Development of inhibitory synaptic inputs on layer 2/3 pyramidal neurons in the rat medial prefrontal cortex.

Inhibitory control of pyramidal neurons plays a major role in governing the excitability in the brain. While spatial mapping of inhibitory inputs onto pyramidal neurons would provide important structural data on neuronal signaling, studying their distribution at the single cell level is difficult due to the lack of easily identifiable anatomical proxies. Here, we describe an approach where in utero electroporation of a plasmid encoding for fluorescently tagged gephyrin into the precursors of pyramidal cells along with ionotophoretic injection of Lucifer Yellow can reliably and specifically detect GABAergic synapses on the dendritic arbour of single pyramidal neurons. Using this technique and focusing on the basal dendritic arbour of layer 2/3 pyramidal cells of the medial prefrontal cortex, we demonstrate an intense development of GABAergic inputs onto these cells between postnatal days 10 and 20. While the spatial distribution of gephyrin clusters was not affected by the distance from the cell body at postnatal day 10, we found that distal dendritic segments appeared to have a higher gephyrin density at later developmental stages. We also show a transient increase around postnatal day 20 in the percentage of spines that are carrying a gephyrin cluster, indicative of innervation by a GABAergic terminal. Since the precise spatial arrangement of synaptic inputs is an important determinant of neuronal responses, we believe that the method described in this work may allow a better understanding of how inhibition settles together with excitation, and serve as basics for further modelling studies focusing on the geometry of dendritic inhibition during development.

K-Cl Cotransporter 2-mediated Cl- Extrusion Determines Developmental Stage-dependent Impact of Propofol Anesthesia on Dendritic Spines.

BACKGROUND: General anesthetics potentiating γ-aminobutyric acid (GABA)-mediated signaling are known to induce a persistent decrement in excitatory synapse number in the cerebral cortex when applied during early postnatal development, while an opposite action is produced at later stages. Here, the authors test the hypothesis that the effect of general anesthetics on synaptogenesis depends upon the efficacy of GABA receptor type A (GABAA)-mediated inhibition controlled by the developmental up-regulation of the potassium-chloride (K-Cl) cotransporter 2 (KCC2). METHODS: In utero electroporation of KCC2 was used to prematurely increase the efficacy of (GABAA)-mediated inhibition in layer 2/3 pyramidal neurons in the immature rat somatosensory cortex. Parallel experiments with expression of the inward-rectifier potassium channel Kir2.1 were done to reduce intrinsic neuronal excitability. The effects of these genetic manipulations (n = 3 to 4 animals per experimental group) were evaluated using iontophoretic injection of Lucifer Yellow (n = 8 to 12 cells per animal). The total number of spines analyzed per group ranged between 907 and 3,371. RESULTS: The authors found a robust effect of the developmental up-regulation of KCC2-mediated Cl transport on the age-dependent action of propofol on dendritic spines. Premature expression of KCC2, unlike expression of a transport-inactive KCC2 variant, prevented a propofol-induced decrease in spine density. In line with a reduction in neuronal excitability, the above result was qualitatively replicated by overexpression of Kir2.1. CONCLUSIONS: The KCC2-dependent developmental increase in the efficacy of GABAA-mediated inhibition is a major determinant of the age-dependent actions of propofol on dendritic spinogenesis.

Effects of morphine on the differentiation and survival of developing pyramidal neurons during the brain growth spurt.

Although morphine is frequently administered to treat procedural pain in neonates and young children, little is known about the effects of this drug on developing neural circuitry during the brain growth spurt. Here we systematically explored the impact of morphine on neuronal survival and differentiation during the peak synaptogenic period. By focusing on the rat medial prefrontal cortex, we show that single bolus ip injections of morphine, although it induces deep sedation and analgesia, do not entrain apoptosis in this cortical region either at postnatal day 7 or at postnatal day 15. Iontophoretic single cell injections of Lucifer Yellow followed by semiautomatic neuronal arbor tracing revealed that repeated daily administration of this drug between postnatal days 7 and 15 or 15 and 20 did not interfere with dendritic development of layer 5 pyramidal neurons. Confocal microscopic analysis of dendritic spines at the aforementioned distinct stages of the brain growth spurt demonstrated that neither single bolus nor repeated administration of morphine affected the density of these postsynaptic structures. Altogether, these preclinical rodent experimental observations argue against overt neurotoxic effects of morphine exposure during the brain growth spurt.