of ethidium bromide in the rat. Locomotor function after transplantation was assessed using a beam‐walking test that has previously been shown able to detect deficits associated with demyelination in the dorsal funiculus of the rat spinal cord. Two groups of animals with transplants were examined. In one group, spontaneous remyelination was prevented by exposure of the lesion to 40 Gy of X‐irradiation; in the other, male glial cells were transplanted into nonirradiated female receipients, permitting their identification by use of a probe specific to the rat Y chromosome. Following transplantation, there was severe axon loss in a large proportion of the irradiated animals and those affected did not recover normal behavioural function. In contrast, both the small proportion of the irradiated group that sustained only mild axon loss and the nonirradiated recipients of transplants recovered normal function on our behavioural test. We conclude that glial cell transplantation is able to reverse the functional deficits associated with demyelination, provided axonal loss is minimal.
Long‐lasting substance‐P‐mediated modulation of NMDA‐induced rhythmic activity in the lamprey locomotor network involves separate RNA‐ and protein‐synthesis‐dependent stages
Authors: Parker David , Grillner Sten
Published: March 2003

Bath application of the tachykinin neuropeptide substance P (1 μm) for 10 min causes long‐lasting (> 24 h) modulation of the frequency and regularity of NMDA‐evoked locomotor bursts in the lamprey. The change in burst frequency has an induction phase (< 2 h), which depends on the potentiation of NMDA responses and an increase in intracellular calcium levels, and a maintenance phase (> 2 h), that is blocked by translational protein synthesis inhibitors. Here, the maintenance phase has been examined further. Unlike translation inhibitors, the transcription inhibitors actinomycin D and 5,6‐dichlorobenzimidazole riboside (DRB) failed to reverse the change in burst frequency 2–3 h after substance P application, suggesting that the protein synthesized at this time does not require de novo RNA synthesis. Transcription inhibitors, however, reversed the change in burst frequency 15–24 h after substance P application, as did brefeldin A, which disrupts the Golgi complex and thus interferes with the post‐translational transport of proteins. The change in burst regularity was unaffected by transcription or translation inhibitors, but was partially reversed by protein kinase A inhibitors applied 2.5–8 h after substance P. The glycoprotein synthesis inhibitor 2‐deoxygalactose did not affect the changes in burst frequency or burst regularity. These results suggest that there are two phases to the maintenance of the change in burst frequency: an intermediate protein‐, but not RNA‐, synthesis‐dependent phase, and a final RNA‐synthesis‐dependent phase. The change in burst regularity is protein‐synthesis‐independent, but may depend on activation of protein kinase A for at least 8 h after substance P application.
Protection and regeneration of nigral dopaminergic neurons by neurturin or GDNF in a partial lesion model of Parkinson's disease after administration into the striatum or the lateral ventricle
Authors: Rosenblad Carl , Kirik Deniz , Devaux Brigitte , Moffat Barbara , Phillips Heidi S. , Björklund Anders
Published: March 2003

Both glial cell line‐derived neurotrophic factor (GDNF) and its recently discovered congener, neurturin (NTN), have been shown to excert neuroprotective effects on lesioned nigral dopamine (DA) neurons when administered at the level of the substantia nigra. In the present study, we have explored the relative in vivo potency of these two neurotrophic factors using two alternative routes of administration, into the striatum or the lateral ventricle, which may be more relevant in a clinical setting. In rats subjected to an intrastriatal (IS) 6‐hydroxydopamine (6‐OHDA) lesion, GDNF and NTN were injected every third day for 3 weeks starting on the day after the 6‐OHDA injection. GDNF provided almost complete (90–92%) protection of the lesioned nigral DA neurons after both IS and intracerebroventricular (ICV) administration. NTN, by contrast, was only partially effective after IS injection (72% sparing) and totally ineffective after ICV injection. Although the trophic factor injections protected the nigral neurons from lesion‐induced cell death, the level of expression of the phenotypic marker, tyrosine hydroxylase (TH), was markedly reduced in the rescued cell bodies. The extent of 6‐OHDA‐induced DA denervation in the striatum was unaffected by both types of treatment; consistent with this observation, the high rate of amphetamine‐induced turning seen in the lesioned control animals was unaltered by either GDNF or NTN treatment. In the GDNF‐treated animals, and to a lesser extent also after IS NTN treatment, prominent axonal sprouting was observed within the globus pallidus, at the level where the lesioned nigrostriatal axons are known to end at the time of onset of the neurotrophic factor treatment. The results show that GDNF is highly effective as a neuroprotective and axon growth‐stimulating agent in the IS 6‐OHDA lesion model after both IS and ICV administration. The lower efficacy of NTN after IS, and particularly ICV, administration may be explained by the poor solubility and diffusion properties at neutral pH.
Mutual regulation between the intercellular messengers nitric oxide and brain‐derived neurotrophic factor in rodent neocortical neurons
Authors: Xiong Huabao , Yamada Kiyofumi , Han Daikenn , Nabeshima Toshitaka , Enikolopov Grigori , Carnahan Josette , Nawa Hiroyuki
Published: March 2003

The diffusible factors, nitric oxide (NO) and brain‐derived neurotrophic factor (BDNF) are both suggested to be intercellular messengers that have similar synaptic activities and developmental influences in the brain. In the present study, we have analysed their mutual regulation with respect to their production in rodent neocortical neurons. Some of the cultured rat neocortical neurons exhibited immunoreactivity for both neuronal NO synthase (NOS) and the BDNF receptor trkB. Neuronal NOS appeared to be activated autonomously and produced NO in culture as monitored by nitrite accumulation. Inhibition of the endogenous NO production in culture by a NOS inhibitor, NG‐monomethyl‐l‐arginine (NMMA), enhanced basal expression of BDNF mRNA and protein. Similarly, cerebroventricular administration of another NOS inhibitor, N‐ω‐nitro‐l‐arginine methylester (l‐NAME), but not d‐NAME or saline, increased BDNF content in the neocortex. In the opposite direction, however, BDNF appeared to function as a positive regulator for NO synthesis. Addition of BDNF upregulated the neuronal NOS expression as well as NO production in neocortical culture. In agreement, BDNF knock‐out mice exhibited significant impairment of neuronal NOS expression in the neocortex. Taken together, these observations suggest that the trans‐synaptic signalling molecules, NO and BDNF, influence the production of each other and mutually regulate the strength of their intercellular communications.
A novel P0 glycoprotein transgene activates expression of lacZ in myelin‐forming Schwann cells
Authors: Feltri M. Laura , D'Antonio Maurizio , Quattrini Angelo , Numerato Rita , Arona Marta , Previtali Stefano , Chiu Shing‐Yan , Messing Albee , Wrabetz Lawrence
Published: March 2003

P0 glycoprotein, the most abundant protein in peripheral nerve, is expressed specifically in the Schwann cell lineage. Upstream of the rat P0 gene 1.1 kb of DNA can activate expression of cDNAs specifically in Schwann cells in transgenic mice. However, the expression of P0 promoter‐based transgenes has been inconsistent. As much as 9 kb of 5′ flanking sequence fused to lacZ never yielded detectable levels of β‐galactosidase in multiple lines of mice. We describe transgenic mice that express lacZ in peripheral nerve, using the complete mouse P0 gene, including 6 kb of 5′ flanking sequence, all exons and introns, and the natural polyadenylation signal. This vector activated lacZ expression specifically in cultured Schwann cells, and myelin‐forming Schwann cells in four out of six transgenic lines. Transgene expression paralleled that of the endogenous P0 gene, both during development and after Wallerian degeneration. lacZ expression was lower than endogenous P0 expression, and was not detected in neural crest or Schwann cell precursors, where low levels of P0 mRNA are present. However, when the same vector contained a small myc tag instead of the 3.2‐kb lacZ insert, the resulting transgenic mRNA was expressed at levels comparable to endogenous P0 mRNA. These data suggest that intragenic or 3′ flanking sequences are necessary to generate the remarkable levels of endogenous P0 gene expression.
Region‐dependent difference in the sleep‐promoting potency of an adenosine A2A receptor agonist
Authors: Satoh Shinsuke , Matsumura , Hitoshi , Koike Nobuyo , Tokunaga , Yoshimitsu , Maeda Toshihiro , Hayaishi Osamu
Published: March 2003

The present study has demonstrated that the sleep‐promoting potency of 2‐[p‐(2‐carboxyethyl)phenethylamino]‐5′‐N‐ethylcarboxamido adenosine (CGS21680), a selective agonist for the adenosine A2A receptor, varies depending on the location of the administration. CGS21680 was continuously administered to rats through a chronically implanted cannula for 6 h during their active phase. The tip of the cannula was located in the subarachnoid space or the brain ventricle neighbouring the established brain areas implicated in the regulation of sleep–wake phenomena, i.e. rostral basal forebrain, medial preoptic area, lateral preoptic area, posterior hypothalamus, and dorsal tegmentum of the pons and medulla. At an infusion rate of 2.0 pmol/min, the magnitude of increase in non‐rapid eye movement sleep varied from 14 min (a 15% increase) to 96 min (a 103% increase), and those of rapid eye movement sleep varied from 6 min (a 40% increase) to 28 min (a 264% increase) from the respective baseline values. The largest increases in both types of sleep occurred when CGS21680 was administered to the subarachnoid space underlying the rostral basal forebrain. These findings were interpreted to mean that the major, if not the only, site responsible for the CGS21680‐inducing sleep was located in or near the rostral basal forebrain. This interpretation was supported by the findings that the administration of CGS21680 to the rostral basal forebrain produced predominant expression of Fos within the shell of the nucleus accumbens and the medial portion of the olfactory tubercle, and that the microdialysis perfusion of CGS21680 into the shell of the nucleus accumbens also exhibited a sleep‐promoting effect.
Neuronal and behavioural abnormalities in striatal function in DARPP‐32‐mutant mice
Authors: Hiroi Noboru , Fienberg Allen A. , Haile Colin N. , Alburges Mario , Hanson Glen R. , Greengard Paul , Nestler Eric J.
Published: March 2003

We investigated the role of the protein phosphatase inhibitor, dopamine‐ and cAMP‐regulated phosphoprotein of 32 kDa (DARPP‐32), in the expression of striatal neuropeptides and in biochemical and behavioural responses to repeated cocaine administration, using DARPP‐32 knock‐out mice. The striatum of DARPP‐32‐mutant mice showed heightened substance‐P‐like immunoreactivity, but normal levels of other neuropeptides. Repeated cocaine administration increased levels of ΔFosB, a Fos family transcription factor, in the striatum of wild‐type mice, and this increase was abolished in DARPP‐32‐mutant mice. Cocaine (20 mg/kg) acutely induced the same level of locomotor activity in the mutant and wild‐type mice, but the mutants showed a higher rate of locomotor sensitization to repeated cocaine exposures. These data show that DARPP‐32 is involved in regulating substance P expression in the striatonigral pathway, and in biochemical and behavioural plasticity with chronic administration of cocaine.
Acetylcholine synthesis and quantal release reconstituted by transfection of mediatophore and choline acetyltranferase cDNAs
Authors: Bloc Alain , Bugnard Elisabeth , Dunant Yves , Falk‐Vairant Jean , Israël Maurice , Loctin Françoise , Roulet Emmanuelle
Published: March 2003

Neuroblastoma N18TG‐2 cells cannot synthesize or release acetylcholine (ACh), and do not express proteins involved in transmitter storage and vesicle fusion. We restored some of these functions by transfecting N18TG‐2 cells with cDNAs of either rat choline acetyltransferase (ChAT), or Torpedo mediatophore 16‐kDa subunit, or both. Cells transfected only with ChAT synthesized but did not release ACh. Cells transfected only with mediatophore expressed Ca2+‐dependent ACh release provided they were previously filled with the transmitter. Cell lines produced after cotransfection of ChAT and mediatophore cDNAs released the ACh that was endogenously synthesized. Synaptic‐like vesicles were found neither in native N18TG‐2 cells nor in ChAT‐mediatophore cotransfected clones, where all the ACh content was apparently cytosolic. Furthermore, restoration of release did not result from enhanced ACh accumulation in intracellular organelles consecutive to enhanced acidification by V‐ATPase, as Torpedo 16 kDa transfection did not increase, but decreased the V‐ATPase‐driven proton transport. Using ACh‐sensitive Xenopus myocytes for real‐time recording of evoked release, we found that cotransfected cells released ACh in a quantal manner. We compared the quanta produced by ChAT‐mediatophore cotransfected clones to those produced by clones transfected with mediatophore alone (artificially filled with ACh). The time characteristics and quantal size of currents generated in the myocyte were the same in both conditions. However, cotransfected cells released a larger proportion of their initial ACh store. Hence, expression of mediatophore at the plasma membrane seems to be necessary for quantal ACh release; the process works more efficiently when ChAT is operating as well, suggesting a functional coupling between ACh synthesis and release.
Neuronal correlates of vestibulo‐ocular reflex adaptation in the alert guinea‐pig
Authors: Serafin Mauro , Ris Laurence , Bernard Patrick , Muhlethaler Michel , Godaux Emile , Vidal Pierre‐Paul
Published: March 2003

The spiking behaviour of 66 second‐order vestibular neurons was studied in alert, chronically prepared guinea‐pigs during the horizontal vestibulo‐ocular reflex (VOR). Among the 66 studied neurons, seven were held long enough (> 1 h) to compare their spiking behaviour before and after a training procedure inducing a decrease in the gain of the VOR. When tested in darkness following adaptation, five of them showed a significant decrease of their sensitivity to head rotation. However, the resting discharge of these five neurons remained unchanged. This suggests that VOR adaptation is mediated not only by changes in synaptic efficacities but also by modifications in the spike generator which transforms synaptic inputs into a pattern of action potentials.
Lithium‐pilocarpine‐induced status epilepticus produces necrotic neurons with internucleosomal DNA fragmentation in adult rats
Authors: Fujikawa Denson G. , Shinmei Steve S. , Cai Baiyuan
Published: March 2003

Prolonged and continuous epileptic seizures [status epilepticus (SE)] produce a widespread pattern of neuronal death, primarily in limbic brain regions. Because it has been suggested that seizure‐induced neuronal death may be apoptotic in nature, we tested the hypothesis that lithium‐pilocarpine‐induced status epilepticus (LPCSE) produces apoptotic neurons. LPCSE lasting 3 h was induced in male Wistar rats which were allowed to recover for 24 or 72 h before perfusion‐fixation. Neuronal death was assessed by light microscopy with the haematoxylin‐and‐eosin stain (H&E), with in situ DNA nick‐end labelling (TUNEL stain), by electron microscopy, and by agarose gel electrophoresis of DNA extracted from vulnerable brain regions. Ultrastructurally, acidophilic neurons identified with H&E were dark, shrunken and necrotic in appearance, exhibiting pyknotic nuclei, irregular, dispersed chromatin clumps and cytoplasmic vacuolization. No cells with apoptotic features were seen. Acidophilic neurons were found in 21 out of 23 brain regions examined, and comprised 26–45% of the total number of neurons examined. A subset of these neurons (< 10% of the total number of neurons) were TUNEL‐positive at 72 h, but not 24 h, after SE. Internucleosomal DNA cleavage (DNA ‘laddering’) was found in the six brain regions examined ultrastructurally 24 and 72 h after SE. These results indicate that, in adult rats, LPCSE produces neuronal injury with the appearance of necrosis rather than apoptosis. The necrotic neurons show nuclear pyknosis, chromatin condensation and internucleosomal DNA fragmentation, confirming the nonspecificity of these nuclear changes. Internucleosomal DNA cleavage and other programmed cell death mechanisms can be activated by SE in neurons which become necrotic.
Cells defective in sphingolipids biosynthesis express low amounts of muscle nicotinic acetylcholine receptor
Authors: Roccamo A. M. , Pediconi M. F. , Aztiria E. , Zanello L. , Wolstenholme A. , Barrantes F. J.
Published: March 2003

The properties of the nicotinic acetylcholine receptor (AChR) are modulated by its lipid microenvironment. Studies of such modulation are hampered by the cell's homeostatic mechanisms that impede sustained modification of membrane lipid composition. We have devised a novel strategy to circumvent this problem and study the effect of changes in plasma membrane lipid composition on the functional properties of AChR. This approach is based on the stable transfection of AChR subunit cDNAs into cells defective in a specific lipid metabolic pathway. In the present work we illustrate this new strategy with the successful transfection of a temperature‐sensitive Chinese hamster ovary (CHO) cell line, SPB‐1, with the genes corresponding to the four adult mouse AChR subunits. The new clone, SPB‐1/SPH, carries a mutation of the gene coding for serine palmitoyl transferase, the enzyme that catalyses the first step in sphingomyelin (Sph) biosynthesis. This defect causes a decrease of Sph de novo synthesis at non‐permissive temperatures. The IC50 for inhibition of α‐BTX binding with the agonist carbamoylcholine exhibited values of 3.6 and 2.7 μm in the wild‐type and Sph‐deficient cell lines, respectively. The corresponding IC50 values for the competitive antagonist d‐tubocurarine (d‐TC) were 2.8 and 3.4 μm, respectively. No differences in single‐channel properties were observed between wild‐type and mutant cell lines grown at the non‐permissive, lipid defect‐expressing temperature using the patch‐clamp technique. Both cells exhibited two open times with mean values of 0.35 ± 0.05 and 1.78 ± 0.2 ms at 12 °C. Taken together, these results suggest that the AChR is expressed as the complete heteroligomer. However, only 10–20% of the total AChR synthesized reached the surface membrane in the mutant cell line and exhibited a higher metabolic turnover, with a half‐life about 50% shorter than the wild‐type cells. When control CHO‐K1/A5 cells were treated with fumonisin B1, an inhibitor of sphingosine (sphinganine) N‐acetyltransferase (ceramide synthase), a 45.5% decrease in cell surface AChR expression was observed. The results suggest that sphingomyelin deficiency conditions AChR targeting to the plasma membrane.
Anatomical and functional demonstration of a multisynaptic suprachiasmatic nucleus adrenal (cortex) pathway
Authors: Buijs Ruud M. , Wortel Joke , Van Heerikhuize Joop J. , Feenstra Matthijs G. P. , Ter Horst Gert J. , Romijn Herms J. , Kalsbeek Andries
Published: March 2003

In view of mounting evidence that the suprachiasmatic nucleus (SCN) is directly involved in the setting of sensitivity of the adrenal cortex to ACTH, the present study investigated possible anatomical and functional connections between SCN and adrenal. Transneuronal virus tracing from the adrenal revealed first order labelling in neurons in the intermedio‐lateral column of the spinal cord that were shown to receive an input from oxytocin fibres and subsequently second‐order labelling in neurons of the autonomic division of the paraventricular nucleus. The latter neurons were shown to receive an input from vasopressin or vasoactive intestinal peptide (VIP) containing SCN efferents. The true character of this SCN input to second‐order neurons was also demonstrated by the fact that third‐order labelling was present within the SCN, vasopressin or VIP neurons. The functional presence of the SCN–adrenal connection was demonstrated by a light‐induced fast decrease in plasma corticosterone that could not be attributed to a decrease in ACTH. Using intact and SCN‐lesioned animals, the immediate decrease in plasma corticosterone was only observed in intact animals and only at the beginning of the dark period. This fast decrease of corticosterone was accompanied by constant basal levels of blood adrenaline and noradrenaline, and is proposed to be due to a direct inhibition of the neuronal output to the adrenal cortex by light‐mediated activation of SCN neurons. As a consequence, it is proposed that the SCN utilizes neuronal pathways to spread its time of the day message, not only to the pineal, but also to other organs, including the adrenal, utilizing the autonomic nervous system.
Alterations in the morphology of dendrites and dendritic spines in the nucleus accumbens and prefrontal cortex following repeated treatment with amphetamine or cocaine
Authors: Robinson Terry E. , Kolb Bryan
Published: March 2003

Repeated treatment with psychostimulant drugs produces changes in brain and behaviour that far outlast their initial neuropharmacological actions. The nature of persistent drug‐induced neurobehavioural adaptations is of interest because they are thought to contribute to the development of dependence and addiction, and other forms of psychopathology, e.g. amphetamine psychosis. There are many reports that psychostimulants produce biochemical adaptations in brain monoamine systems, especially dopamine systems. The purpose of the present study was to determine if they might also alter the morphology of neurons in brain regions that receive monoaminergic innervation. Rats were given repeated injections of either amphetamine or cocaine, or, to control for general motor activity, allowed access to a running wheel. They were then left undisturbed for 24–25 days before their brains were processed for Golgi–Cox staining. Treatment with either amphetamine or cocaine (but not wheel running experience) increased the number of dendritic branches and the density of dendritic spines on medium spiny neurons in the shell of the nucleus accumbens, and on apical dendrites of layer V pyramidal cells in the prefrontal cortex. Cocaine also increased dendritic branching and spine density on the basilar dendrites of pyramidal cells. In addition, both drugs doubled the incidence of branched spines on medium spiny neurons. It is suggested that some of the persistent neurobehavioural consequences of repeated exposure to psychostimulant drugs may be due to their ability to reorganize patterns of synaptic connectivity in the nucleus accumbens and prefrontal cortex.
Apoptotic cell death of proliferating neuroepithelial cells in the embryonic retina is prevented by insulin
Authors: Díaz Begoña , Pimentel Belén , De Pablo Flora , De La Rosa Enrique J.
Published: March 2003

The role of programmed cell death is well established for connecting neurons. Conversely, much less is known about apoptosis affecting proliferating neuroepithelial cells. Chick retina from day 4 to day 6 of embryonic development (E), essentially proliferative, presented a defined distribution of apoptotic cells during normal in vivo development, as visualized by TdT‐mediated dUTP nick end labelling (TUNEL). Insulin, expressed in the early chick embryonic retina as proinsulin, attenuated apoptosis in growth factor‐deprived organotypic culture of E5 retina. This effect was demonstrated both by TUNEL and by staining of pyknotic nuclei, as well as by release of nucleosomes. Application of a 1 h [methyl‐3H]thymidine pulse in ovo at E5, followed by organotypic culture in the presence or absence of insulin, showed that this factor alone decreased the degradation of labelled DNA to nucleosomes by 40%, as well as the proportion of labelled pyknotic nuclei. Both features are a consequence of apoptosis affecting neuroepithelial cells, which were in S‐phase or shortly after. In addition, when the E5 embryos were maintained in ovo after the application of [methyl‐3H]thymidine, 70% of the apoptotic retinal cells were labelled, indicating the in vivo prevalence of cell death among actively proliferating neuroepithelial cells. Apoptotic cell death is thus temporally and spatially regulated during proliferative stages of retinal neurogenesis, and embryonic proinsulin is presumably an endogenous protective factor.
Calcium‐dependent mechanisms involved in presynaptic long‐term depression at the hippocampal mossy fibre–CA3 synapse
Authors: Kobayashi Katsunori , Manabe Toshiya , Takahashi Tomoyuki
Published: March 2003

Long‐term potentiation (LTP) and long‐term depression (LTD) are induced presynaptically at the hippocampal mossy fibre–CA3 synapse. Activation of presynaptic metabotropic glutamate receptors (mGluRs) is necessary, but not sufficient for the LTD induction. Using mouse hippocampal slices, we attempted to identify additional presynaptic factors involved in the induction of mossy fibre LTD. Suppression of a rise in the presynaptic intracellular Ca2+ concentration ([Ca2+]i) with a membrane‐permeable Ca2+ chelator, 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid tetraacetoxymethyl ester (BAPTA‐AM), reduced the magnitude of LTD, whereas an increase in Ca2+ influx induced LTD, suggesting that an elevation of presynaptic [Ca2+]i is crucial for the LTD induction. A broad‐spectrum protein kinase inhibitor, H‐7, blocked LTD without affecting a presynaptic inhibition induced by an mGluR agonist. Furthermore, LTD was reduced by an inhibitor of calmodulin or Ca2+/calmodulin‐dependent protein kinases. Thus, we conclude that mossy fibre LTD requires an increase in presynaptic [Ca2+]i and subsequent activation of Ca2+/calmodulin‐dependent protein kinases. Because mossy fibre LTP may also require a rise in presynaptic [Ca2+]i, bidirectional long‐term plasticity at the mossy fibre synapse is likely to be regulated by presynaptic Ca2+‐dependent processes.
Adrenalectomy increases neurogenesis but not PSA‐NCAM expression in aged dentate gyrus
Authors: Montaron M. F. , Petry K. G. , Rodriguez J. J. , Marinelli M. , Aurousseau C. , Rougon G. , Le Moal M. , Abrous D. N.
Published: March 2003

Ageing is accompanied by a decline in neurogenesis and in polysialylated isoforms of neural cell adhesion molecule (PSA‐NCAM) expression within the hippocampus and by elevated basal levels of circulating corticosterone. In a companion study, we demonstrated that suppression of corticosterone by adrenalectomy increased neurogenesis and PSA‐NCAM expression in the dentate gyrus of adult rats. Here we show that adrenalectomy increased neurogenesis in this structure in old rats, as measured by the incorporation of 5‐bromo‐2′‐deoxyuridine in neuronal progenitors. This effect was prevented by corticosterone replacement. In contrast, PSA‐NCAM expression remained unchanged in comparison with controls. Thus, in the aged brain, stem cells are still present and able to enter the cell cycle. This may point to ways of protecting or treating age‐related cognitive impairments.
BDNF stimulates expression, activity and release of tissue‐type plasminogen activator in mouse cortical neurons
Authors: Fiumelli Hubert , Jabaudon Denis , Magistretti Pierre J. , Martin Jean‐Luc
Published: March 2003

Brain‐derived neurotrophic factor (BDNF) is a neurotrophic factor involved in neuronal development and synaptic plasticity. Although the physiological effects of BDNF have been examined in detail, target proteins which mediate its actions remain largely unknown. Here, we report that BDNF stimulates the expression of tissue‐type plasminogen activator (tPA) in primary cultures of cortical neurons in a time‐ and concentration‐dependent manner. Among the other members of the neurotrophin family, neurotrophin‐4 (NT‐4) and to a lesser extent neurotrophin‐3 (NT‐3) also increased tPA mRNA expression, while nerve growth factor (NGF) was devoid of any effect. Induction of tPA expression by BDNF is accompanied by an increase in the proteolytic activity of tPA associated with cortical neurons and a release of tPA into the extracellular space. Release of tPA induced by BDNF depends on extracellular Ca2+ since it is markedly reduced in the presence of ethylene glycol‐bis(β‐aminoethylether)‐N,N,N′,N′‐tetraacetic acid (EGTA). Up‐regulation of tPA expression by BDNF is followed by the induction of plasminogen activator inhibitor 2 (PAI‐2), an inhibitor of tPA. Together these results suggest that activation of tPA by BDNF may contribute to structural changes associated with neuronal development or synaptic plasticity.
Alterations in the expression of GABAA receptor subunits in cerebellar granule cells after the disruption of the α6 subunit gene
Authors: Nusser Zoltan , Ahmad Zahida , Tretter Verena , Fuchs Karoline , Wisden William , Sieghart Werner , Somogyi Peter
Published: March 2003

Any given subunit of the heteromultimeric type‐A γ‐aminobutyric acid (GABA) GABAA receptor may be present in several receptor subtypes expressed by individual neurons. Changes in the expression of a subunit may result in differential changes in the expression of other subunits depending on the subunit composition of the receptor subtype, leading to alterations in neuronal responsiveness to GABA. We used the targeted disruption of the α6 subunit gene to test for changes in the expression of other GABAA receptor subunits. Immunoprecipitation and ligand binding experiments indicated that GABAA receptors were reduced by ≈ 50% in the cerebellum of α6 –/– mice. Western blot experiments indicated that the α6 subunit protein completely disappeared from the cerebellum of α6 –/– mice, which resulted in the disappearance of the δ subunit from the plasma membrane of granule cells. The amount of β2, β3 and γ2 subunits was reduced by ≈ 50%, 20% and 40%, respectively, in the cerebella of α6 –/– mice. A comparison of the reduction in the level of α1, β2, β3, γ2, or δ‐subunit‐containing receptors in α6 –/– cerebellum with those observed after removal of α6‐subunit‐containing receptors from the cerebella of α6 +/+ mice by immuno‐affinity chromatography demonstrated the presence of a significantly higher than expected proportion of receptors containing β3 subunits in α6 –/– mice. The receptors containing α1, β2, β3 and γ2 subunits were present in the plasma membrane of granule cells of α6 –/– mice at both synaptic and extrasynaptic sites, as shown by electron microscopic immunocytochemistry. Despite the changes, the α1 subunit content of Golgi‐cell‐to‐granule‐cell synapses in α6 –/– animals remained unaltered, as did the frequency of α1 immunopositive synapses in the glomeruli. Furthermore, no change was apparent in the expression of the α1, β2 and γ2 subunits in Purkinje cells and interneurons of the molecular layer.

These results demonstrate that in α6 –/– mice, the cerebellum expresses only half of the number of GABAA receptors present in wild‐type animals. Since these animals have no gross motor deficits, synaptic integration in granule cells is apparently maintained by α1‐subunit‐containing receptors with an altered overall subunit composition, and/or by changes in the expression of other ligand and voltage gated channels.
BDNF and NT‐4 differentiate two pathways in the modulation of neuropeptide protein levels in postnatal hippocampal interneurons
Authors: Marty Serge , Onténiente Brigitte
Published: March 2003

Neuropeptide protein levels in hippocampal interneurons exhibit a considerable maturation in postnatal animals. This study characterizes the role of neuronal activity in determining neuropeptide protein levels in postnatal hippocampal interneurons, and the involvement of neurotrophins. In hippocampal slices from 7‐day‐old rats cultured for 2 weeks, treatment with the γ‐aminobutyric acidA (GABAA) receptor antagonist bicuculline increased the staining intensity and the number of neurons immunoreactive for neuropeptide Y (NPY). An opposite effect was observed when non‐N‐methyl‐d‐aspartate (non‐NMDA) excitatory transmission was blocked. The effects of either treatment were reversed after return to control medium. These findings were similar to those previously obtained on the effects of activity on somatostatin immunostaining. Blockade of endogenous tyrosine kinase neurotrophin receptors using K252a prevented the effects of bicuculline on NPY‐ and somatostatin‐immunoreactive neurons. Application of exogenous neurotrophin‐3 (NT‐3) increased NPY and somatostatin protein levels in long‐term but not short‐term cultures, while nerve growth factor (NGF) had no effect. In contrast, brain‐derived neurotrophic factor (BDNF) or neurotrophin‐4 (NT‐4) did not affect equally NPY and somatostatin immunoreactivity: they mimicked the effects of bicuculline treatment on NPY‐immunoreactive neurons, but exerted no conspicuous effect on somatostatin immunostaining. These results indicate that although neuronal activity plays a major role in determining neuropeptide protein levels in postnatal hippocampal interneurons, its effects on different neuropeptides might be exerted through different mechanisms, with or without the mediation of BDNF or NT‐4.