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 spur

Effect of Ketamine on Dendritic Arbor Development and Survival of Immature GABAergic Neurons In Vitro

Ketamine, a noncompetitive antagonist of the N-methyl-D-aspartate type of glutamate receptors, was reported to induce neuronal cell death when administered to produce anesthesia in young rodents and monkeys. Subanesthetic doses of ketamine, as adjuvant to postoperative sedation and pain control, are also frequently administered to young children. However, the effects of these low concentrations of ketamine on neuronal development remain unknown. The present study was designed to evaluate the effects of increasing concentrations (0.01-40 μg/ml) and durations (1-96 h) of ketamine exposure on the differentiation and survival of immature γ-aminobutyric acidergic (GABAergic) interneurons in culture. In line with previous studies (Scallet et al., 2004), we found that a 1-h-long exposure to ketamine at concentrations ≥ 10 μg/ml was sufficient to trigger cell death. At lower concentrations of ketamine, cell loss was only observed when this drug was chronically (> 48 h) present in the culture medium. Most importantly, we found that a single episode of 4-h-long treatment with 5 μg/ml ketamine induced long-term alterations in dendritic growth, including a significant (p 24 h) of neurons to ketamine at concentrations as low as 0.01 μg/ml also severely impaired dendritic arbor development. These results suggest that, in addition to its dose-dependent ability to induce cell death, even very low concentrations of ketamine could interfere with dendritic arbor development of immature GABAergic neurons and thus could potentially interfere with the development neural network

The Polysialylated Neural Cell Adhesion Molecule Promotes Neurogenesis in vitro

A characteristic feature of neurogenic sites in the postnatal brain is the expression of the polysialylated forms of the neural cell adhesion molecule (PSA-NCAM). To investigate the role of PSA-NCAM in generation of neuronal populations, we developed an in vitro model where neurogenesis occurs in primary cortical cultures following serum withdrawal. We show that removal or inactivation of the PSA tail of NCAM in these cultures leads to a significant decrease in the number of newly generated neurons. Similarly, cultures prepared from NCAM knock-out mice exhibit a significantly reduced neurogenesis. Pulse-chase experiments using the proliferation marker BrdU reveal that the lack of PSA does not affect the mitotic rate of neural progenitors but rather, it reduces the early survival of newly generated neurons. These results suggest that, in addition to its role in the migration of neuronal progenitors, PSA-NCAM is required for the adequate survival of these cell

VEGF is a chemoattractant for FGF-2–stimulated neural progenitors

Mmigration of undifferentiated neural progenitors is critical for the development and repair of the nervous system. However, the mechanisms and factors that regulate migration are not well understood. Here, we show that vascular endothelial growth factor (VEGF)-A, a major angiogenic factor, guides the directed migration of neural progenitors that do not display antigenic markers for neuron- or glia-restricted precursor cells. We demonstrate that progenitor cells express both VEGF receptor (VEGFR) 1 and VEGFR2, but signaling through VEGFR2 specifically mediates the chemotactic effect of VEGF. The expression of VEGFRs and the chemotaxis of progenitors in response to VEGF require the presence of fibroblast growth factor 2. These results demonstrate that VEGF is an attractive guidance cue for the migration of undifferentiated neural progenitors and offer a mechanistic link between neurogenesis and angiogenesis in the nervous system

Loss of non-canonical KCC2 functions promotes developmental apoptosis of cortical projection neurons

KCC2, encoded in humans by the SLC12A5 gene, is a multifunctional neuron-specific protein initially identified as the chloride (Cl-) extruder critical for hyperpolarizing GABA(A) receptor currents. Independently of its canonical function as a K-Cl cotransporter, KCC2 regulates the actin cytoskeleton via molecular interactions mediated through its large intracellular C-terminal domain (CTD). Contrary to the common assumption that embryonic neocortical projection neurons express KCC2 at non-significant levels, here we show that loss of KCC2 enhances apoptosis of late-born upper-layer cortical projection neurons in the embryonic brain. In utero electroporation of plasmids encoding truncated, transport-dead KCC2 constructs retaining the CTD was as efficient as of that encoding full-length KCC2 in preventing elimination of migrating projection neurons upon conditional deletion of KCC2. This was in contrast to the effect of a full-length KCC2 construct bearing a CTD missense mutation (KCC2(R952H)), which disrupts cytoskeletal interactions and has been found in patients with neurological and psychiatric disorders, notably seizures and epilepsy. Together, our findings indicate ion transport-independent, CTD-mediated regulation of developmental apoptosis by KCC2 in migrating cortical projection neurons.Peer reviewe

Plasma Concentrations of Brain-derived Neurotrophic Factor in Patients Undergoing Minor Surgery: A Randomized Controlled Trial

We measured perioperative plasma concentrations of brain-derived neurotrophic factor (BDNF), a major mediator of synaptic plasticity in the central nervous system, in males, 30-65 years old, undergoing lumbar or cervical discotomy. Patients were randomly allocated to a general anesthetic with propofol induction and maintenance or with thiopental induction and isoflurane maintenance. BDNF plasma concentrations were measured before induction (baseline), 15min after induction but before start of surgery, at skin closure, in the post-anesthetic care unit, and 24h postoperatively. Data from 26 patients (13 in each group) were analyzed. At each time point, BDNF plasma concentrations showed large variability. At baseline, concentrations were 631±337 (mean±SD)pgml−1 in the propofol group and were 549±512pgml−1 in the thiopental-isoflurane group (P=0.31). At 15min, concentrations significantly decreased in the propofol group (247±219pgml−1, P=0.0012 compared with baseline) but remained unchanged in the thiopental-isoflurane group (597±471pgml−1, P=0.798 compared with baseline). At skin closure and in the post-anesthetic care unit, concentrations were not different from baseline in both groups. At 24h, concentrations significantly decreased below baseline in both groups (propofol: 232±129pgml−1, P=0.0015; thiopental-isoflurane: 253±250pgml−1, P=0.016). In the propofol group, there was a weak but statistically significant positive correlation (R 2=0.38, P=0.026) between the duration of surgery and BDNF plasma concentrations at skin closure. These data suggest that in males undergoing elective minor surgery, BDNF plasma concentrations show a specific pattern that is influenced by the anesthetic technique and, possibly, by the duration of surger

Early Postnatal Migration and Development of Layer II Pyramidal Neurons in the Rodent Cingulate/Retrosplenial Cortex

The cingulate and retrosplenial regions are major components of the dorsomedial (dm) limbic cortex and have been implicated in a range of cognitive functions such as emotion, attention, and spatial memory. While the structure and connectivity of these cortices are well characterized, little is known about their development. Notably, the timing and mode of migration that govern the appropriate positioning of late-born neurons remain unknown. Here, we analyzed migratory events during the early postnatal period from ventricular/subventricular zone (VZ/SVZ) to the cerebral cortex by transducing neuronal precursors in the VZ/SVZ of newborn rats/mice with Tomato/green fluorescent protein-encoding lentivectors. We have identified a pool of postmitotic pyramidal precursors in the dm part of the neonatal VZ/SVZ that migrate into the medial limbic cortex during the first postnatal week. Time-lapse imaging demonstrates that these cells migrate on radial glial fibers by locomotion and display morphological and behavioral changes as they travel through the white matter and enter into the cortical gray matter. In the granular retrosplenial cortex, these cells give rise to a Satb2+ pyramidal subtype and develop dendritic bundles in layer I. Our observations provide the first insight into the patterns and dynamics of cell migration into the medial limbic corte

An Ion Transport-Independent Role for the Cation-Chloride Cotransporter KCC2 in Dendritic Spinogenesis In Vivo

The neuron-specific K-Cl cotransporter, KCC2, is highly expressed in the vicinity of excitatory synapses in pyramidal neurons, and recent in vitro data suggest that this protein plays a role in the development of dendritic spines. The in vivo relevance of these observations is, however, unknown. Using in utero electroporation combined with post hoc iontophoretic injection of Lucifer Yellow, we show that premature expression of KCC2 induces a highly significant and permanent increase in dendritic spine density of layer 2/3 pyramidal neurons in the somatosensory cortex. Whole-cell recordings revealed that this increased spine density is correlated with an enhanced spontaneous excitatory activity in KCC2-transfected neurons. Precocious expression of the N-terminal deleted form of KCC2, which lacks the chloride transporter function, also increased spine density. In contrast, no effect on spine density was observed following in utero electroporation of a point mutant of KCC2 (KCC2-C568A) where both the cotransporter function and the interaction with the cytoskeleton are disrupted. Transfection of the C-terminal domain of KCC2, a region involved in the interaction with the dendritic cytoskeleton, also increased spine density. Collectively, these results demonstrate a role for KCC2 in excitatory synaptogenesis in vivo through a mechanism that is independent of its ion transport functio

Anesthetics Rapidly Promote Synaptogenesis during a Critical Period of Brain Development

Experience-driven activity plays an essential role in the development of brain circuitry during critical periods of early postnatal life, a process that depends upon a dynamic balance between excitatory and inhibitory signals. Since general anesthetics are powerful pharmacological modulators of neuronal activity, an important question is whether and how these drugs can affect the development of synaptic networks. To address this issue, we examined here the impact of anesthetics on synapse growth and dynamics. We show that exposure of young rodents to anesthetics that either enhance GABAergic inhibition or block NMDA receptors rapidly induce a significant increase in dendritic spine density in the somatosensory cortex and hippocampus. This effect is developmentally regulated; it is transient but lasts for several days and is also reproduced by selective antagonists of excitatory receptors. Analyses of spine dynamics in hippocampal slice cultures reveals that this effect is mediated through an increased rate of protrusions formation, a better stabilization of newly formed spines, and leads to the formation of functional synapses. Altogether, these findings point to anesthesia as an important modulator of spine dynamics in the developing brain and suggest the existence of a homeostatic process regulating spine formation as a function of neural activity. Importantly, they also raise concern about the potential impact of these drugs on human practice, when applied during critical periods of development in infants

A multiparameter panel method for outcome prediction following aneurysmal subarachnoid hemorrhage

Purpose: Accurate early anticipation of long-term irreversible brain damage during the acute phase of patients with aneurysmal subarachnoid hemorrhage (aSAH) remains difficult. Using a combination of clinical scores together with brain injury-related biomarkers (H-FABP, NDKA, UFD1 and S100β), this study aimed at developing a multiparameter prognostic panel to facilitate early outcome prediction following aSAH. Methods: Blood samples of 141 aSAH patients from two separated cohorts (sets of 28 and 113 patients) were prospectively enrolled and analyzed with 14months of delay. Patients were admitted within 48h following aSAH onset. A venous blood sample was withdrawn within 12h after admission. H-FABP, NDKA, UFD1, S100β and troponin I levels were determined using classical immunoassays. The World Federation of Neurological Surgeons (WFNS) at admission and the Glasgow Outcome Score (GOS) at 6months were evaluated. Results: In the two cohorts, blood concentration of H-FABP, S100β and troponin I at admission significantly predicted unfavorable outcome (GOS 1-2-3). A multivariate analysis identified a six-parameter panel, including WFNS, H-FABP, S100β, troponin I, NDKA and UFD-1; when at least three of these parameters were simultaneously above cutoff values, prediction of unfavorable outcome reached around 70% sensitivity in both cohorts for 100% specificity. Conclusion: The use of this panel, including four brain injury-related proteins, one cardiac marker and a clinical score, could be a valuable tool to identify aSAH patients at risk of poor outcom