Astroglial type-1 cannabinoid (CB ) receptors are involved in synaptic transmission, plasticity and behavior by interfering with the so-called tripartite synapse formed by pre- and post-synaptic neuronal elements and surrounding astrocyte processes. However, little is known concerning the subcellular distribution of astroglial CB receptors. In particular, brain CB receptors are mostly localized at cells' plasmalemma, but recent evidence indicates their functional presence in mitochondrial membranes. Whether CB receptors are present in astroglial mitochondria has remained unknown. To investigate this issue, we included conditional knock-out mice lacking astroglial CB receptor expression specifically in glial fibrillary acidic protein (GFAP)-containing astrocytes (GFAP-CB -KO mice) and also generated genetic rescue mice to re-express CB receptors exclusively in astrocytes (GFAP-CB -RS). To better identify astroglial structures by immunoelectron microscopy, global CB knock-out (CB -KO) mice and wild-type (CB -WT) littermates were intra-hippocampally injected with an adeno-associated virus expressing humanized renilla green fluorescent protein (hrGFP) under the control of human GFAP promoter to generate GFAPhrGFP-CB -KO and -WT mice, respectively. Furthermore, double immunogold (for CB ) and immunoperoxidase (for GFAP or hrGFP) revealed that CB receptors are present in astroglial mitochondria from different hippocampal regions of CB -WT, GFAP-CB -RS and GFAPhrGFP-CB -WT mice. Only non-specific gold particles were detected in mouse hippocampi lacking CB receptors. Altogether, we demonstrated the existence of a precise molecular architecture of the CB receptor in astrocytes that will have to be taken into account in evaluating the functional activity of cannabinergic signaling at the tripartite synapse.

This study examines the axonal projections of so-called inverted pyramids and other neurons with their major dendritic shaft oriented in the direction of the white matter ('inverted cells') in the adult rabbit cortex. Single injections of horseradish peroxidase wheat germ agglutinin were made into cortical or subcortical sites. The resulting retrograde labelling in the cortex was analysed and the distribution across areas and layers of inverted cells contributing to each of these projections was estimated. In addition, the radial distribution of inverted cells was independently determined from rapid Golgi-impregnated and Nissl-stained material. All three procedures revealed that inverted cells lay overwhelmingly in infragranular layers, but congregated at the border between layers 5 and 6. Inverted cells, identified by retrograde labelling, seldom furnished non-telencephalic centres; in contrast, these cells constituted a major source for the projections to the ipsi- or the contralateral cortex, the claustrum or the nucleus caudatus. In general, each set of inverted cells (when defined by its specific destination as a group) was located below the typically oriented cells whose axons were aimed at the same target. Thus, the inverted cells of the rabbit cortex are characterized not only by their unique morphology and their corticocortical, corticoclaustral and corticostriatal projections, but also by their distinctive radial locations. These findings suggest that inverted cells, even though possibly composed of different cell types, are a specific class of projection neurons.

The potential neuroprotection of nicotinamide on the consequences of perinatal asphyxia was investigated with triple organotypic cultures. Perinatal asphyxia was induced in vivo by immersing foetuses-containing uterine horns removed from ready-to-deliver rats into a water bath for 20 min. Sibling caesarean-delivered pups were used as controls. Three days later tissue from substantia nigra, neostriatum and neocortex was dissected and placed on a coverslip. After a month, the cultures were processed for immunocytochemistry and phenotyped with markers against the NMDA receptor subunit NR1, tyrosine hydroxylase (TH), or neuronal nitric oxide synthase (nNOS). Some cultures were analysed for cell viability. Nicotinamide (0.8 mmol/kg, i.p.) or saline was administered to asphyxia-exposed and caesarean-delivered control pups 24, 48 and 72 h after birth. Perinatal asphyxia produced a decrease of cell viability in substantia nigra, but not in neostriatum or neocortex. Immunocytochemistry confirmed the vulnerability of the substantia nigra, demonstrating that there was a significant decrease in the number of NR1 and TH-positive (+) cells/mm2, as well as a decrease in the length of TH+ processes, suggesting neurite atrophy. In control cultures, many nNOS+ cells were seen, with different features, regional distribution and cell body sizes. Following perinatal asphyxia, there was an increase in the number of nNOS+ cells/mm2 in substantia nigra, versus a decrease in neostriatum including reduced neurite length, and no apparent changes in neocortex. The main effect of nicotinamide was seen in the neostriatum, preventing the asphyxia-induced decrease in the number of nNOS+ cells and neurite length. Nicotinamide also prevented the effect of perinatal asphyxia on TH-positive neurite length. The present results support the idea that nicotinamide can prevent the effects produced by a sustained energy-failure condition, as occurring during perinatal asphyxia.

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