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The alteration of the endocannabinoid tone usually associates with changes in the expression and/or function of the cannabinoid CB

The cannabinoid CB

The use and abuse of cannabis can be associated with significant pathophysiology, however, it remains unclear whether (1) acute administration of Δ-9-tetrahydrocannabinol (THC) during early adulthood alters the cannabinoid type 1 (CB1 ) receptor localization and expression in cells of the brain, and (2) THC produces structural brain changes. Here we use electron microscopy and a highly sensitive pre-embedding immunogold method to examine CB1 receptors in the hippocampus cornu ammonis subfield 1 (CA1) 30 min after male mice were exposed to a single THC injection (5 mg/kg). The findings show that acute exposure to THC can significantly decrease the percentage of CB1 receptor immunopositive terminals making symmetric synapses, mitochondria, and astrocytes. The percentage of CB1 receptor-labeled terminals forming asymmetric synapses was unaffected. Lastly, CB1 receptor expression was significantly lower at terminals of symmetric and asymmetric synapses as well as in mitochondria. Structurally, CA1 dendrites were significantly larger, and contained more spines and mitochondria following acute THC administration. The area of the dendritic spines, synaptic terminals, mitochondria, and astrocytes decreased significantly following acute THC exposure. Altogether, these results indicate that even a single THC exposure can have a significant impact on CB1 receptor expression, and can alter CA1 ultrastructure, within 30 min of drug exposure. These changes may contribute to the behavioral alterations experienced by young individuals shortly after cannabis intoxication.

The cannabinoid CB1 receptor localizes to the glutamatergic parallel fiber (PF) terminals of the cerebellar granule cells and participates in synaptic plasticity, motor control and learning that are impaired in CB1 receptor knockout (CB 1 -KO) mice. However, whether ultrastructural changes at the PF-Purkinje cell (PC) synapses occur in CB 1 -KO remains unknown. We studied this in the vermis of the spinocerebellar lobule V and the vestibulocerebellar lobule X of CB 1 -KO and wild-type (CB 1 -WT) mice by electron microscopy. Lobule V, but not lobule X, of CB 1 -KO had significantly less and longer synapses than in CB 1 -WT. PF terminals were significantly larger in both lobules of CB 1 -KO with no changes in PC dendritic spines. The PF terminals in lobule V of CB 1 -KO contained less synaptic vesicles and lower vesicle density; by contrast, vesicle density in lobule X of CB 1 -KO remained unchangeable relative to CB 1 -WT. There were as many vesicles in lobule V of CB 1 -KO as in CB 1 -WT, but their distribution decreased drastically at 300 nm of the active zone. In lobule X of CB 1 -KO, less vesicles were found within 150 nm from the presynaptic membrane; however, no vesicles were at 450-600 nm of the active zone. A significant higher amount of synaptic vesicles close to the active zone in lobule V and X of CB 1 -KO was observed. In conclusion, the absence of CB1 receptors strikingly and distinctively impacts on the ultrastructural architecture of the PF-PC synapses located in cerebellar lobules that differ in vulnerability to damage and motor functions.

Binge drinking (BD) is a serious health concern in adolescents as high ethanol (EtOH) consumption can have cognitive sequelae later in life. Remarkably, an enriched environment (EE) in adulthood significantly recovers memory in mice after adolescent BD, and the endocannabinoid, 2-arachydonoyl-glycerol (2-AG), rescues synaptic plasticity and memory impaired in adult rodents upon adolescent EtOH intake. However, the mechanisms by which EE improves memory are unknown. We investigated this in adolescent male C57BL/6J mice exposed to a drinking in the dark (DID) procedure four days per week for a duration of 4 weeks. After DID, the mice were nurtured under an EE for 2 weeks and were subjected to the Barnes Maze Test performed the last 5 days of withdrawal. The EE rescued memory and restored the EtOH-disrupted endocannabinoid (eCB)-dependent excitatory long-term depression at the dentate medial perforant path synapses (MPP-LTD). This recovery was dependent on both the cannabinoid CB1 receptor and group I metabotropic glutamate receptors (mGluRs) and required 2-AG. Also, the EE had a positive effect on mice exposed to water through the transient receptor potential vanilloid 1 (TRPV1) and anandamide (AEA)-dependent MPP long-term potentiation (MPP-LTP). Taken together, EE positively impacts different forms of excitatory synaptic plasticity in water- and EtOH-exposed brains. This research was funded by ISCIII (“RD16/0017/0012” to P.G.), co-funded by ERDF/ESF, “Investing in your future”; The Basque Government (IT1230-19 to P.G.); Ministry of Science and Innovation (PID2019-107548RB-I00 to P.G.); Ph.D. contract from MINECO (BES-2013-065057 to S.P.); Ph.D. contract from UPV/EHU (PIF 18/315 to L.L.), and Ph.D. contract from UPV/EHU (PIF 19/164 to M.S.).

The use and abuse of cannabis can be associated with significant pathophysiology, however, it remains unclear whether (1) acute administration of Δ-9-tetrahydrocannabinol (THC) during early adulthood alters the cannabinoid type 1 (CB

Binge drinking (BD) is a common pattern of ethanol (EtOH) consumption by adolescents. The brain effects of the acute EtOH exposure are well-studied; however, the long-lasting cognitive and neurobehavioral consequences of BD during adolescence are only beginning to be elucidated. Environmental enrichment (EE) has long been known for its benefits on the brain and may serve as a potential supportive therapy following EtOH exposure. In this study, we hypothesized that EE may have potential benefits on the cognitive deficits associated with BD EtOH consumption. Four-week-old C57BL/6J male mice were exposed to EtOH following an intermittent 4-day drinking-in-the-dark procedure for 4 weeks. Then they were exposed to EE during EtOH withdrawal for 2 weeks followed by a behavioral battery of tests including novel object recognition, novel location, object-in-place, rotarod, beam walking balance, tail suspension, light-dark box and open field that were run during early adulthood. Young adult mice exposed to EE significantly recovered recognition, spatial and associative memory as well as motor coordination skills and balance that were significantly impaired after adolescent EtOH drinking with respect to controls. No significant permanent anxiety or depressive-like behaviors were observed. Taken together, an EE exerts positive effects on the long-term negative cognitive deficits as a result of EtOH consumption during adolescence.

Cannabinoid type-1 (CB1 ) receptors are widely distributed in the brain and play important roles in astrocyte function and the modulation of neuronal synaptic transmission and plasticity. However, it is currently unknown how CB1 receptor expression in astrocytes is affected by long-term exposure to stressors. Here we examined CB1 receptors in astrocytes of ethanol (EtOH)-exposed adolescent mice to determine its effect on CB1 receptor localization and density in adult brain. 4-8-week-old male mice were exposed to 20 percent EtOH over a period of 4 weeks, and receptor localization was examined after 4 weeks in the hippocampal CA1 stratum radiatum by pre-embedding immunoelectron microscopy. Our results revealed a significant reduction in CB1 receptor immunoparticles in astrocytic processes of EtOH-exposed mice when compared with controls (positive astrocyte elements: 21.50 ± 2.80 percent versus 37.22 ± 3.12 percent, respectively), as well as a reduction in particle density (0.24 ± 0.02 versus 0.35 ± 0.02 particles/μm). The majority of CB1 receptor metal particles were in the range of 400-1200 nm from synaptic terminals in both control and EtOH. Altogether, the decrease in the CB1 receptor expression in hippocampal astrocytes of adult mice exposed to EtOH during adolescence reveals a long lasting effect of EtOH on astrocytic CB1 receptors. This deficiency may also have negative consequences for synaptic function.

The cannabinoid CB

Type 1 cannabinoid (CB1 ) receptors are widely distributed in the brain. Their physiological roles depend on their distribution pattern, which differs remarkably among cell types. Hence, subcellular compartments with little but functionally relevant CB1 receptors can be overlooked, fostering an incomplete mapping. To overcome this, knockin mice with cell-type-specific rescue of CB1 receptors have emerged as excellent tools for investigating CB1 receptors' cell-type-specific localization and sufficient functional role with no bias. However, to know whether these rescue mice maintain endogenous CB1 receptor expression level, detailed anatomical studies are necessary. The subcellular distribution of hippocampal CB1 receptors of rescue mice that express the gene exclusively in dorsal telencephalic glutamatergic neurons (Glu-CB1 -RS) or GABAergic neurons (GABA-CB1 -RS) was studied by immunoelectron microscopy. Results were compared with conditional CB1 receptor knockout lines. As expected, CB1 immunoparticles appeared at presynaptic plasmalemma, making asymmetric and symmetric synapses. In the hippocampal CA1 stratum radiatum, the values of the CB1 receptor-immunopositive excitatory and inhibitory synapses were Glu-CB1 -RS, 21.89% (glutamatergic terminals); 2.38% (GABAergic terminals); GABA-CB1 -RS, 1.92% (glutamatergic terminals); 77.92% (GABAergic terminals). The proportion of CB1 receptor-immunopositive excitatory and inhibitory synapses in the inner one-third of the dentate molecular layer was Glu-CB1 -RS, 53.19% (glutamatergic terminals); 2.30% (GABAergic terminals); GABA-CB1 -RS, 3.19% (glutamatergic terminals); 85.07% (GABAergic terminals). Taken together, Glu-CB1 -RS and GABA-CB1 -RS mice show the usual CB1 receptor distribution and expression in hippocampal cell types with specific rescue of the receptor, thus being ideal for in-depth anatomical and functional investigations of the endocannabinoid system. J. Comp. Neurol. 525:302-318, 2017. © 2016 Wiley Periodicals, Inc.

Binge drinking (BD) is a common pattern of ethanol (EtOH) consumption by adolescents. The brain effects of the acute EtOH exposure are well-studied; however, the long-lasting cognitive and neurobehavioral consequences of BD during adolescence are only beginning to be elucidated. Environmental enrichment (EE) has long been known for its benefits on the brain and may serve as a potential supportive therapy following EtOH exposure. In this study, we hypothesized that EE may have potential benefits on the cognitive deficits associated with BD EtOH consumption. Four-week-old C57BL/6J male mice were exposed to EtOH following an intermittent 4-day drinking-in-the-dark procedure for 4 weeks. Then they were exposed to EE during EtOH withdrawal for 2 weeks followed by a behavioral battery of tests including novel object recognition, novel location, object-in-place, rotarod, beam walking balance, tail suspension, light-dark box and open field that were run during early adulthood. Young adult mice exposed to EE significantly recovered recognition, spatial and associative memory as well as motor coordination skills and balance that were significantly impaired after adolescent EtOH drinking with respect to controls. No significant permanent anxiety or depressive-like behaviors were observed. Taken together, an EE exerts positive effects on the long-term negative cognitive deficits as a result of EtOH consumption during adolescence.

Cannabinoid type-1 (CB

The hypothalamus and the endocannabinoid system are important players in the regulation of energy homeostasis. In a previous study, we described the ultrastructural distribution of CB1 receptors in GABAergic and glutamatergic synaptic terminals of the dorsomedial region of the ventromedial nucleus of the hypothalamus (VMH). However, the specific localization of the enzymes responsible for the synthesis of the two main endocannabinoids in the hypothalamus is not known. The objective of this study was to investigate the precise subcellular distribution of N-arachidonoylphospatidylethanolamine phospholipase D (NAPE-PLD) and diacylglycerol lipase α (DAGL-α) in the dorsomedial VMH of wild-type mice by a high resolution immunogold electron microscopy technique. Knock-out mice for each enzyme were used to validate the specificity of the antibodies. NAPE-PLD was localized presynaptically and postsynaptically but showed a preferential distribution in dendrites. DAGL-α was mostly postsynaptic in dendrites and dendritic spines. These anatomical results contribute to a better understanding of the endocannabinoid modulation in the VMH nucleus. Furthermore, they support the idea that the dorsomedial VMH displays the necessary machinery for the endocannabinoid-mediated modulation of synaptic transmission of brain circuitries that regulate important hypothalamic functions such as feeding behaviors.

The cannabinoid type 1 (CB1) receptor is widely distributed in the brain and peripheral organs where it regulates cellular functions and metabolism. In the brain, CB1 is mainly localized on presynaptic axon terminals but is also found on mitochondria (mtCB1), where it regulates cellular respiration and energy production. Likewise, CB1 is localized on muscle mitochondria, but very little is known about it. The aim of this study was to further investigate in detail the distribution and functional role of mtCB1 in three different striated muscles. Immunoelectron microscopy for CB1 was used in skeletal muscles (gastrocnemius and rectus abdominis) and myocardium from wild-type and CB1-KO mice. Functional assessments were performed in mitochondria purified from the heart of the mice and the mitochondrial oxygen consumption upon application of different acute delta-9-tetrahydrocannabinol (Δ9-THC) concentrations (100 nM or 200 nM) was monitored. About 26% of the mitochondrial profiles in gastrocnemius, 22% in the rectus abdominis and 17% in the myocardium expressed CB1. Furthermore, the proportion of mtCB1 versus total CB1 immunoparticles was about 60% in the gastrocnemius, 55% in the rectus abdominis and 78% in the myocardium. Importantly, the CB1 immunolabeling pattern disappeared in muscles of CB1-KO mice. Functionally, acute 100 nM or 200 nM THC treatment specifically decreased mitochondria coupled respiration between 12 and 15% in wild-type isolated mitochondria of myocardial muscles but no significant difference was noticed between THC treated and vehicle in mitochondria isolated from CB1-KO heart. Furthermore, gene expression of key enzymes involved in pyruvate synthesis, tricarboxylic acid (TCA) cycle and mitochondrial respiratory chain was evaluated in the striated muscle of CB1-WT and CB1-KO. CB1-KO showed an increase in the gene expression of Eno3, Pkm2, and Pdha1, suggesting an increased production of pyruvate. In contrast, no significant difference was observed in the Sdha and Cox4i1 expression, between CB1-WT and CB1-KO. In conclusion, CB1 receptors in skeletal and myocardial muscles are predominantly localized in mitochondria. The activation of mtCB1 receptors may participate in the mitochondrial regulation of the oxidative activity probably through the relevant enzymes implicated in the pyruvate metabolism, a main substrate for TCA activity.

Type 1 cannabinoid (CB

Various animals (bacteria, bees, fishes, birds, etc.) show the ability to find orientation in the geomagnetic field. This magnetoreception effect can be explained by the presence of small biogenic magnetite crystals in their organisms that interact with the geomagnetic field. Some studies carried out on rodents show that they respond to magnetic stimulation in the earth’s magnetic field by the expression of activity genes like C-fos, but the mechanism of magnetoreception for them it is still unknown. In this paper, the amount of magnetite of two brains and two cerebellums of Sprague-Dawley rats was measured using ferromagnetic resonance spectroscopy. No presence of magnetite (with a limit of a few picograms) was found. This means that either biogenic magnetite is not located in the brain, but somewhere else, or that the magnetic field sensibility in rats is not related to biogenic magnetite.

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.

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. J. Comp. Neurol. 468:410–424, 2004. © 2003 Wiley-Liss, Inc.

We have recently shown that the transient receptor potential vanilloid type 1 (TRPV1), a non-selective cation channel in the peripheral and central nervous system, is localized at postsynaptic sites of the excitatory perforant path synapses in the hippocampal dentate molecular layer (ML). In the present work, we have studied the distribution of TRPV1 at inhibitory synapses in the ML. With this aim, a preembedding immunogold method for high resolution electron microscopy was applied to mouse hippocampus. About 30% of the inhibitory synapses in the ML are TRPV1 immunopositive, which is mostly localized perisynaptically (similar to 60% of total immunoparticles) at postsynaptic dendritic membranes receiving symmetric synapses in the inner 1/3 of the layer. This TRPV1 pattern distribution is not observed in the ML of TRPV1 knock-out mice. These findings extend the knowledge of the subcellular localization of TRPV1 to inhibitory synapses of the dentate molecular layer where the channel, in addition to excitatory synapses, is present. P. Grandes' laboratory is provided by Ministerio de Economia y Competitividad (BFU2012-33334); Basque Government grant IT764-13; University of the Basque Country (UFI11/41) and by Red de Trastornos Adictivos, RETICS, Instituto de Salud Carlos III, grant number: RD07/0001/2001, RD12/0028/0004.

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.

The effects of neonatal enucleation on the final adult pattern of retrospleniocollicular connection in the rat was studied using the anterograde tracer biotin-dextranamine 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.

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.

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.

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.

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.

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.

Astrocytes release factors like cholesterol, apoE, and pleiotropic molecules that influence synaptogenesis in the central nervous system. In vitro studies have shown that guanosine elicits the production and further release of these synaptogenic factors. To demonstrate that such astrocytic factors are synaptogenic in vivo, osmotic pumps were implanted in primary visual cortex (VC) of Sprague-Dawley rats to deliver guanosine. Simultaneous injection of dextran amine as an anterograde tracer at the same site where the osmotic pumps were implanted enabled the morphology of the fibers emerging from the VC to be visualized as well. The guanosine-treated efferent connections from these animals showed a significant increase in the number and size of synaptic boutons along the efferent fibers when compared with controls. A similar increase in the number and size of synaptic boutons was also detected when the cortico–cortical connection to the lateral secondary visual area was studied in more detail. The ensuing morphological changes to the synapses did not show a clear preference for any particular type or site of the axonal branches that integrates this cortical connection. Moreover, the distribution of boutons along the fibers was clearly stochastic according to their size. Thus, guanosine administration appears to open up the possibility of manipulating connections to compensate for total or partial denervation. Anat Rec, 292:1968–1975, 2009. © 2009 Wiley-Liss, Inc.

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.

Synaptic plasticity is defined as any sort of change in intensity of the synaptic response or in the number of synapses that occurs in nervous structures. Long-lasting increase or decrease of synaptic efficacy — termed long-term potentiation (LTP) and long-term depression (LTD), respectively — has been related to the establishment of memory traces. Experimental induction of both types of long-lasting changes has contributed effectively to the study of the mechanisms that underlie the learning processes. Although the wealth of mechanisms related to synaptic plasticity still remains partially unveiled, at the core of this phenomenon lies the glutamate receptor system together with neurotrophins and their receptors, second-messenger cascades, and changes of gene expression. An increase in the numbers of synapses is termed structural plasticity, and is governed mainly by molecules belonging to the neurotrophin and cytokine families. This form of plasticity participates in the establishment of the intricate arrays of nervous connections during normal development, and in rearrangements of the neuronal circuitries that occur after nervous lesions.

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.

Quantification of presynaptic terminals often requires laborious techniques that involve tissue preparation for ultrastructural analysis [Science 171 (1971) 804; Vision Res. Suppl. 3 (1971) 281]. 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 (Da) 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.

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.

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 acetyltranferase (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 (SGSu) 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, SGSu and SGSl. The density of boutons was particularly high in the SGSu, 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.

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.