bluuebunny/crossref_metadata_2025 huggingface dataset:
Cholecystokinin in Cortico‐striatal Neurons in the Rat: Immunohistochemical Studies at the Light and Electron Microscopical Level
Authors: Morino P. , Herrera‐Marschitz M. , Castel M. N. , Ungerstedt U. , Varro A. , Dockray G. , Hökfelt T.
Published: April 2006

Using immunohistochemical techniques we have analysed the occurrence of cholecystokinin‐like immunoreactivity (CCK‐LI) in the cortex and striatum of the rat. In the cortex few CCK‐immunoreactive cell bodies, mainly interneurons, could be visualized in normal brains, and a moderately dense network of CCK fibres was also observed. Injections of colchicine into the striatum led to an accumulation, in the surrounding cortex, of CCK‐LI in the initial segment of the axon of numerous cells. In addition, with an antibody to pro‐CCK several cell bodies, many of which with pyramidal shape, could be visualized. Furthermore, retrograde staining of cortical cells after unilateral injection of wheat germ agglutinin into the striatum revealed bilaterally in the cortex a number of labelled cells that also contained pro‐CCK‐LI. In the striatum CCK‐LI was diffusely distributed in fine fibres as well as in patches of fibres located in the medial aspects. After decortication followed by callosotomy these patches disappeared on the side ipsilateral to the lesion, while the pattern of immunoreactivity of several other peptides in the striatum was unaffected. No change was observed on the contralateral side. Decortication or callosotomy alone did not affect the pattern of CCK‐LI. At the ultrastructural level several CCK‐immunoreactive terminals could be observed, mostly with clear, densely packed vesicles and straight asymmetric synaptic contacts with small spines, characteristic for terminals of cortical origin. The results are consistent with the presence of a major, partly crossed, CCK‐containing cortico‐striatal pathway.
Hydrophilic and Amphiphilic Forms of Drosophila Choline Acetyltransferase are Encoded by a Single mRNA
Authors: Salem Natalie , Medilanski Jana , Pellegrinelli Nathalie , Eder‐Colli Lorenza
Published: April 2006

We have previously shown that the enzyme choline‐O‐acetyltransferase (ChAT) exists in a hydrophilic and an amphiphilic form in Drosophila head. A complementary DNA clone of 4.2 kb containing the entire coding region of ChAT was isolated from a cDNA library of Drosophila heads. The cDNA was subcloned in an expression vector and injected into the nucleus of Xenopus oocytes. Injected oocytes expressed high levels of ChAT activity. This activity was inhibited by bromoacetylcholine, a specific inhibitor of the enzyme. In the present study the non‐ionic detergent Triton X‐114 was used to analyse whether the expression of hydrophilic and amphiphilic ChAT was or was not directed by a single cDNA. The two forms of ChAT were found to be synthesized in injected oocytes. Approximately 9% of the recombinant enzyme partitioned as amphiphilic activity. This value was similar to that found for native amphiphilic ChAT in Drosophila heads. Sedimentation in sucrose gradients of amphiphilic enzyme was found to be influenced by the type of detergent present in the gradient whereas this was not the case for hydrophilic ChAT. Hydrophilic and amphiphilic enzyme activities differed in some of their biochemical properties. Amphiphilic ChAT was less sensitive to inhibition by the product acetylcholine than was hydrophilic ChAT. Moreover, amphiphilic ChAT was found to be more resistant than hydrophilic ChAT to heat inactivation at 45°C. These properties were observed for the native as well as for recombinant ChAT. These results demonstrate that the hydrophilic and amphiphilic forms of ChAT are derived from one mRNA.
Transient Production of TGF‐β2 by Postnatal Cerebellar Neurons and its Effect on Neuroblast Proliferation
Authors: Constam Daniel B. , Schmid Peter , Aguzzi Adriano , Schachner Melitta , Fontana Adriano
Published: April 2006

The beta transforming growth factors (TGF‐β) are suggested to regulate developmental processes since they are distinctly expressed during embryogenesis and exert pleiotropic effects on cell growth and differentiation. In the present study the expression of TGF‐β isoforms was investigated in the postnatal and adult mouse brain. As shown by in situ hybridization, TGF‐β2 was expressed in the choroid plexus, hippocampus, dentate gyrus and cerebellar Purkinje neurons, both postnatally and in adults. Furthermore, TGF‐β2 expression was observed postnatally in immature cerebellar neurons of both the external and internal granule cell layers. In the external granule cell layer, the frequency of TGF‐β2 transcripts increased until postnatal day 10 and declined thereafter. In contrast to TGF‐β2, no TGF‐β1 mRNA was detected in cerebellar granule cells. TGF‐β3 expression was widely distributed in postnatal brains although at very low levels. The significance of TGF‐β2 production by cerebellar granule cells was further investigated using cultures of small cerebellar neurons. In these cultures reverse polymerase chain reaction analysis revealed expression of TGF‐β2 but low or almost undetectable levels of TGF‐β1 or ‐β3 mRNAs. Likewise, only TGF‐β2 protein in its latent form was identified in the culture supernatant; the release of TGF‐β2 was maximal during the second day in vitro. Furthermore, TGF‐β was found to inhibit the proliferation of cultured small cerebellar neurons. Taken together, these data suggest that TGF‐β2 is involved in the regulation of postnatal development of the cerebellum.
Is Protein Kinase C Activity Required for the N‐Methyl‐d‐Aspartate‐evoked Rise in Cytosolic Ca2+ in Mouse Striatal Neurons?
Authors: Murphy Nuala P. , Cordier Jocelyne , Glowinski Jacques , Prémont Joël
Published: April 2006

The present study investigates the roles of protein kinase C (PKC) and A (PKA) activities in NMDA‐mediated Ca2+ entry in primary cultures of mouse striatal neurons. Inhibitors of protein kinases, such as sphingosine, RO 31 – 8220 and staurosporine inhibited the NMDA‐ but also the KCI‐induced rise in cytosolic Ca2+. However, the PKA antagonist Rp‐adenosine‐3′,5′monophosphothioate (Rp‐cAMPS) did not alter the NMDA +d‐serine response, whereas it completely suppressed the KCI response. The NMDA +d‐serine‐evoked rise in cytosolic Ca2+, observed in the absence of external Mg2+, was potentiated by the PKC activator phorbol 12‐myristate 13‐acetate (PMA) only when submaximal effective concentrations of this agonist and co‐agonist were used. In addition, the PKC activator did not alter the NMDA +d‐serine‐evoked response in the presence of varying concentrations of Mg2+. Confirming the dependence on PKC activity, desensitization of PKC resulting from long‐term PMA treatment led to an impairment of the NMDA response, leaving the KCI‐induced response intact. We therefore propose that PKC not only potentiates but is also required for the NMDA‐evoked elevation in cytosolic Ca2+ in mouse striatal neurons.
The NO‐cGMP Pathway in Neonatal Rat Dorsal Horn
Authors: Morris R. , Southam E. , Gittins S. R. , de Vente J. , Garthwaite J.
Published: April 2006

Incubation of slices of neonatal rat spinal cord with nitric oxide donor compounds produced marked elevations in cyclic guanosine 3′,5’monophosphate (cGMP) levels. The excitatory amino acid receptor agonists N‐methyl‐d‐aspartate (NMDA) and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate (AMPA) produced smaller increases, which were blocked by the nitric oxide synthase (NOS) inhibitor Ml‐NG‐nitroarginine (NOArg), indicating that these cGMP responses were mediated by nitric oxide. Immunocytochemistry revealed that, in response to NMDA, cGMP accumulated in a population of small cells and neuropil in laminae II and III of the dorsal horn. This area was also shown, by reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry, to contain NOS. These observations suggest that, in the rat spinal cord, NMDA receptor activation is linked to the formation of NO and, hence, of cGMP. This pathway is located selectively in the superficial dorsal horn, consistent with a role in the processing of nociceptive signals.
In Situ Hybridization of trkB and trkC Receptor mRNA in Rat Forebrain and Association with High‐affinity Binding of [125I]BDNF, [125I]NT‐4/5 and [125I]NT‐3
Authors: Altar C. Anthony , Siuciak Judith A. , Wright Paul , Ip Nancy Y. , Lindsay Ronald M. , Wiegand Stanley J.
Published: April 2006

The TrkB and TrkC receptor tyrosine kinases have been identified as high‐affinity receptors for the neurotrophic factors brain‐derived neurotrophic factor (BDNF) and neurotrophin‐4/5 (NT‐4/5) and NT‐3 respectively. These receptor classes were identified and mapped by the in situ hybridization of antisense riboprobes complementary to portions of the intracellular (tyrosine kinase) or extracellular (ligand‐binding) domains of trkB and trkC mRNA, and by the distribution of high‐affinity [125I]BDNF, [125I]NT‐4/5 and [125I]NT‐3 binding sites in adjacent rat brain sections. Both methods showed that TrkB and TrkC receptors are abundant and widely expressed throughout the brain. Kinase or extracellular domain trkC probes labelled neuronal somata in a qualitatively similar manner in virtually every major area of the forebrain. Neither trkC probe labelled non‐neuronal cells except for elements within cerebral arteries and arterioles. The kinase domain trkB probe hybridized exclusively to neurons. Neurons expressing trkB were even more widely distributed than those expressing trkC. The extracellular domain trkB probe labelled neurons with the same relative distribution as the trkB kinase domain probe, but also hybridized extensively with non‐neural cells, particularly astrocytes, ependyma and choroid epithelium cells. The distribution of [125I]NT‐3 binding sites generally resembled that of trkC hybridization, particularly in the neocortex, striatum and thalamus. [125I]BDNF and [125I]NT‐4/5 binding sites were more widely distributed and denser than those for [125I]NT‐3, and resembled the trkB hybridization pattern. These patterns are consistent with the preferential binding in the brain of TrkC receptors by [125I]NT‐3 and of TrkB receptors by [125I]BDNF and [125I]NT‐4/5. That the predominantly neuronal patterns of hybridization obtained with kinase and extracellular domain probes for trkC are qualitatively indistinguishable suggests that truncated and full‐length forms of TrkC are expressed within extensively overlapping populations of neurons. In marked contrast to TrkC, expression of the full‐length and truncated forms of TrkB appears to be largely segregated, being expressed principally on neurons and non‐neuronal cells respectively. The abundant and widespread neuronal distribution of full‐length, signal‐transducing forms of TrkB and TrkC predict that their cognate ligands, BDNF, NT‐4/5 and NT‐3, may exert direct effects on a large proportion of neurons within the mature brain.
Preserved Recognition Memory for Small Sets, and Impaired Stimulus Identification for Large Sets, Following Rhinal Cortex Ablations in Monkeys
Authors: Eacott M. J. , Gaffan D. , Murray E. A.
Published: April 2006

Seven cynomolgus monkeys (Macaca fascicularis) performed a series of tasks designed to assess their visual memory and their ability to identify visual stimuli. Preoperatively they were trained and tested in delayed and simultaneous matching‐to‐sample, both with a large stimulus set and with a small stimulus set; there were ∼500 million possible stimuli in the large set, which effectively means that stimuli were trial‐unique with this set, while in the small set there were only four stimuli, which appeared repeatedly in every session of training with the small set. Three of the monkeys then had the cortex within and adjacent to the rhinal sulcus removed bilaterally, while the other four served as an unoperated control group. Postoperatively, the animals with ablation of the rhinal cortex showed severe impairment in delayed matching‐to‐sample with the large set. With the large set they were also impaired, however, in matching‐to‐sample with no delay between sample and test (0 s delay) and in simultaneous matching‐to‐sample, in which the sample and the two choice patterns were simultaneously present for inspection. The impairment in simultaneous matching‐to‐sample was particularly clear when the task was made more difficult by reducing the physical discriminability of the trial‐unique stimuli. With the small set of four stimuli, the animals with rhinal cortex ablation were not significantly impaired in overall performance level in delayed matching‐to‐sample, though their level was on average below that of the normal control animals. The stimulus set was then further restricted, so that there were now only two stimuli used throughout; in this condition, the animals with rhinal cortex ablation performed delayed matching‐to‐sample without any suggestion of impairment, showing indistinguishable performance levels from those of the control animals over a range of forgetting intervals. Subsequently, the animals were trained in trial‐unique non‐matching‐to‐sample with 0 s delay, which required reversal of the matching‐to‐sample rule they had previously learned; animals with rhinal cortex ablation showed a clear impairment in this rule‐reversal learning. The final experimental task was a concurrent discrimination learning task in which 20 pairs of stimuli were presented once per session; the animals with rhinal cortex ablation learned more slowly than the control animals on average, but the difference between the groups did not attain statistical significance. Overall, this pattern of deficits and of preserved abilities is clearly inconsistent with the idea that rhinal cortex ablation produces an impairment in all forms of visual recognition memory, and only in visual recognition memory. Instead, the present results indicate a general impairment in the capacity for knowledge about visual stimuli.
Cortical Layer‐specific Effects of the Metabotropic Glutamate Receptor Agonist 1S,3R‐ACPD in Rat Primary Somatosensory Cortex In Vivo
Authors: Cahusac Peter M. B.
Published: April 2006

The effects of iontophoretically applied (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid (1S,3R‐ACPD), a metabotropic glutamate receptor (mGluR) agonist, were studied on extracellularly recorded neurons throughout the depth of the primary somatosensory cortex in the anaesthetized adult rat. Distinct excitatory effects were found almost exclusively in neurons recorded in layer V. Postsynaptic depressant effects dominated neurons recorded in layers I–IV. In layer VI, neurons were equally divided as to excitation and depression. Both the excitatory and postsynaptic depressant effects could be antagonized by the mGluR antagonist (RS)‐α‐methyl‐4‐carboxyphenylglycine (MCPG). Experiments using bicuculline and several lines of analysis suggested that the postsynaptic depressant effects were mediated directly, rather than through disfacilitation. In a proportion of neurons 1S,3R‐ACPD selectively depressed synaptically evoked responses (produced by vibrissa deflections), with little or no effect on the postsynaptic level of firing. Comparing the depressant effects of 1S,3R‐ACPD with those of GABA supported a presynaptic mGluR site. Responses to centre and surround receptive field stimulation were depressed to the same extent, suggesting that thalamocortical and intracortical axon terminals are equally endowed with presynaptic receptors. In contrast to previous studies, the actions of l‐2‐amino‐4‐phosphonobutyric acid (L‐AP4) were shown to be qualitatively different to those of 1S,3R‐ACPD, in particular suggesting that the presynaptic depression produced by 1S,3R‐ACPD is not mediated by l‐AP4‐type receptors. The functional implications of different mGluR actions in the primary somatosensory cortex are discussed.
Uptake of Acetylcholinesterase by Neurons in the Substantia Nigra
Authors: Dickie B. G. M. , Budd T. C. , Vaux D. , Greenfield S. A.
Published: April 2006

It is known that acetylcholinesterase is secreted by the dopaminergic neurons of the substantia nigra and has a subsequent action independent of cholinergic transmission. Although non‐cholinergic actions of this protein have been demonstrated, the subsequent fate of acetylcholinesterase is unknown. One possibility is that acetylcholinesterase is taken up following secretion into the extracellular space. This hypothesis has been tested in vivo, in both conscious and anaesthetized guinea‐pigs. Exogenous acetylcholinesterase (2–20 pM) was infused via a push‐pull cannula implanted into either the substantia nigra or the surrounding extranigral regions; the amount subsequently recovered in the perfusate was then compared with control values. Only when the push‐pull cannulae were implanted in the substantia nigra was there a marked decrease in the amount of acetylcholinesterase recovered; this selective retention was abolished when the perfusion medium was cooled to 4°C or when the experiment was performed post mortem. Direct visualization of immunocytochemically identified nigral dopaminergic cells revealed co‐localized deposits of labelled, exogenous acetylcholinesterase. Moreover, when exogenous acetylcholinesterase was boiled to prevent detection by the assay system and to eliminate any classical enzymatic action, an enhancement in perfusate levels of endogenous acetylcholinesterase was observed from nigral but not from extranigral sites, indicating that endogenous and exogenous acetylcholinesterases were in competition. These results suggest that, within the substantia nigra, secreted acetylcholinesterase may be subject to a temperature‐ and energy‐dependent uptake mechanism.
Clearance of Myelin Constituents and Axonal Sprouting in the Transected Postcommissural Fornix of the Adult Rat
Authors: Stichel Christine C. , Wunderlich Gilbert , Schwab Martin E. , Müller Hans Werner
Published: April 2006

Following transection of the postcommissural fornix in the adult rat, fibres retract from the lesion zone but then start to regrow within their former pathway up to the lesion site, where they terminate. The fibres neither penetrate nor bypass this region. In order to define the molecular mechanisms that cause regenerative failure at the lesion site, we analysed the spatiotemporal relationship between clearance/re‐expression of myelin constituents and axon sprouting. Using immunocytochemical methods, we investigated the distribution of myelin‐associated growth inhibitor (Nl‐35/250) and myelin basic protein after transection of the postcommissural fornix. In the studies described here we demonstrate the sequential removal of neurofilaments and myelin constituents in a perilesion zone and at the lesion site. The removal of myelin constituents was followed by the extensive regrowth of fornix fibres in the proximal segment. However, these fibres stopped at the lesion site, an area that lacked immunostaining for Nl‐35/250 and. In the distal stump we observed the disappearance of neurofilament along the entire fornix segment but spatial differences in the removal of myelin constituents. While both NI‐35/250 and myelin basic protein disappeared in the perilesion zone, they persisted in the more distal segment for at least 28 months after lesion. In conclusion, our study indicates that the onset of axon sprouting is correlated with the removal of myelin basic protein and NI‐35/250. Furthermore, we suggest that it seems unlikely that the myelin growth inhibitor NI‐35/250 constitutes the stop signal of the axon growth barrier in the transected fornix.
The Effects of Early Postnatal Modification of Body Shape on the Somatosensory—Visual Organization in Mouse Superior Colliculus
Authors: Benedetti F. , Ferro I.
Published: April 2006

Different regions of the body of an animal have their own shape and location within visual space. Accordingly, in the superior colliculus there are somatosensory‐visual bimodal neurons receiving tactile and visual input from the same region of space. In newborn mice, we changed the position of some body parts within visual space in order to see what happened to the alignment of the somatosensory and visual receptive fields of superior colliculus bimodal neurons. To do this, we modified the shape of the head by displacing the superior vibrissae and the ears, normally in the superior portion of visual space, into the inferior visual space. Analogously, we bent the inferior vibrissae into the superior visual space. At the sixth postnatal week we recorded from somatosensory‐visual bimodal neurons of the deep layers of the superior colliculus and found that the tactile and visual receptive fields were aligned. Neurons receiving tactile input from the downward‐displaced superior vibrissae and ears showed visual receptive fields in the inferior portion of visual space, whereas neurons receiving input from the upward‐displaced inferior vibrissae showed visual receptive fields in the superior visual space. These results show that an experience‐dependent interaction between visual and somatosensory inputs occurs during development, and that early exposure to abnormal visual‐somatosensory experience modifies the organization of multisensory neurons in the superior colliculus.
Postnatal Development of GABA Neurons in the Rat Somatosensory Barrel Cortex: A Quantitative Study
Authors: Micheva Kristina D. , Beaulieu Clemont
Published: April 2006

As an estimate of the numerical importance of GABA‐containing neurons during development, their quantitative distribution was analysed in the primary somatosensory cortex of rats between postnatal days (P) 5 and 60, using the disector method and GABA postembedding immunocytochemistry. In relation to the overall number of neurons in the barrel field cortex, the proportion of GABA neurons showed an early significant decrease between P5 and P10 from 14 to 11%, most likely due to termination of transient expression of GABA by some cells. It then remained stable until P20, after which it started slowly but steadily to increase, reaching 14% of the total at P60. The absolute number of GABA neurons also increased by nearly 50% during that period, whereas the number of all neurons remained constant. These changes are seemingly due to a subpopulation of neurons, shown to be of small size, which express GABA late in development. Thus, anatomical adjustments of the cortical GABA system may be observed at least until the end of the second postnatal month, reflecting both delayed maturation and adaptation of this inhibitory circuitry. We suggest the existence of three subpopulations of cortical GABA neurons depending on the time of onset and the regulation of their GABA expression: (i) neurons which express GABA before completion of migration and thus provide for its neurotrophic influence. (ii) neurons which express GABA immediately after completion of migration and build up the cortical inhibitory circuitry, and (iii) neurons which express GABA later in development and represent a substrate of experience dependent plasticity.
Temporal Coding of Amplitude and Frequency Modulation in the Rat Auditory Cortex
Authors: Gaese Bernhard H. , Ostwald Joachim
Published: April 2006

The rat primary auditory cortex was explored for neuronal responses to pure tones and sinusoidally amplitude‐modulated (SAM) and frequency‐modulated (SFM) stimuli. Units showed phase‐locked responses to SAM stimulation (55%) and SFM stimulation (80%), with modulation frequencies up to 18 Hz. Tuning characteristics to the modulation frequency were mainly band‐pass with best modulation frequencies (BMFs) between 4 and 15 Hz. Units with synchronized activity to SFM stimulation showed three response types with respect to the direction of the frequency modulation: 52% were selective to the upward direction, 30% to the downward direction, and 18% had no preference. Triangular frequency modulations were used to test if units were tuned to specific modulation frequencies or to specific rates of frequency change. In the vast majority of units tested the response characteristics were strongly influenced by varying the modulation frequency, whereas varying the rate of frequency change had little effect in the stimulus range used. Units that showed phase‐locked responses to SAM and SFM stimulation had similar activity patterns in response to both types of stimuli. BMFs for SAM and SFM stimulation were significantly correlated. Intrinsic oscillations of up to 20 Hz could be seen in the spontaneous activity and after the stimuli independent of the stimulus type. Oscillation frequencies were significantly correlated with the BMFs of the respective units. The results are discussed in terms of a mechanism for periodicity detection based on a temporal code. This could be important for the recognition of complex acoustic signals.
Nerve‐Dependent Plasticity of the Golgi Complex in Skeletal Muscle Fibres: Compartmentalization Within the Subneural Sarcoplasm
Authors: Jasmin Bernard J. , Antony Claude , Changeux Jean‐Pierre , Cartaud Jean
Published: April 2006

Several recent reports have highlighted the plasticity of the Golgi apparatus during myogenesis, yet the organization of this specialized organelle in innervated skeletal muscle fibres remains poorly understood. Using four bona fide anti‐Golgi antibodies, directed against a 210 kDa protein, a 160 kDa sialoglycoprotein, the small GTP‐binding protein rabop, and TGN38, the localization of which covers the various compartments of the Golgi complex, we show by immunofluorescence microscopy that the Golgi complex undergoes considerable reorganization in the course of myogenic differentiation and motor endplate formation in the rat. Unlike the typical perinuclear distribution of the Golgi stacks associated with every nucleus in myotubes, a striking subneural compartmentalization is observed in adult innervated myofibres. In short‐term denervated adult muscle fibres, we noticed the presence of the perinuclear Golgi apparatus in extrajunctional regions, a pattern reminiscent of that of developing myotubes. At variance with anti‐Golgi antibodies, antibodies to the rough endoplasmic reticulum label structures dispersed throughout the entire sarcoplasm, hence suggesting that it is not the entire membrane/secretory protein synthesis machinery which is compartmentalized, but only the Golgi apparatus. Also, an unexpected lack of immunoreactivity with the TGN38 and α‐mannosidase II antibodies points to biochemical differentiation of the subneural Golgi apparatus at the adult motor endplate. These new data extend our previous observations on the compartmentalization of the Golgi apparatus in the postsynaptic sarcoplasm of chick muscle fibres, and further illustrate the plasticity of the Golgi apparatus in muscle cells. The specialization of the Golgi apparatus within the subneural compartment provides this particular region with a compartmentalized secretory pathway, and these observations highlight the notion that the level of differentiation of this domain is not only maintained via transcriptional regulation but also by post‐translational control mechanisms
Ectopic Substance P and Calcitonin Gene‐related Peptide Immunoreactive Fibres in the Spinal Cord of Transgenic Mice Over‐expressing Nerve Growth Factor
Authors: Ma Weiya , Ribeiro‐da‐Silva A. , Noel G. , Julien J.‐P. , Cuello A. Claudio
Published: April 2006

The aim of this study was to investigate the in vivo effects of CNS over‐expression of nerve growth factor (NGF) on primary sensory neurons. To achieve this objective a transgenic mouse model was generated which bore a chick NGF gene driven by the myelin basic protein promoter. Northern blot analysis demonstrated that high levels of NGF mRNA were detected in the spinal cord of adult transgenic mice. Using immunocytochemistry NGF‐immunoreactive (IR) oligodendrocytes were observed throughout the white matter. Furthermore, numerous ectopic substance P (SP)‐ and calcitonin gene‐related peptide (CGRP)‐IR fibres were detected in the white matter of the spinal cord of transgenic mice. NGF‐IR oligodendrocytes and ectopic SP‐ and CGRP‐ fibres were entirely absent from control mice. In the cervical and lumbar dorsal root ganglia, the percentages of SP‐IR neurons were significantly higher in transgenic mice when compared with controls. At the electron microscope level, ectopic SP‐ and CGRP‐IR fibres were characterized as unmyelinated axons and axonal boutons. SP co‐localized with CGRP in some of those axonal boutons and fibres. Capsaicin treatment of adult mice completely abolished the ectopic SP‐IR fibres, confirming their primary sensory origin. Our results indicate that primary sensory neurons are responsive to NGF over‐expression in the CNS. Ectopic SP‐ and CGRP‐IR fibres in the white matter are likely to represent collateral sprouts of the central processes of the dorsal root ganglion cells which were triggered by NGF over‐expressed in the myelinating oligodendrocytes in the spinal cord of transgenic mice.
Interactive Events Subserving the Pupillary Light Reflex in Pigmented and Albino Rats
Authors: Chan K. M. C. , Young Michael J. , Lund Raymond D.
Published: April 2006

The consensual pupilloconstrictor response requires the intensity information that is delivered to each eye to be integrated to produce an averaged response such that the pupillary diameter in each eye is equal. Here we have examined how luminance information from each eye is integrated in the rat and where the integration occurs, using a pupillometry system to record pupillary diameter after illumination of one or both eyes. In albino rats the size of the uncrossed optic pathway to the pupilloconstrictor centre, the olivary pretectal nucleus, is reduced three‐fold and there is a concomitant but less dramatic reduction in the size of the consensual response. Unilateral lesions of the olivary pretectal nucleus in pigmented rats restrict all optic input to the opposite pretectum. Stimulation of one or both eyes in these animals suggests that integration occurs not only at the level of the olivary pretectal nucleus, but also downstream in the Edinger‐Westphal nucleus. Parallels with human studies and the opportunity to perform secondary lesions suggests the rat may provide a good model in which to study the substrates of the pupilloconstrictor response and mechanisms of integration of convergent parallel inputs in general.
The Critical Period for Repair of CNS of Neonatal Opossum (Monodelphis domestica) in Culture: Correlation with Development of Glial Cells, Myelin and Growth‐inhibitory Molecules
Authors: Varga Z. M. , Bandtlow C. E. , Eurlkar S. D. , Schwab M. E. , Nicholls J. G.
Published: April 2006

A comparison was made of neurite growth across spinal cord lesions in the isolated central nervous system (CMS) of newborn opossums (Monodelphis domestica) at various stages of development. The aim was to define the critical period at which growth after injury ceases to occur, with emphasis on growth‐inhibitory proteins, myelin and glial cells. In postnatal opossums 3–6 days old (P3–6), repair was observed 5 days after lesions were made in culture at the cervical level (C7) by crushing with forceps. Through‐conduction of action potentials was re‐established and axons stained by Oil grew into and beyond the crush. In a series of 66 animals 29 showed repair. In 28 animals at P11–12 with comparable lesions repair was observed in five preparations. At P13–14, the CMS was still viable in culture, but none of the 25 preparations examined showed any axonal growth into the crush or conduction through it. The rostra‐caudal gradient of development permitted lesions to be made in mature cervical and immature lumbar regions of P11–12 spinal cord. Growth across crushes occurred in lumbar but not in cervical segments of the same preparation. The development of glial cells and myelin was assessed by electron microscopy and by staining with specific antibodies (Rip‐1 and myelin‐associated glycoprotein) in cervical segments of neonatal P6–14 opossums. At P8, oligodendrocytes and thin myelin sheaths started to appear followed at P9 by astrocytes stained with antibody against glial fibrillary acidic protein. By P14, astrocytes, oligodendrocytes and well‐developed myelin sheaths were abundant. The cervical crush sites of P12 cords contained occasional astrocytes but no oligodendrocytes. Specific antibodies (IN‐1) to neurite growth‐inhibiting proteins (NI‐35/250) associated with oligodendrocytes and myelin in the rat CNS cross‐reacted with opossum proteins. Assays using the spreading of 3T3 fibroblasts and IN‐1 showed that by P7 inhibitory proteins became apparent, particularly in the hindbrain and cervical spinal cord. The concentrations of NI‐35/250 thereafter increased and became abundant in the adult opossum. Our finding of a well‐defined critical period, encompassing only 5 days, in CNS preparations that can be maintained in culture offers advantages for analysing mechanisms that promote or prevent CNS repair.
Purification of a Meningeal Cell‐derived Chondroitin Sulphate Proteoglycan with Neurotrophic Activity for Brain Neurons and its Identification as Biglycan
Authors: Junghans Ulrich , Koops Antje , Westmeyer Annette , Kappler Joachim , Meyer Helmut E. , Müller Hans Werner
Published: April 2006

Serum‐free cultures of meningeal fibroblasts synthesize and release a chondroitin sulphate proteoglycan (CSPG) that markedly enhances survival but not adhesion of embryonic rat (embryonic day 15) neocortical neurons in vitro. The active molecule was purified from conditioned medium (meningeal cell‐conditioned medium, MCM) in three steps by means of fast‐performance liquid chromatography fractionation combined with a quantitative microphotometric bioassay: (i) preparative Q‐Sepharose anion exchange chromatography under native conditions; (ii) rechromatography of biologically active Q‐Sepharose fractions on a MonoQ column in the presence of 8 M urea; and (iii) final gel filtration of active MonoQ fractions on Superose 6 in the presence of 4 M guanidinium hydrochloride. Analytical sodium dodecyl sulphate‐polyacrylamide gradient gel electrophoresis of active Superose 6 fractions revealed a single broad glycoprotein band with a molecular mass in the range of 220–340 kDa. Further characterization of the purified molecule with glycosaminoglycan:lyases revealed a core protein of 50 kDa and the nearly complete loss of neurotrophic activity after chondroitinase digestion, whereas heparitinase treatment changed neither electrophoretic mobility nor biological activity. Amino‐terminal sequencing of the purified CSPG core protein revealed identity with the amino acid sequence of rat biglycan. Biglycan purified from bovine cartilage supported neuron survival with virtually the same activity as the CSPG purified from MCM (half‐maximal activity ∼10‐8 M). In conclusion, we isolated a neurotrophic CSPG from meningeal cells with strong survival‐enhancing activity for brain neurons that was identified as biglycan, a molecule not previously related to neural functions.
Actions of Glycine on Non‐dopaminergic Neurons of the Rat Substantia Nigra
Authors: Mercuri Nicola B. , Bonci Antonello , Pisani Antonio , Calabresi Paolo , Bernardi Giorgio
Published: April 2006

The effects of glycine on non‐dopaminergic cells in rat substantia nigra pars compacta and pars reticulata maintained in vitro were investigated using intracellular recording techniques. Glycine, superfused at a concentration between 30 μM and 1 mM, reversibly blocked the spontaneous firing of these neurons. The inhibition of firing discharge was associated with a hyperpolarization of the membrane (potassium acetate‐filled electrodes) and an increase in conductance. Under voltage‐clamp experiments (holding potential between ‐57 and ‐65 mV), glycine produced an outward response which reversed polarity at about ‐74 mV. However, when the recording electrodes were filled with KCI, the glycinergic response was mainly depolarizing/inward and reversed at about ‐43 mV. Thus, it appeared to be due to an increase in chloride permeability. Furthermore, the effects of glycine were reversibly antagonized by strychnine (between 300 nM and 1 μM). Our findings demonstrate that glycine is a potent inhibitory agent on non‐dopaminergic cells of the substantia pars compacta and pars reticulata that acts by activating strychnine‐sensitive receptors.
Serotonin Modulation of Inferior Olivary Oscillations and Synchronicity: A Multiple‐electrode Study in the Rat Cerebellum
Authors: Sugihara I. , Lang E. J. , Llinás R.
Published: April 2006

Simultaneous recording of complex spikes from multiple Purkinje cells (up to 44) in the rat cerebellum was used to examine the effects of 5‐hydroxytryptamine (serotonin, 5‐HT) on olivocerebellar function. Microinjection into the inferior olive was found to increase the average firing rate of inferior olivary neurons while slowing their oscillation frequency and increasing the coherence of their oscillations. Indeed, while the normal rostrocaudal band of synchronous activity remained unchanged, the degree of synchrony between Purkinje cell complex spikes within this band was enhanced following the 5‐HT injections. Multiple‐electrode recordings obtained from crus Ha and vermal lobule Vlb yielded qualitatively similar results; however, the effects on vermal activity were more pronounced. The effects of the 5‐HT microinjection decayed with a time course of 75 min. The half‐maximum effective concentration of 5‐HT was between 10 and 100 μM. Injections of various 5‐HT agonists and antagonists demonstrated that a 5‐HT type‐2A (5‐HT2A) receptor is the main mediator for the 5‐HT effect, which was very similar to the effect produced by injections of harmaline. However, 5‐HT and harmaline appear to have independent mechanisms since the action of harmaline was not blocked by the 5‐HT2A antagonist LY53857. A possible role for 5‐HT, as a physiological enhancer of the timing of motor function of the olivocerebellar system, is discussed.
Age‐related Changes in the NMDA Receptor/Nitric Oxide/cGMP Pathway in the Hippocampus and Cerebellum of Freely Moving Rats Subjected to Transcerebral Microdialysis
Authors: Vallebuona F. , Raiteri M.
Published: April 2006

The N‐methyl‐d‐aspartate (NMDA) receptor/nitric oxide synthase/guanylate cyclase pathway was studied during aging by monitoring extracellular cGMP in the rat hippocampus and cerebellum during in vivo microdialysis. In the hippocampus the basal cGMP efflux decreased by 50% from 3 to 12 months of age, whereas it remained constant with age in the cerebellum. Locally perfused NMDA (1 mM) evoked remarkable cGMP responses in 3‐month‐old rats; in the hippocampus the cGMP production was already dramatically reduced at 12 months, whereas in the cerebellum a similar impairment occurred much later (24 months). The nitric oxide donor S‐nitroso‐N‐penicillamine (1 mM) elicited cGMP responses which slightly decreased from 3 to 12–24 months in the hippocampus, while no significant decrement with age could be seen in the cerebellum. Local perfusion of the phosphodiesterase inhibitor 3‐isobutyl‐1 methylxanthine (IBMX, 1 mM) produced large increases in hippocampal cGMP levels. The response decreased at 12 and 24 months, apparently in parallel with the fall in the basal level of cGMP. No significant differences across ages were observed following IBMX infusion in the cerebellum. The decreases in basal outflow and in the NMDA‐evoked cGMP response seen in the aged hippocampus were not compensated for by supplying l‐arginine. Infusion of d‐serine (1 mM) enhanced (150–200%) extracellular cGMP in the cerebellum with no age‐related differences. The activity in vitro, of hippocampal nitric oxide synthase at 24 months was 33% lower than at 3 months, whereas the cerebellar enzyme did not show any age‐related decay. Aging seems therefore to affect differentially the NMDA receptor/nitric oxide synthase/cGMP pathway in the rat hippocampus versus the cerebellum. In the hippocampus the early fall in the NMDA‐evoked cGMP response seems to originate from deficits in NMDA receptor function and nitric oxide synthase and guanylate cyclase activities; in the cerebellum, the decreased response to NMDA in the old animals seems essentially to be due to impairment of NMDA receptor function.
Double In Situ Hybridization Reveals Overlapping Neuronal Populations Expressing the Low Molecular Weight GTPases Rab3a and Rab3b in Rat Brain
Authors: Stettler Olivier , Nothias Fatiha , Tavitian Bertrand , Vernier Philippe
Published: April 2006

The ras‐related Rab3 gene subfamily codes for small GTP‐binding proteins which control a late step of exocytosis during which vesicles become docked to the plasma membrane. Rab3a and Rab3b are the most abundant Rab3 isoforms expressed in the CNS of mammals. We have shown previously that the Rab3a protein was selectively distributed and expressed in various regions of the rat brain. Here we have determined the pattern of expression of Rab3b mRNA in the brain and compared it with that of Rab3a mRNA. In addition, we examined the co‐expression of these two Rab within individual neurons. In general the Rab3b transcript was detected in many regions which also express Rab3a mRNA but at a lower level than Rab3a, except in the olfactory bulb and in the pituitary where the Rab3b hybridization signal was similar and higher respectively. Double in situ hybridization revealed that Rab3a and Rab3b mRNAs were co‐localized in most neurons, in all brain areas examined. However, in each of these areas, subsets of neurons appeared to preferentially express either Rab3b or Rab3a, or some neurons did not express either Rab3 homologue at detectable levels. These data support the view of a functional specialization of Rab3a and Rab3b in the control of exocytosis in neuronal and neuroendocrine cells.
Neurophysiological Investigations of a Recognition Memory System for Imprinting in the Domestic Chick
Authors: Nicol A. U. , Brown M. W. , Horn G.
Published: April 2006

The responsiveness of neurons in a region of the chick brain involved in the learning process of imprinting, the right intermediate and medial hyperstriatum ventrale (right IMHV), has been investigated in unanaesthetized, trained and untrained chicks. The results demonstrate that neuronal responsiveness in this region reflects a variety of behavioural consequences of imprinting and is markedly altered as a result of the learning process. Groups of chicks (nine in each group) were either dark‐reared or trained (imprinted) by exposure to a rotating red box or a rotating blue cylinder. Recordings of single or small groups of neurons were subsequently made from 156 sites in the right IMHV while the 2‐day‐old chicks were free to move in a running wheel. There was a highly significant increase in the proportion of sites responsive to the stimulus used to train the birds compared to the proportion responsive to that stimulus in dark‐reared birds (30 and 9% respectively). These changes were found when either the red box or the blue cylinder was used to train the bird, the changes being similar for both stimuli. There was also a significant increase in the mean magnitude of the change in neuronal activity on stimulus presentation for the training stimulus compared to the same stimulus when not used in the bird's training. No significant effects of the training experience of the chicks were found upon either the magnitude of evoked activity or the proportion of sites responsive to a rotating stuffed jungle fowl or the sound of the maternal call. The presence of the training stimulus was selectively signalled by the response at certain sites. At other sites there was response generalization across stimulus shape or colour. A comparison with results for the left IMHV demonstrates both similarities and differences in neuronal responsiveness between the two regions. In both regions imprinting selectively enhances neuronal responsiveness to the training stimulus. However, for trained birds the mean proportion of sites responding to whichever of the red box or the blue cylinder was not used in the bird's training was significantly lower in the right than the left IMHV. These results are discussed in relationship to previously reported asymmetries in the response of the right and left IMHV regions to imprinting. A model is introduced to explain the physiological findings. The effects of training on right IMHV neuronal function are consistent with a long‐term role for this region in the recognition memory of imprinting.
Up‐regulation of a Chondroitin Sulphate Epitope during Regeneration of Mouse Sciatic Nerve: Evidence that the Immunoreactive Molecules are Related to the Chondroitin Sulphate Proteoglycans Decorin and Versican
Authors: Braunewell Karl‐Heinz , Martini Rudolf , LeBaron Richard , Kresse Hans , Faissner Andreas , Schmitz Brigitte , Schachner Melitta
Published: April 2006

After transection of adult mouse sciatic nerve, the expression of a chondroitin sulphate epitope recognized by the monoclonal antibody 473‐HD (mAb 473‐HD) was found to be up‐regulated. The epitope was localized immunocytochemically mainly in Schwann cell basal laminae and, more weakly, also in the endoneurium. In cultures of mouse dorsal root ganglion cells, Schwann cells expressed high levels but fibroblasts only low levels of the epitope. To identify the molecule(s) carrying this chondroitin sulphate epitope, human sciatic nerves were extracted with phosphate‐buffered saline and shown to contain two chondroitin sulphate proteoglycans of apparent molecular weights of 130 and 900 kDa. The 900 kDa and, more weakly, the 130 kDa proteoglycan were reactive with mAb 473‐HD, which was found to recognize chondroitin‐6‐sulphate as epitope. Following chondroitinase ABC treatment of the 130 kDa proteoglycan, a core protein of ∼45 kDa was seen and shown to react with polyclonal antibodies against the chondroitin‐dermatan sulphate proteoglycan decorin from human fibroblasts. Chondroitinase ABC treatment of the 900 kDa proteoglycan yielded a core protein with a molecular weight of ∼400 kDa that was recognized by polyclonal antibodies against recombinantly expressed fusion proteins from human versican. After transection of adult mouse sciatic nerves, the distal nerve stumps showed up‐regulation of the chondroitin‐6‐sulphate epitope of the 900 kDa proteoglycan, whereas the core protein of this proteoglycan did not show any detectable change in the level of expression. In contrast, the core protein of the 130 kDa proteoglycan was up‐regulated in expression. These observations suggest that versican‐ and decorinlike molecules may contribute to successful regeneration in the peripheral nervous system of mammals.
Anatomical Distribution and Postnatal Changes in Endogenous Free D‐Aspartate and D‐Serine in Rat Brain and Periphery
Authors: Hashimoto Atsushi , Oka Takae , Nishikawa Toru
Published: April 2006

We have investigated the anatomical distribution and postnatal development of D‐aspartate and D‐serine in the rat brain and periphery using HPLC techniques. D‐Serine was confined predominantly to the brain throughout postnatal life. At birth, a substantial quantity of D‐serine was observed throughout the brain areas. The cerebral D‐serine content increased from birth to postnatal week (PW) 3 and remained constant thereafter, whereas the cerebellar D‐serine content peaked at PW1. In contrast, the transient emergence of D‐aspartate was found in almost all brain and peripheral organs. A substantial quantity of D‐aspartate was also seen in all brain areas at birth, whereas the D‐aspartate content in the cerebrum and cerebellum decreased dramatically by PW1 and 7 respectively. Further, the D‐aspartate content and the ratio of D‐aspartate to total aspartate were highest in the adrenal at PW3 (608 ± 70 nmol/g, 45.9%) and in the testis at PW14 (221 ± 7 nmol/g, 57.8%) respectively. Because D‐serine potentiates N‐methyl‐D‐aspartate receptor‐mediated transmission through the strychnine‐insensitive glycine site and because D‐serine exhibits an N‐methyl‐D‐aspartate receptor‐related distribution and development, D‐serine may be a tenable candidate for an intrinsic ligand for the glycine site. In contrast, because the periods of maximal emergence of D‐aspartate in the brain and periphery occur during critical periods of morphological and functional maturation of organs, D‐aspartate could participate in the regulation of these developmental processes of organs.