Functional and morphological characterization of glutamate transporters in the rat locus coeruleus.

BACKGROUND AND PURPOSE: Excitatory amino acid transporters (EAATs) in the CNS contribute to the clearance of glutamate released during neurotransmission. The aim of this study was to explore the role of EAATs in the regulation of locus coeruleus (LC) neurons by glutamate. EXPERIMENTAL APPROACH: We measured the effect of different EAAT subtype inhibitors/enhancers on glutamate- and KCl-induced activation of LC neurons in rat slices. EAAT2-3 expression in the LC was also characterized by immunohistochemistry. KEY RESULTS: The EAAT2-5 inhibitor DL-threo-ß-benzyloxaspartic acid (100 µM), but not the EAAT2, 4, 5 inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (100 µM) or the EAAT2 inhibitor dihydrokainic acid (DHK; 100 µM), enhanced the glutamate- and KCl-induced activation of the firing rate of LC neurons. These effects were blocked by ionotropic, but not metabotrobic, glutamate receptor antagonists. DHK (100 µM) was the only EAAT inhibitor that increased the spontaneous firing rate of LC cells, an effect that was due to inhibition of EAAT2 and subsequent AMPA receptor activation. Chronic treatment with ceftriaxone (200 mg·kg(-1) i.p., once daily, 7 days), an EAAT2 expression enhancer, increased the actions of glutamate and DHK, suggesting a functional impact of EAAT2 up-regulation on the glutamatergic system. Immuhistochemical data revealed the presence of EAAT2 and EAAT3 surrounding noradrenergic neurons and EAAT2 on glial cells in the LC. CONCLUSIONS AND IMPLICATIONS: These results remark the importance of EAAT2 and EAAT3 in the regulation of rat LC by glutamate. Neuronal EAAT3 would be responsible for terminating the action of synaptically released glutamate, whereas glial EAAT2 would regulate tonic glutamate concentrations in this nucleus.

Plastic response of the retrospleniocollicular connection after removal of retinal inputs in neonatal rats. An anterograde tracing study.

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.

Changes of the cholinergic input to the superior colliculus following enucleation in neonatal and adult rats.

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.

Morphology and topographical organization of the retrospleniocollicular connection: a pathway to relay contextual information from the environment to the superior colliculus.

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.

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 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.

Expression of the genes for alpha-type and beta-type calcitonin gene-related peptide during rat embryogenesis.

Throughout rat embryogenesis we analysed the expression patterns of the three mature transcripts generated from the two calcitonin gene-related peptide genes: calcitonin, alpha-calcitonin gene-related peptide, and beta-calcitonin gene-related peptide messenger RNAs. In addition, we examined in parallel the distribution of calcitonin gene-related peptide and calcitonin immunoreactivity. Of the three transcripts, beta-calcitonin gene-related peptide messenger RNA was first detected in sensory ganglia on embryonic day 14, and by embryonic day 15 was seen to a lesser degree in motor neurons and autonomic ganglia. Starting at embryonic day 16, however, the highest levels of beta-calcitonin gene-related peptide messenger RNA were found in motor neurons rather than sensory ganglia. Alpha-calcitonin gene-related peptide messenger RNA was first detected on embryonic day 16 in both sensory ganglia and motor neurons, but at lower levels than beta-calcitonin gene-related peptide, particularly in the motor neurons of the spinal cord. By embryonic day 20, transcripts for alpha- and beta-calcitonin gene-related peptide were expressed in distinct brain regions. High levels of alpha-calcitonin gene-related peptide messenger RNA were detected in hypoglossal, facial, and parabrachial nuclei, and moderate levels in the trigeminal motor and ambiguus nuclei. By contrast, beta-calcitonin gene-related peptide messenger RNA was detected at low levels in hypoglossal, ambiguus, facial, and parabrachial nuclei, and at high levels in the trigeminal nucleus. In the oculomotor-trochlear nucleus, beta-calcitonin gene-related peptide messenger RNA was the sole isotype expressed. Low levels of messenger RNA for both calcitonin gene-related peptide transcripts were appreciated in the inferior olive. Outside the nervous system, alpha-calcitonin gene-related peptide messenger RNA was weakly expressed in the thyroid gland and beta-calcitonin gene-related peptide messenger RNA in the thymus. Throughout embryogenesis, calcitonin gene-related peptide immunoreactivity usually followed the expression of either alpha- or beta-calcitonin gene-related peptide messenger RNA. Calcitonin messenger RNA and protein were detected only in the thyroid gland from embryonic day 18 onward. This work shows that of the three mature transcripts produced by the two calcitonin gene-related peptide genes, beta-calcitonin gene-related peptide messenger RNA is the predominant transcript produced early in rat embryogenesis. However, by perinatal stages alpha-calcitonin gene-related peptide shows the highest expression in the brain and spinal cord. In autonomic ganglia, beta-calcitonin gene-related peptide is either the sole or the predominant transcript. Unlike the chick embryo in which calcitonin messenger RNA is expressed early in the CNS, in rat it was only expressed outside the nervous system in the thyroid gland during the last days of embryogenesis.

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 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.

The two mature transcripts of the chick calcitonin gene are expressed within the central nervous system during embryogenesis.

Calcitonin mRNA and calcitonin gene-related peptide (CGRP) mRNA both are generated from the calcitonin gene because of tissue-specific alternative splicing of the primary transcript. It is currently established that, of the two mature transcripts, calcitonin mRNA is far the predominant transcript produced in thyroid C-cells whereas only CGRP mRNA is produced in the nervous system. However, here we provide evidence that the two splicing forms of the chick calcitonin primary transcript are found within the developing central nervous system, although displaying specific patterns of expression. While CGRP mRNA is first expressed in motor neurons at rather advanced stages of embryogenesis, calcitonin mRNA is expressed in the floor plate and dorsal rhombencephalon from earliest stages.

Expression of the genes for alpha-type and beta-type calcitonin gene-related peptide during postnatal rat brain development.

In this study we have analysed the expression of the genes for both alpha-type and beta-type calcitonin gene-related peptide (CGRP) during postnatal development of the rat brain and compared it with the expression of CGRP-like immunoreactivity. At birth both alpha-type and beta-type CGRP messenger RNA were present in the parabrachial nucleus, inferior olive and motor nuclei (except for abducens nucleus), and only alpha-type CGRP messenger RNA in some posterior thalamic nuclei. As development advanced, new nuclei started to express either only alpha-CGRP gene (superior olive, parabigeminal, sagulum, and some hypothalamic and cranial thalamic nuclei) or both genes (abducens nucleus). In the inferior olive both genes were transiently expressed. Beta-CGRP messenger RNA disappeared by postnatal day 10 and alpha-CGRP messenger RNA by postnatal day 20. During the whole postnatal development beta-CGRP gene expression predominated over that of alpha-CGRP in the trigeminal and eye motor nuclei, while in the remainder nuclei alpha-CGRP messenger RNA was either the predominant isoform or the sole one. CGRP-like immunoreactivity, which does not distinguish between alpha-type and beta-type CGRP, was detected in those nuclei containing either alpha-CGRP messenger RNA or beta-CGRP messenger RNA. However, no CGRP messenger RNA was detected in areas such as superior colliculus, lateral pontine nucleus, pars reticulata of the substantia nigra, perifornical area, or zona incerta in which CGRP-like immunoreactivity was prominent. CGRP-like immunoreactivity, but not CGRP messenger RNA, was also transiently detected by postnatal day 5 in some cells of the globus pallidus. In the adult brain, the levels of alpha- and beta-CGRP messenger RNA as well as those of CGRP-like immunoreactivity were considerably reduced. This fact, similar to that of other growth- and development-associated factors, suggests a role for CGRP as a neuron-derived neurotrophic factor. The transient expression in neurons of the inferior olive, matching the period when climbing fibres and cerebellar cortex are developing, seems to support such an idea. The results of this study show that those nuclei expressing beta-CGRP gene also express alpha-CGRP gene. However, there are a number of nuclei that only express alpha-CGRP gene. On the other hand, CGRP-like immunoreactivity is detected in some nuclei which express no CGRP messenger RNA. It suggests that such nuclei express any CGRP-related protein (identified by the antibodies against CGRP) or, if they really contain CGRP protein, this is produced from undetectable amounts (using our in situ hybridization histochemistry procedure) of CGRP messenger RNA or it comes from other nuclei that connect with them in which CGRP protein is synthesized and then transferred.

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

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.