Abstract: 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 (Na(v)), NF186 (neurofascin 186), NrCAM (neuron glia-related cell adhesion molecule), and cytoskeleton linkers ankyrin G (AnkG) and betaIV-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 Ca(2+) 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 betaIV-spectrin. We suggest that IQCJ-SCHIP-1 might participate, along with AnkG and betaIV-spectrin, in the stabilization or function of the multimolecular complexes of AISs and NRs, possibly by participating in Ca(2+)-mediated responses.

Abstract: Calcitonin gene-related peptide (CGRP) is a widespread neuropeptide with multiple central and peripheral targets. In an analysis on the expression of this peptide throughout the rat brain during postnatal development, we observed a discrepancy between results obtained by immunohistochemistry and by in situ hybridization. In the superior colliculus (SC), only the immunohistochemical signal could be detected (Terrado et al. [1997] Neuroscience 80:951-970). Here we focus our attention on this structure because the temporal pattern of CGRP immunoreactivity observed in the SC suggested the participation of this peptide in the postnatal maturation of the SC. In the present study, we describe in detail the postnatal development of collicular CGRP-immunoreactive structures and their spatiotemporal relationship with cholinergic modules and definitively demonstrate the local expression of CGRP in the SC. CGRP-immunopositive axons and neurons were distributed within the most ventral part of superficial strata and in the intermediate strata of the SC, showing a peak in staining intensity and density at the end of the first postnatal week. At P14, CGRPergic terminal fibers are arranged in small, clearly defined patches in a complementary manner with respect to the cholinergic modules, which start forming at this stage. By using Western blot and RT-PCR analyses, and by means of injections of antisense oligonucleotides, both the presence of CGRP peptide in the SC and the local expression of alpha-CGRP transcripts in collicular neurons were demonstrated. A possible role of CGRP is discussed in the context of postnatal modular compartmentalization of collicular afferents.

Abstract: 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.

Abstract: Somatosensory stimuli from the body to deep and intermediate strata of the superior colliculus (SC) are relayed from the dorsal column nuclei (DCN), gracile (GrN) and cuneate (CuN). Electrophysiological studies have shown that the somatosensory representation in SC is arranged into a map-like pattern. However, there is a lack of studies confirming a morphological correlate of such an organization. On the other hand, after neonatal enucleation in rodents, somatosensory inputs ascend from their normal termination territory in intermediate and deep collicular strata to invade the more dorsally located visual strata. However, the origin of these reactive afferents has not been specified. By using anterograde (biotinylated dextran amine 10,000; BDA) and retrograde (Fluoro-Gold; FG) tracers, we studied separately the connection from GrN and CuN to the intact and neonatally deafferented SC. GrN-collicular afferents were found to terminate mainly within the periphery of the caudomedial SC quadrant, whereas CuN-collicular fibers innervated primarily the lateral part of the rostrolateral and caudolateral collicular quadrants, in a way consistent with previously described functional data. Retrograde tracing experiments using FG injected in SC confirmed this topographical arrangement. Injections of BDA in GrN or CuN of neonatally enucleated rats showed that reactive fibers reaching superficial strata are only those CuN-collicular fibers innervating the caudolateral SC quadrant, where the forelimb is represented. The present results provide an anatomical substrate for the known somatotopic organization of tactile representation in SC and further reinforce the previous proposal that the plastic reorganization of DCN-collicular afferents following neonatal enucleation constitutes a functional compensatory response.

Abstract: Quantification of presynaptic terminals often requires laborious techniques that involve tissue preparation for ultrastructural analysis. Modern preembedding immunohistochemical techniques provide a high morphological resolution at the light microscope level, thus allowing us to identify immunostained presynaptic boutons using specific antibodies. When absolute density of boutons (D(a)) is analysed for comparison between control and deafferented nervous tissue, quantification may be distorted due to tissue shrinkage that follows deafferentiation. The magnitude of this effect must be, therefore, estimated to correct quantitative data. Using the superior colliculus (SC) as a model, an easily applicable protocol to quantify the density of small size labelled particles in control and deafferented nervous tissue is described. This protocol was used to analyse the effect of neonatal and adult enucleation on the adult pattern of cholinergic input to the rat SC. Statistical treatment of data demonstrated that neonatal enucleation caused a drastic increase in bouton density in the visual collicular layers, stratum zonale (SZ) and stratum griseum superficiale (SGS). The same lesion carried out in adult animals caused an increase in the bouton density exclusively in the SZ.

Abstract: 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.

Abstract: The effect of chronic amphetamine administration on the expression of D2dopamine receptor was immunolhistochenmically analyzed in the caudate-putamen and the lateral habenular nucleus, which are important links of the dorsal diencephalic conduction system. Significant decrease in the numbers of immunopositive neural cells was observed in both regions, suggesting that chronically administered amphetamine alters D2 dopamine receptor function in the dorsal diencephalic conduction svstem. This could be involved in the development of schizophrenia. stereotypies and neural impairment in psychostimulant abusers.

Abstract: The effects of neonatal and adult enucleation on the adult pattern of cholinergic inputs to the rat superior colliculus (SC) was analysed. In the superficial layers immunohistochemical labelling revealed that choline acetyltransferase (ChAT) was predominantly confined to single boutons which were almost continuously distributed throughout the rostrocaudal and lateromedial axes. In these layers a higher density of boutons was observed in the stratum zonale (SZ) and lower stratum griseum superficiale (SGSl) than in the upper stratum griseum superficiale (SGS(u)) and stratum opticum (SO). In intermediate collicular layers ChAT-immunostaining was mainly found in axonal profiles which were arranged in a patchy fashion. Neonatal enucleation caused a drastic increase in bouton density in the SZ, SGS(u) and SGSl. The density of boutons was particularly high in the SGS(u), giving the appearance of an almost homogeneous distribution of boutons from the collicular surface down to the upper limit of SO. Visual deafferentiation at the adult stage was followed by an increase in the bouton density exclusively in the SZ. Neonatal enucleation produced a dorsoventral enlargement of the region containing patches of ChAT staining which was slightly greater following adult deafferentiation. The results described here show that after visual deafferentiation an increase in ChAT innervation to superficial and intermediate collicular layers occurs, providing new information regarding plasticity in the visual system. In view of previous data on cholinergic function in the central nervous system, such an increase could compensate for the loss of retinal excitatory input by facilitating neuronal responses in the SC.

Abstract: The effects of neonatal enucleation on the final adult pattern of retrospleniocollicular connection in the rat was studied using the anterograde tracer biotindextranamine 10,000 (BDA) iontophoretically injected in different anteroposterior locations of the retrosplenial cortex. Retrosplenial afferents are normally distributed in all collicular layers beneath the stratum griseum superficiale (SGS) throughout almost the entire rostrocaudal and lateromedial collicular axes. Neonatal enucleation caused an invasion of lower SGS by abundant retrosplenial afferents, whose distribution remained unaltered in intermediate and deep collicular layers. Axons entering the deafferented SGS showed variable morphologies and arborization patterns. Some of them ran lateromedially close to the SGS-stratum opticum (-SO) limit, giving rise to many collaterals which invaded the lower part of the SGS; whereas others formed narrow terminal arbors, mostly branching in the SO. In the intermediate layers, synaptic profiles were mainly found close to the borders of acetylcholinesterase (AChE) patches in both control and enucleated animals, indicating that neonatal enucleation does not alter the final pattern of retrospleniocollicular afferents to these collicular regions. The results presented here demonstrate that neonatal enucleation leads to the development of an aberrant projection from the retrosplenial cortex to the deafferented superficial layers of the superior colliculus. These results provide new information regarding the reorganization of connections subsequent to neonatal enucleation and suggest that, in enucleated animals, nonvisual multisensorial information could be relayed to central circuits which in intact animals belong to the visual system.

Abstract: The retrospleniocollicular connection is of interest because it constitutes one link between the limbic system, which is considered the anatomical substrate of emotional experience, and the superior colliculus (SC), which mediates approach and avoidance behavior. The morphology, topography, and origin of the retrospleniocollicular connections were studied by using anterograde [biotinylated dextranamine 10,000 (BDA)] and retrograde [Fluoro-Gold (FG)] tracers. After BDA injections involving retrosplenial granular and agranular cortices, terminal fibers innervating all collicular layers except stratum griseum superficiale were found throughout nearly the entire colliculi. Axons branched within restricted portions of the dorsoventral collicular axis with variable morphologies, suggesting functional heterogeneity. Terminal fields originating in anterior and posterior regions of the retrosplenial cortex were preferentially distributed in laterodorsal and medioventral collicular regions, respectively, but there were also large, densely innervated regions in which the terminal fields overlapped. FG injections in the SC confirmed the retrospleniocollicular topography and demonstrated that this connection originated from layer V pyramidal cells of all retrosplenial areas. The distribution of retrospleniocollicular boutons was related to that of the AChE modules, which are associated with connections in the intermediate layers of the SC. In lateral portions of the SC intermediate layers, most retrospleniocollicular boutons were found in medium AChE stained regions, whereas in medial portions, they terminated in AChE-poor domains. The present results demonstrate that the retrosplenial cortex is the origin of a broad and dense network of axonal branches that may modulate SC-mediated motor and physiological responses involved in emotional behavior.

Abstract: 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 alpha-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 recovery in the hypoglossal nucleus.

Abstract: The expression of ionotropic glutamate receptor subunits in the motoneuronal pools of the hypoglossal nucleus was studied using specific antibodies against subunits of the alpha-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 Ca(2+)-entry through AMPA channels) strongly suggests that AMPA receptors in hypoglossal motoneurons are Ca(2+)-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.

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.