Complex neuronal circuitries such as those present in the mammalian cerebral cortex have evolved as balanced networks of excitatory and inhibitory neurons. Although the establishment of appropriate numbers for these cells is essential for brain function and behaviour, our understanding of this fundamental process is very fragmentary. Here we show that interneuron cell survival in mice depends on the activity of pyramidal cells during a critical window of postnatal development, in which excitatory synaptic input to individual interneurons predicts their outcome. Pyramidal cells regulate interneuron survival through the negative modulation of PTEN signalling, which effectively drives interneuron cell death during this period. Taken together, our findings indicate that activity-dependent mechanisms dynamically adjust the number of inhibitory cells in nascent local cortical circuits, ultimately establishing the appropriate proportions of excitatory and inhibitory neurons in the cerebral cortex.

The striking differences between the clinical symptoms of tetanus and botulism have been ascribed to the different fate of the parental neurotoxins once internalised in motor neurons. Tetanus toxin (TeNT) is known to undergo transcytosis into inhibitory interneurons and block the release of inhibitory neurotransmitters in the spinal cord, causing a spastic paralysis. In contrast, botulinum neurotoxins (BoNTs) block acetylcholine release at the neuromuscular junction, therefore inducing a flaccid paralysis. Whilst overt experimental evidence supports the sorting of TeNT to the axonal retrograde transport pathway, recent findings challenge the established view that BoNT trafficking is restricted to the neuromuscular junction by highlighting central effects caused by these neurotoxins. These results suggest a more complex scenario whereby BoNTs also engage long-range trafficking mechanisms. However, the intracellular pathways underlying this process remain unclear. We sought to fill this gap by using primary motor neurons either in mass culture or differentiated in microfluidic devices to directly monitor the endocytosis and axonal transport of full length BoNT/A and BoNT/E and their recombinant binding fragments. We show that BoNT/A and BoNT/E are internalised by spinal cord motor neurons and undergo fast axonal retrograde transport. BoNT/A and BoNT/E are internalised in non-acidic axonal carriers that partially overlap with those containing TeNT, following a process that is largely independent of stimulated synaptic vesicle endo-exocytosis. Following intramuscular injection in vivo, BoNT/A and TeNT displayed central effects with a similar time course. Central actions paralleled the peripheral spastic paralysis for TeNT, but lagged behind the onset of flaccid paralysis for BoNT/A. These results suggest that the fast axonal retrograde transport compartment is composed of multifunctional trafficking organelles orchestrating the simultaneous transfer of diverse cargoes from nerve terminals to the soma, and represents a general gateway for the delivery of virulence factors and pathogens to the central nervous system.

Tetanus neurotoxin (TeNT) is among the most poisonous substances on Earth and a major cause of neonatal death in nonvaccinated areas. TeNT targets the neuromuscular junction (NMJ) with high affinity, yet the nature of the TeNT receptor complex remains unknown. Here, we show that the presence of nidogens (also known as entactins) at the NMJ is the main determinant for TeNT binding. Inhibition of the TeNT-nidogen interaction by using small nidogen-derived peptides or genetic ablation of nidogens prevented the binding of TeNT to neurons and protected mice from TeNT-induced spastic paralysis. Our findings demonstrate the direct involvement of an extracellular matrix protein as a receptor for TeNT at the NMJ, paving the way for the development of therapeutics for the prevention of tetanus by targeting this protein-protein interaction.

The type II classic cadherin subfamily contains a number of extensively studied genes (cdh6, cdh8, cdh11); however, the expression and function of the other members have only been partially described. Here we employed reverse-transcription polymerase chain reaction (RT-PCR) and in situ hybridization to characterize cortical and hippocampal expression of all type II cadherins (with the exception of the nonneural Cdh5) in the developing and adult mouse brain. Many of these genes have ubiquitous mRNA distribution patterns throughout development, indicating high functional redundancy, which might be necessary for safe production of the strictly laminated structure of these regions. A few of the genes examined, however, exhibit a unique spatiotemporal pattern of expression, particularly during cortical development, indicating a potentially specific function. In the developing and adult hippocampus, almost all of these genes are strongly expressed in glutamatergic neurons of the CA1-CA3 pyramidal cell layer and the granular layer of the dentate gyrus. In contrast, there are significant expression differences within the GABAergic cells of the adult hippocampus. Our results indicate that selective expression of type II cadherins may generate a flexible cell-adhesion machinery for developing neurons to selectively bind to each other, but can also provide a high level of security due to the multiple overlaps in the expression domains.

Axonal transport ensures long-range delivery of essential components and signals between proximal and distal areas of the neuron, and it is crucial for neuronal homeostasis and survival. Several pathogens and virulence factors use this route to gain access to the central nervous system, exploiting the complex and still poorly understood trafficking mechanisms that regulate the dynamics of their cellular receptors. Studying the intracellular transport of neurotropic pathogens is therefore instrumental to glean new insights into these important molecular events. Botulinum (BoNT) and tetanus (TeNT) neurotoxins bind with high affinity to a variety of neurons and are internalised by specialised endocytic pathways leading to specific intracellular fates. Whereas BoNT trafficking is largely confined to the neuromuscular junction, TeNT is internalised in signalling endosomes shared with neurotrophins and their receptors, which are recruited to the fast axonal retrograde transport pathway. Recently, important paradigms regarding the mechanisms by which BoNT and TeNT interact with their cellular targets and are transported in neurons have been challenged. In this review, we summarise new findings concerning the uptake and intracellular trafficking of these neurotoxins, and discuss their implications in terms of the physiological effects of BoNT and TeNT in the central nervous system.

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Significance Brain function requires appropriate numbers of different neuronal subtypes, but how these numbers are established remains poorly understood. Here, we identified a key role for the cerebral cortex in remotely controlling interneuron survival and thus establishing appropriate numbers of the two main types of interneurons in another brain region, the striatum. While cortical pyramidal cells directly control the survival of parvalbumin-expressing GABAergic neurons, the survival of cholinergic interneurons is indirectly controlled through the activity of the striatal medium spiny neurons. Our results demonstrate that the input-specific modulation of neuronal activity is universally required to control the survival of interneurons across very different brain circuits. This is important for the assembly of balanced neural networks.

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