Gontzal García del Caño

Ionotropic glutamate receptor subunit distribution on hypoglossal motoneuronal pools in the rat

The expression of ionotropic glutamate receptor subunits in the motoneuronal pools of the hypoglossal nucleus was studied using specific antibodies against subunits of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), kainate and N-methyl-D-aspartate (NMDA) subtypes. The highest numbers of intensely immunolabelled motoneurons were found in the dorsal tier and caudoventromedial part of the hypoglossal nucleus with all antibodies except that against the GluR1 AMPA subunit. Labelling for the GluR1 subunit was weak except for caudally located groups of motoneurons which innervate tongue muscles related to respiratory activity. By contrast, most motoneurons were intensely immunostained with antibodies against GluR2/3 and GluR4 subunits of the AMPA subtype. The low staining observed using an antibody specific for the GluR2 subunit (which prevents Ca2+-entry through AMPA channels) strongly suggests that AMPA receptors in hypoglossal motoneurons are Ca2+-permeable. Immunolabelling for the GluR5/6/7 kainate receptor subunits was found in many motoneuronal somata as well as in thin axon-like profiles and puncta that resembled synaptic boutons. Most motoneurons were intensely immunostained for the NMDA receptor subunit NR1. These results show that the hypoglossal nucleus contains five heterogeneous pools of motoneurons which innervate functionally defined groups of tongue muscles. The uneven expression of the different receptor subunits analysed here could reflect diverse phenotypic properties of hypoglossal motoneurons which might be expected to generate different patterns of motor responses under different physiological or pathological conditions.

Schwannomin-Interacting Protein-1 Isoform IQCJ-SCHIP-1 Is a Late Component of Nodes of Ranvier and Axon Initial Segments

Axon initial segments (AISs) and nodes of Ranvier (NRs) are essential regions for saltatory conduction of the action potential along the axon. These two domains are enriched in similar multimolecular complexes, which include voltage-gated sodium channels (Nav), NF186 (neurofascin 186), NrCAM (neuron glia-related cell adhesion molecule), and cytoskeleton linkers ankyrin G (AnkG) and βIV-spectrin. Identification of novel members of these complexes is critical to better understand their formation, function, and maintenance. Here we report that IQCJ-SCHIP-1, a recently identified isoform of schwannomin-interacting protein-1 (SCHIP-1), is a novel component of both AISs and NRs in the central and peripheral nervous systems. We show that IQCJ-SCHIP-1 binds calmodulin in the absence of Ca2+ and is highly enriched at AISs and NRs. IQCJ-SCHIP-1 accumulation at AISs and NRs is a late event, suggesting that IQCJ-SCHIP-1 is likely to play a role in mature AISs and NRs rather than during their formation. IQCJ-SCHIP-1 was not detected at AISs in the absence of AnkG and interacted in vitro with this protein. IQCJ-SCHIP-1 was also absent from central NRs and AISs of quivering mice, which have a mutation of βIV-spectrin. We suggest that IQCJ-SCHIP-1 might participate, along with AnkG and βIV-spectrin, in the stabilization or function of the multimolecular complexes of AISs and NRs, possibly by participating in Ca2+-mediated responses.

Plastic reaction of the rat visual corticocollicular connection after contralateral retinal deafferentiation at the neonatal or adult stage: Axonal growth versus reactive synaptogenesis

The effects of neonatal or adult enucleation on the final adult pattern of the rat visual corticocollicular (C‐Co) connection were studied using the anterograde tracer biotinylated dextranamine 10,000 (BDA) iontophoretically injected in the primary visual cortex. In control animals, column‐shaped terminal fields limited to a small portion of the collicular surface were observed. Synaptic boutons were present in all superficial strata of the superior colliculus (SC), with the highest density in the ventral part of the stratum griseum superficiale (SGS). Neonatal enucleation caused a considerable expansion of the contralateral visual C‐Co terminal fields, which occupied almost the entire collicular surface, suggesting that axonal sprouting had occurred. In addition, terminal boutons tended to localize more dorsally in these cases compared with controls. Following enucleation in adult animals, no changes were observed with respect to the extension of the terminal fields, although a plastic reaction leading to an increase in the bouton density in the stratum zonale (SZ) and upper SGS was found, reflecting a process of reactive synaptogenesis at these levels. These results show that both neonatal and adult visual C‐Co fibers react in response to retinal ablation, although this reaction shows distinct characteristics. Molecular factors, such as growth‐associated cytoskeletal proteins operating in the cortical origin, and extracellular matrix components and myelin‐associated axonal growth inhibitors acting on the collicular target very likely account for these differences. J. Comp. Neurol. 446:166–178, 2002.

Selective impairment of spinal mu-opioid receptor mechanism by plasticity of serotonergic facilitation mediated by 5-HT2A and 5-HT2B receptors

Summary Plasticity of serotonergic neurotransmission mediated by 5‐HT2A and 5‐HT2B receptors impairs spinal mu‐opioid of receptor efficacy during neuropathic pain, including upregulation of receptor expression in mu‐opioid receptor containing neurons. ABSTRACT Opioid analgesia is compromised by intracellular mediators such as protein kinase C (PKC). The phosphatidylinositol hydrolysis‐coupled serotonin receptor 5‐HT2 is ideally suited to promote PKC activation. We test the hypothesis that 5‐HT2A and 5‐HT2B receptors, which have been previously shown to become pro‐excitatory after spinal nerve ligation (SNL), can negatively influence the ability of opioids to depress spinal excitation evoked by noxious input. Spinal superfusion with (100 nM) mu‐opioid receptor (MOR)‐agonist DAMGO significantly depressed C fiber‐evoked spinal field potentials. Simultaneous administration of subclinical 5‐HT2AR antagonist 4F 4PP (100 nM) or 5‐HT2BR antagonist SB 204741 (100 nM) significantly reduced the IC50 value for DAMGO in nerve‐ligated rats (97.56 nM ± 1.51 and 1.20 nM ± 1.28 respectively, relative to 104 nM ± 1.08 at the baseline condition), but not in sham‐operated rats. Both antagonists failed to alter depression induced by delta‐opioid receptor (DOR)‐agonist D‐ala2‐deltorphin II after SNL as well as in the sham condition. Western blot analysis of dorsal horn homogenates revealed bilateral upregulation of 5‐HT2AR and 5‐HT2BR protein band densities after SNL. As assessed from double immunofluorescence labeling for confocal laser scanning microscopy, scarce dorsal horn cell processes showed co‐localization color overlay for 5‐HT2AR/MOR, 5‐HT2BR/MOR, 5‐HT2AR/DOR, or 5‐HT2BR/DOR in sham‐operated rats. Intensity correlation‐based analyses showed significant increases in 5‐HT2AR/MOR and 5‐HT2BR/MOR co‐localizations after SNL. These results indicate that plasticity of spinal serotonergic neurotransmission can selectively reduce spinal MOR mechanisms via 5‐HT2A and 5‐HT2B receptors, including upregulation of the latter and increased expression in dorsal horn neurons containing MOR.

Nuclear phospholipase C-β1 and diacylglycerol LIPASE-α in brain cortical neurons

Phosphoinositide (PtdIns) signaling involves the generation of lipid second messengers in response to stimuli in a receptor-mediated manner at the plasma membrane. In neuronal cells of adult brain, the standard model proposes that activation of metabotropic receptors coupled to Phospholipase C-β1 (PLC-β1) is linked to endocannabinoid signaling through the production of diacylglycerol (DAG), which could be systematically metabolized by 1,2-diacylglycerol Lipases (DAGL) to produce an increase of 2-arachidonoyl-glycerol (2-AG), the most abundant endocannabinoid in the brain. However, the existence of a nuclear PtdIns metabolism independent from that occurring elsewhere in the cell is now widely accepted, suggesting that the nucleus constitutes both a functional and a distinct compartment for PtdIns metabolism. In this review, we shall highlight the main achievements in the field of neuronal nuclear inositol lipid metabolism with particular attention to progress made linked to the 2-AG biosynthesis. Our aim has been to identify potential sites of 2-AG synthesis other than the neuronal cytoplasmic compartment by determining the subcellular localization of PLC-β1 and DAGL-α, which is much more abundant than DAGL-β in brain. Our data show that PLC-β1 and DAGL-α are detected in discrete brain regions, with a marked predominance of pyramidal morphologies of positive cortical cells, consistent with their role in the biosynthesis and release of 2-AG by pyramidal neurons to control their synaptic inputs. However, as novelty, we showed here an integrated description of the localization of PLC-β1 and DAGL-α in the neuronal nuclear compartment. We discuss our comparative analysis of the expression patterns of PLC-β1 and DAGL-α, providing some insight into the potential autocrine role of 2-AG production in the neuronal nuclear compartment that probably subserve additional roles to the recognized activation of the CB1 cannabinoid receptor.

Organization and origin of the connection from the inferior to the superior colliculi in the rat

The inferior colliculus (IC) is the main ascending auditory relay station prior to the superior colliculus (SC). The morphology and origin of the connection from inferior to superior colliculus (I‐SC) was analyzed both by anterograde and retrograde tracing. Irrespective of the subregion of the IC in which they originate, the terminal fields of these connections formed two main tiers in the SC. While the dorsal one primarily involved the stratum opticum and the stratum griseum intermediale, the ventral one innervated the deep strata, although some fibers did connect these tiers. While the dorsal tier occupied almost the whole extension of the SC, the ventral one was mostly confined to its caudomedial quadrant. The fiber density in these tiers decreased gradually in a rostral gradient and the terminal fields became denser as the anterograde tracer at the injection site was distributed more externally in the cortex of the IC. Retrograde tracing confirmed this result, although it did not reveal any topographic ordering for the I‐SC pathway. Most presynaptic boutons of the I‐SC terminal field were located either inside or close to the patches of acetylcholinesterase activity. Together with previous anatomical and physiological studies, our results indicate that the I‐SC connection relays behaviorally relevant information for sensory‐motor processing. Our observation that this pathway terminates in regions of the superior colliculus, where neurons involved in fear‐like responses are located, reinforce previous suggestions of a role for the IC in generating motor stereotypes that occur during audiogenic seizures. J. Comp. Neurol. 499:716–731, 2006.

Transient, 5-HT2B receptor–mediated facilitation in neuropathic pain: Up-regulation of PKCγ and engagement of the NMDA receptor in dorsal horn neurons

Summary 5‐HT2B receptor activation promotes transient translocation of protein kinase C &ggr; (PKC&ggr;) and phosphorylation of NR1, subserving hyersensitivity to thermal and mechanical pain in the rat after spinal nerve ligation. ABSTRACT Spinal nociception can be facilitated by 5‐HT2 receptors in neuropathic pain. We investigated the involvement of glutamate receptors in dorsal neuron hyperexcitation that is promoted by 5‐HT2B receptor (5‐HT2BR) after spinal nerve ligation (SNL) in the rat. Augmentation of C‐fiber–evoked potentials by spinal superfusion with 5‐HT2BR agonist BW 723C86 in nerve‐ligated rats was impeded by co‐administration of NMDA receptor (NMDAR) antagonist D‐AP5, but not by mGluR1/5 antagonist AIDA or mGluR2/3 antagonist LY 341495. Evoked potentials were increased by cis‐ACPD in nerve‐injured rats, irrespective of simultaneous 5‐HT2BR blockade by SB204741. In uninjured rats, NMDAR agonist cis‐ACPD enhanced evoked potentials in the presence of BW 723C86 but not if administered alone or during exposure to protein kinase C &ggr; (PKC&ggr;) inhibitor peptide. Triple immunofluorescence labelings revealed co‐localization of NMDAR and 5‐HT2BR in PKC&ggr;‐expressing perikarya in lamina II neurons. As a result of SNL, PKC&ggr; was transiently and bilaterally up‐regulated in synaptic fraction from dorsal horn homogenates, peaking at day 2 and returning to basal levels by day 9. Chronic blockade of 5‐HT2BR with selective antagonist SB 204741 after SNL bilaterally decreased the following: (i) PKC&ggr; up‐regulation in synaptic fraction, (ii) phosphorylation of NMDAR subunit NR1 (serine 889) in synaptic fraction, and (iii) co‐localization of both PKC&ggr; and phosphorylated NR1 with postsynaptic marker PSD‐95. Chronic delivery of SB 204741 bilaterally attenuated thermal and mechanical allodynia occurring after SNL, particularly at day 2 post injury. These findings suggest that transient activation of the PKC&ggr;/NMDAR pathway is critically involved in 5‐HT2BR‐mediated facilitation in the SNL model of neuropathic pain.

Ionotropic glutamate receptor subunits are differentially regulated in the motoneuronal pools of the rat hypoglossal nucleus in response to axotomy

Unilateral hypoglossal nerve axotomy was used as a model to analyse immunohistochemically the expression of the GluR1, GluR2, GluR3, and GluR4 glutamate receptor subunits of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype and the NR1 subunit of the N-methyl-D-aspartate (NMDA) subtype in the different morphofunctional hypoglossal pools from 1 to 45 days postaxotomy. Following hypoglossal nerve axotomy, the percentage of motoneurons that were GluR1-immunopositive and the labeling intensity for this subunit was increased in some hypoglossal pools. Immunolabeling for the GluR2 subunit was undetectable. These results contrast with the unchanged pattern for these two subunits after sciatic nerve axotomy previously described. Image analysis showed a significant decrease in the intensity of immunohistochemical labeling for the GluR2/3 and GluR4 subunits in motoneurons, although most motoneurons were still immunopositive for these 2 subunits after axotomy. The intensity of immunolabeling for the NR1 subunit was slightly decreased postlesion, whereas the percentage of NR1-immunopositive motoneurons increased. Immunoreactivity returned to basal levels 45 days postlesion. These findings show that in axotomized hypoglossal motoneurons, i) AMPA and NMDA receptor subunits are still expressed, ii) the composition of the ionotropic glutamate receptor subunit pool is subjected to continuous changes during the regeneration process, iii) AMPA receptors, if functional, would have physiological properties different to those in intact motoneurons, and iv) the various AMPA receptor subunits are differentially regulated. The present results also suggest a faster recovery of basal levels of immunoreactivity for caudally localised groups of motoneurons which could reflect a caudo-rostral sequential functional revovery in the hypoglossal nucleus.

Sprouting of the visual corticocollicular terminal field after removal of contralateral retinal inputs in neonatal rabbits

Abstract The morphological changes occurring in the visual corticocollicular projection following removal of the contralateral retina (within the first 48 h of postnatal life) were studied using New Zealand rabbits. At 45–50 days after lesion, the corticocollicular terminal field was examined by anterograde transport of Phaseolus vulgaris leucoagglutinin, which was applied iontophoretically in the central region of the contralateral striate cortex. In contrast to normal intact rabbits of the same age, the corticocollicular terminal field was markedly enlarged in experimental animals. In the centre of the field we found abundant oblique fibres which sent out branches. These collateral fibres coursed over long distances, parallel to the pial surface, in the stratum zonale and in the upper part of the stratum griseum superficiale. The presence of these fibres, together with an increased density of synaptic boutons at more superficial levels of the sprouted terminal field, suggest that corticocollicular fibres tended to occupy territories left vacant when retinocollicular axons degenerated after enucleation. The high density and extensive distribution of these corticocollicular fibres may be due to the continued growth of the fibres, which occupy an extensive territory during the early postnatal stages and which, under normal circumstances are retracted during the process of postnatal maturation. Despite the expansion of the field occupied by corticocollicular synapses, its centre coincided topographically with the field centres in normal animals, indicating the existence of intrinsic positional cues that persisted after enucleation and determined the arrangement of visual cortical afferents. This model, which involves substantial changes in terminal field organization, should prove useful in elucidating the cellular and molecular processes underlying regeneration and plasticity in the visual system.

Chronic effects of corticosterone on GIRK1-3 subunits and 5-HT1A receptor expression in rat brain and their reversal by concurrent fluoxetine treatment.

Dysregulation of the serotonergic system and abnormalities of the hypothalamic-pituitary-adrenal axis have been demonstrated in major depression. Animal studies indicate that 5-HT1A receptor expression may be reduced by long-term administration of corticosterone. However, similar studies on the regulation of GIRK channels, one of the most important effectors of the neuronal 5-HT1A receptor, are limited. In order to address these issues, slow-release corticosterone pellets were implanted subcutaneously to adrenal intact male rats (200mg pellets, 35 days release). Starting on day 15, animals were treated for 21 days with fluoxetine (5mg/kg/day, i.p.), or vehicle. Using in situ hybridization histochemistry and receptor autoradiography, we found that chronic corticosterone treatment was accompanied by a significant decrease on the mRNAs coding for mineralocorticoid receptors in hippocampal areas. Under these conditions, 5-HT1A receptor mRNA expression decreased in dorsal raphe nucleus and dentate gyrus. However, 5-HT1A receptor levels, as measured by [(3)H]-8-OH-DPAT binding, diminished significantly only in dentate gyrus. It is noteworthy that chronic treatment with fluoxetine reversed the alterations on 5-HT1A receptor mRNA levels only in dorsal raphe. Finally, chronic corticosterone treatment produced an increase on the mRNA coding for the GIRK2 subunit in several hypothalamic and thalamic areas, which was reversed by fluoxetine. Measurements of cell density and volume of the granular layer of the dentate gyrus did not reveal significant changes after corticosterone or corticosterone plus fluoxetine treatments. These data are relevant for a better understanding of the differential regulation of pre- and postsynaptic 5-HT1A receptors by corticosterone flattened rhythm.