Galantamine ameliorates hyperoxia-induced brain injury in neonatal mice.

Introduction: Prolonged oxygen therapy in preterm infants often leads to cognitive impairment. Hyperoxia leads to excess free radical production with subsequent neuroinflammation, astrogliosis, microgliosis and apoptosis. We hypothesized that Galantamine, an acetyl choline esterase inhibitor and an FDA approved treatment of Alzheimer's disease, will reduce hyperoxic brain injury in neonatal mice and will improve learning and memory. Methods: Mouse pups at postnatal day 1 (P1) were placed in a hyperoxia chamber (FiO2 95%) for 7 days. Pups were injected IP daily with Galantamine (5 mg/kg/dose) or saline for 7 days. Results: Hyperoxia caused significant neurodegeneration in cholinergic nuclei of the basal forebrain cholinergic system (BFCS), laterodorsal tegmental (LDT) nucleus and nucleus ambiguus (NA). Galantamine ameliorated this neuronal loss. Treated hyperoxic group showed a significant increase of choline acetyl transferase (ChAT) expression and a decrease of acetyl choline esterase activity, thus increasing acetyl choline levels in hyperoxia environment. Hyperoxia increased pro-inflammatory cytokines namely IL -1ß, IL-6 and TNF α, HMGB1, NF-κB activation. Galantamine showed its potent anti- inflammatory effect, by blunting cytokines surges among treated group. Treatment with Galantamine increased myelination while reducing apoptosis, microgliosis, astrogliosis and ROS production. Long term neurobehavioral outcomes at P60 showed improved locomotor activity, coordination, learning and memory, along with increased hippocampal volumes on MRI with Galantamine treated versus non treated hyperoxia group. Conclusion: Together our findings suggest a potential therapeutic role for Galantamine in attenuating hyperoxia-induced brain injury.

Caffeine inhibits hypoxia-induced nuclear accumulation in HIF-1α and promotes neonatal neuronal survival.

Apnea of prematurity (AOP) defined as cessation of breathing for 15-20 s, is commonly seen in preterm infants. Caffeine is widely used to treat AOP due to its safety and effectiveness. Caffeine releases respiratory arrest by competing with adenosine for binding to adenosine A1 and A2A receptors (A1R and A2AR). Long before its use in treating AOP, caffeine has been used as a psychostimulant in adult brains. However, the effect of caffeine on developing brains remains unclear. We found that A1R proteins for caffeine binding were expressed in the brains of neonatal rodents and preterm infants (26-27 weeks). Neonatal A1R proteins colocalized with PSD-95, suggesting its synaptic localization. In contrast, our finding on A2R expression in neonatal neurons was restricted to the mRNA level as detected by single cell RT/PCR due to the lack of specific A2AR antibody. Furthermore, caffeine (200 µM) at a dose twice higher than the clinically relevant dose (36-130 µM) had minor or no effects on several basic neuronal functions, such as neurite outgrowth, synapse formation, expression of A1R and transcription of CREB-1 and c-Fos, further supporting the safety of caffeine for clinical use. We found that treatment with CoCl2 (125 µM), a hypoxia mimetic agent, for 24 h triggered neuronal death and nuclear accumulation of HIF-1α in primary neuronal cultures. Subsequent treatment with caffeine at a concentration of 100 µM alleviated CoCl2-induced cell death and prevented nuclear accumulation of HIF-1α. Consistently, caffeine treatment in early postnatal life of neonatal mice (P4-P7) also prevented subsequent hypoxia-induced nuclear increase of HIF-1α. Together, our data support the utility of caffeine in alleviating hypoxia-induced damages in developing neurons.

Timing of Caffeine Therapy and Neonatal Outcomes in Preterm Infants: A Retrospective Study.

Background. Caffeine is widely used to treat apnea of prematurity. Here, we evaluated the efficacy of early caffeine (1-2 DOL) in decreasing the incidence of adverse neonatal outcomes. Methods. A retrospective cohort was used to compare the neonatal morbidity of 150 preterm neonates with gestational age ≤29 weeks. Infants were divided into 3 groups based on the initiation timing of caffeine therapy; (1) early caffeine (1-2 DOL), (2) late caffeine (3-7 DOL), and (3) very late caffeine (≥8 DOL). Results. The neonatal outcomes of early caffeine were comparable with those of the late caffeine group. Moreover, when comparing the neonatal morbidity of the very late caffeine group with that of the early caffeine group, multivariable logistic regression analyses were performed. We found that the timing of caffeine did not influence the risk of BPD (OR, 0.393; CI, 0.126-1.223; p = 0.107), but birthweight did (OR, 0.996; CI, 0.993-0.999; p = 0.018) in these infants. Conclusion. Neonatal outcomes of preterm infants were comparable whether caffeine was administered early or late in the first 7 DOL. The risk of BPD in infants receiving caffeine after 8 DOL was irrespective of delayed treatment with caffeine. Our results clearly demonstrate the need for further studies before caffeine prophylaxis can be universally recommended.

Neurexin-neuroligin transsynaptic interaction mediates learning-related synaptic remodeling and long-term facilitation in aplysia.

Neurexin and neuroligin, which undergo heterophilic interactions with each other at the synapse, are mutated in some patients with autism spectrum disorder, a set of disorders characterized by deficits in social and emotional learning. We have explored the role of neurexin and neuroligin at sensory-to-motor neuron synapses of the gill-withdrawal reflex in Aplysia, which undergoes sensitization, a simple form of learned fear. We find that depleting neurexin in the presynaptic sensory neuron or neuroligin in the postsynaptic motor neuron abolishes both long-term facilitation and the associated presynaptic growth induced by repeated pulses of serotonin. Moreover, introduction into the motor neuron of the R451C mutation of neuroligin-3 linked to autism spectrum disorder blocks both intermediate-term and long-term facilitation. Our results suggest that activity-dependent regulation of the neurexin-neuroligin interaction may govern transsynaptic signaling required for the storage of long-term memory, including emotional memory that may be impaired in autism spectrum disorder.

Developmental plasticity of descending motor pathways.

Juvenile animal brains are highly plastic and thus often achieve better functional recovery after injury compared with adult brains. Recently, Umeda et al. (Umeda T, Takahashi M, Isa K, Isa T. J Neurophysiol 104: 1707-1716, 2010) have shown that the remodeling of both corticospinal and extra-pyramidal pathways can contribute to the recovery of grasping and reaching ability in hemidecorticated juvenile rats. They have further unveiled the strengthening of the cortico-reticulo-spinal pathway after injury, that mediates the fast excitation of ipsilateral motoneurons for functional recovery.

A presynaptic role for FMRP during protein synthesis-dependent long-term plasticity in Aplysia.

Loss of the Fragile X mental retardation protein (FMRP) is associated with presumed postsynaptic deficits in mouse models of Fragile X syndrome. However, the possible presynaptic roles of FMRP in learning-related plasticity have received little attention. As a result, the mechanisms whereby FMRP influences synaptic function remain poorly understood. To investigate the cellular locus of the effects of FMRP on synaptic plasticity, we cloned the Aplysia homolog of FMRP and find it to be highly expressed in neurons. By selectively down-regulating FMRP in individual Aplysia neurons at the sensory-to-motor neuron synapse reconstituted in co-cultures, we demonstrate that FMRP functions both pre- and postsynaptically to constrain the expression of long-term synaptic depression induced by repeated pulses of FMRF-amide. In contrast, FMRP has little to no effect on long-term synaptic facilitation induced by repeated pulses of serotonin. Since other components of signaling pathways involved in plasticity appear to be conserved between Aplysia and mammalian neurons, our findings suggest that FMRP can participate in both pre- and postsynaptic regulation of enduring synaptic plasticity that underlies the storage of certain types of long-term memory.

Dscam mediates remodeling of glutamate receptors in Aplysia during de novo and learning-related synapse formation.

Transsynaptic interactions between neurons are essential during both developmental and learning-related synaptic growth. We have used Aplysia neuronal cultures to examine the contribution of transsynaptic signals in both types of synapse formation. We find that during de novo synaptogenesis, specific presynaptic innervation is required for the clustering of postsynaptic AMPA-like but not NMDA-like receptors. We further find that the cell adhesion molecule Dscam is involved in these transsynaptic interactions. Inhibition of Dscam either pre- or postsynaptically abolishes the emergence of synaptic transmission and the clustering of AMPA-like receptors. Remodeling of both AMPA-like and NMDA-like receptors also occurs during learning-related synapse formation and again requires the reactivation of Dscam-mediated transsynaptic interactions. Taken together, these findings suggest that learning-induced synapse formation recapitulates, at least in part, aspects of the mechanisms that govern de novo synaptogenesis.

Serotonin-induced regulation of the actin network for learning-related synaptic growth requires Cdc42, N-WASP, and PAK in Aplysia sensory neurons.

Application of Clostridium difficile toxin B, an inhibitor of the Rho family of GTPases, at the Aplysia sensory to motor neuron synapse blocks long-term facilitation and the associated growth of new sensory neuron varicosities induced by repeated pulses of serotonin (5-HT). We have isolated cDNAs encoding Aplysia Rho, Rac, and Cdc42 and found that Rho and Rac had no effect but that overexpression in sensory neurons of a dominant-negative mutant of ApCdc42 or the CRIB domains of its downstream effectors PAK and N-WASP selectively reduces the long-term changes in synaptic strength and structure. FRET analysis indicates that 5-HT activates ApCdc42 in a subset of varicosities contacting the postsynaptic motor neuron and that this activation is dependent on the PI3K and PLC signaling pathways. The 5-HT-induced activation of ApCdc42 initiates reorganization of the presynaptic actin network leading to the outgrowth of filopodia, some of which are morphological precursors for the learning-related formation of new sensory neuron varicosities.

Presynaptic activation of silent synapses and growth of new synapses contribute to intermediate and long-term facilitation in Aplysia.

The time course and functional significance of the structural changes associated with long-term facilitation of Aplysia sensory to motor neuron synaptic connections in culture were examined by time-lapse confocal imaging of individual sensory neuron varicosities labeled with three different fluorescent markers: the whole-cell marker Alexa-594 and two presynaptic marker proteins-synaptophysin-eGFP to monitor changes in synaptic vesicle distribution and synapto-PHluorin to monitor active transmitter release sites. Repeated pulses of serotonin induce two temporally, morphologically, and molecularly distinct presynaptic changes: (1) a rapid activation of silent presynaptic terminals by filling of preexisting empty varicosities with synaptic vesicles, which parallels intermediate-term facilitation, is completed within 3-6 hr and requires translation but not transcription and (2) a slower generation of new functional varicosities which occurs between 12-18 hr and requires transcription and translation. Enrichment of empty varicosities with synaptophysin accounts for 32% of the newly activated synapses at 24 hr, whereas newly formed varicosities account for 68%.