Phillip G. Nelson

Neonatal Cytokines and Cerebral Palsy in Very Preterm Infants

To examine the relationship of cytokines in blood of very preterm neonates with later diagnosis of spastic cerebral palsy (CP) compared with infants of similar gestational age without CP, we measured concentrations of inflammatory cytokines and other substances in archived neonatal blood by recycling immunoaffinity chromatography. Subjects were surviving children born before 32 wk gestational age (GA) to women without preeclampsia, 64 with later diagnoses of CP and 107 control children. The initial analyses were augmented by measurement of 11 cytokines by a bead-based flow analytic system (Luminex) in an additional 37 children with CP and 34 control children from the same cohort. Concentrations of examined substances did not differ by presence of indicators of infection in mother, infant, or placenta. On ANOVA, concentrations of a number of cytokines were significantly related to neonatal ultrasound abnormalities (periventricular leukomalacia, ventricular enlargement, or moderate or severe germinal matrix hemorrhage). None of the substances measured either by immunoaffinity chromatography or flow analytic methods, including IL-1, -6, and -8 and tumor necrosis factor-α, was related to later diagnosis of CP or its subtypes. Inflammatory cytokines in neonatal blood of very premature infants did not distinguish those with later diagnoses of CP from control children.

Selected neurotrophins, neuropeptides, and cytokines : developmental trajectory and concentrations in neonatal blood of children with autism or down syndrome

Using a double‐antibody immunoaffinity assay (Luminex) and ELISA technology, we measured concentrations of certain neurotrophins, neuropeptides, and cytokines in pooled samples (one to three subjects per sample) eluted from archived neonatal blood of children with later‐diagnosed autism, Down syndrome, very preterm birth, or term control infants. We also measured analytes in blood from healthy adult controls. Case or control status for infant subjects was ascertained by retrospective review of service agency medical records. We observed inhibitory substances in eluates from archived bloodspots, especially marked for measurement of BDNF. Concentrations in control subjects differed by age: BDNF rose markedly with age, while NT‐3 and NT‐4/5 concentrations were lower in adults than in newborn infants. IL‐8 concentrations were higher in newborn infants, preterm and term, than in adults. Considered by diagnostic group, total protein was higher in Down syndrome than in either autism or control subjects. In infants with Down syndrome, concentrations of IL‐8 levels were higher than in controls, whether or not corrected for total protein; NT‐3 and CGRP were lower and VIP higher. In samples from autistic subjects, NT‐3 levels were significantly lower than controls and an increase in VIP approached statistical significance. Concentrations of NT‐4/5 and CGRP were correlated in infants with autism but not in Down syndrome or controls. Some of these results differ from earlier findings using a single‐antibody recycling immunoaffinity chromatography (RIC) system. We discuss interrelationships of VIP, NT‐3 and IL‐8 and their potential relevance to features of the neuropathology of autism or Down syndrome.

Neurons from fetal rat brain in a new cell culture system: A multidisciplinary analysis

A new culture system for cells from the mammalian brain was developed by a modification of a previously established technique. This modification involved the use of fluorodeoxyuridine and adult horse serum. The cultures contained large, easily visualized neurons both isolated from other neurons and in networks of varying complexity. These cells were large enough to permit reliable intracellular electrophysiologic recording and were often sufficiently dispersed to allow examination of membrane responses to iontophoretically applied neurotransmitter candidates. Many responses characteristic of central neurons in situ were seen, including evoked and spontaneous action potentials, complex patterns of inhibitory and excitatory post-synaptic potentials, and neurotransmitter-induced membrane responses. These preparations were examined by phase contrast microscopy, by light microscopy after silver impregnation and by Nomarski interference optics. Total choline acetyltransferase (CAT) activity was little changed and specific activity was increased in the new culture system as compared with the earilier system. Conditions which gave the highest specific activity of CAT also provided the best cultures from the standpoint of electrophysiologic and morphologic analysis. This new approach will allow, in culture, detailed multidisciplinary analyses of individual neurons and small networks of neurons from the mammalian brain.

Choline Acetyltransferase Activity Is Increased in Combined Cultures of Spinal Cord and Muscle Cells from Mice

The activity of choline acetyltransferase was more than tenfold greater in combined cultures of spinal cord and muscle cells than in cultures of spinal cord cells alone. This increase was associated with the formation of functional neuromuscular junctions in culture. Counts of silver-stained cells and determinations of other enzyme activities indicated that the increased choline acetyltransferase activity was not due to nonspecific neuronal survival but reflected greater activity in the surviving neurons. Hence, muscle had a marked, highly specific trophic effect on the cholinergic neurons that innervated it.

Electrophysiologic study of cultured neurons dissociated from spinal cords and dorsal root ganglia of fetal mice

Abstract Spontaneously occurring action potentials and postsynaptic potentials were recorded intracellularly from mouse spinal cord (SC) neurons and dorsal root ganglion (DRG) neurons in mixed SC and DRG cell cultures. In some SC cells, excitatory postsynaptic potentials were evoked by electrical stimulation of a nearby SC or DRG cell. SC and DRG neurons had distinguishing morphologic and electrophysiologic properties. SC neurons usually were elliptical or stellate and had several branched processes whereas DRG cells were most commonly round and had on the average only one process, but occasionally 3 or 4. Calculations from cell measurements revealed that SC neurons had less soma surface area and more process surface area than DRG cells, with a similar total surface area for each class. Lower resting membrane potentials were recorded from SC neurons, but when the capability for action potential generation was tested at comparable steady membrane potentials, most SC and less than half of DRG neurons fired repetitively to electrical stimulation. During the depolarizing and repolarizing phases of SC cell action potentials the rates of change of membrane potential were lower than for DRG cells, which had rapidly rising action potentials and a markedly negative afterpotential. An initially delayed repolarization phase was characteristic of the DRG cell action potential. Cell cultures were prepared by trypsin dissociation of spinal cords with attached spinal ganglia from fetuses of 10, 11, 12, 13, 14, and 17 days gestational age. Cell cultures grown on plastic or collagen were studied electrophysiologically at times from 16 to 94 days.

SEQUENCE DIVERSITY STUDIES OF RAT BRAIN RNA: EFFECTS OF ENVIRONMENTAL COMPLEXITY ON RAT BRAIN RNA DIVERSITY

The sequence complexities of rat brain RNAs were measured by RNA‐driven hybridization reactions with nonrepetitive rat DNA. The total sequence complexity of rat brain HnRNA was estimated to be 6.61 x 108 nucleotides while rat brain poly(A)‐mRNA sequence complexity was 1.32 x 108 nucleotides. Up to 33.7% of the total transcribable nonrepetitive DNA was expressed in the nuclear RNA. The nuclear RNAs reacted with complex kinetics over at least 4.5 decades of equivalent Rot (product of RNA concentration and time), with an apparent division into three major RNA abundance classes. The abundances of average nuclear RNA species in these classes ranged from 2.9 x 109 copies per brain (18 copies per cell) to 2.4 x 105 copies per brain (1.5 x 10−3 copies per cell). Poly(A)‐mRNA diversity was sufficient to code for 8.8 x 104 polypeptides of 50,000 daltons. There were also three distinguishable abundance classes of poly(A)‐mRNA with frequencies which ranged from 8.9 x 108 copies per brain (5.5 copies per cell) to 3.2 x 105 copies per brain (2 x 10−3 copies per cell). Evidence for compartmentalization of expressed RNA sequences supports the concept that the extensive morphological and physiological specialization evident in brain parallels extensive transcriptional specialization at the cellular level.

Delayed depolarization in cat spinal motoneurons

The delayed depolarization (D-D) following antidromic or orthodromic AB spikes in motoneurons, described by others, has been studied by intracellular recording in cat motoneurons. The D-D was usually either present or absent in any particular cell, but in certain exceptional instances all-or-none appearance of a D-D was observed. The D-D could be decreased and in some instances completely blocked with IPSP conditioning, but there was little or no observable interaction between monosynaptic EPSP and the D-D. The “humped” appearance of the D-D appears to be explained by the time course of changes in the soma membrane conductance immediately following the passage of a B spike. The hypothesis that the D-D, when present, is generated by an active spike process propagated into the dendrites for a variable distance is supported, and the extent of propagation appears to be modified by some types of synaptic activity.

Prevention of activity‐dependent neuronal death: Vasoactive intestinal polypeptide stimulates astrocytes to secrete the thrombin‐inhibiting neurotrophic serpin, protease nexin I

Neuronal cell death occurs as a programmed, naturally occurring mechanism and is the primary regressive event in central nervous system development. Death of neurons also occurs on an injury-induced basis after trauma and in human neurodegenerative diseases. Classical neurotrophic factors can reverse this phenomenon in experimental models prompting initiation of clinical trials in conditions such as amyotrophic lateral sclerosis and Alzheimers disease. The glial-derived protease nexin I (PNI), a known promoter of neurite outgrowth in cell culture and a potent inhibitor of serine proteases, also enhances neuronal cell survival. PNI, in nanomolar concentrations, rescues spinal cord motor neurons from both naturally-occurring programmed cell death in the chick embryo as well as following injury in the neonatal mouse. The potent neuromodulator, vasoactive intestinal polypeptide (VIP), influences neuronal survival through glial-mediated factors and also induces secretion of newly synthesized astrocyte PNI. We now report that subnanomolar amounts of PNI enhance neuronal survival in mixed spinal cord cell culture, especially when neuronal cells were made electrically silent by administration of tetrodotoxin. The mediation of this effect is by inhibition of the multifunctional serine protease, thrombin, because hirudin, a thrombin-specific inhibitor, has the same effect. In addition, spinal cord neurons are exquisitely sensitive to thrombin because picomolar and lower levels of the coagulation factor causes neuronal death. Thus, PNI is an astrocyte-derived, thrombin-inhibiting, activity-dependent neurotrophic agent, enhanced secretion of which by VIP may be one approach to treat neurological disorders.

Synaptic excitation in cultures of mouse spinal cord neurones: receptor pharmacology and behaviour of synaptic currents.

Fast monosynaptic excitatory post‐synaptic potentials between spinal cord neurones in cell culture (s.c.‐s.c. e.p.s.p.s) were studied with current‐clamp and two‐electrode voltage‐clamp methods. The reversal potential, response to acidic amino acid antagonists, and behaviour of the synaptic current were examined. The amplitude of the e.p.s.p. increased with membrane potential hyperpolarization and decreased with depolarization. The reversal potential of the e.p.s.p. was +3.8 +/‐ 2.5 mV (mean +/‐ S.E. of mean). The reversal potential for responses to ionophoretically applied L‐glutamate and L‐aspartate was also near 0 mV. The acidic amino acid antagonist, cis‐2,3‐piperidine dicarboxylic acid (PDA, 0.25‐1.0 mM) reversibly antagonized the monosynaptic e.p.s.p.s as well as responses to kainate (KA) or quisqualate (QA). The selective N‐methyl‐D‐aspartate antagonist, (+/‐) 2‐amino‐5‐phosphonovaleric acid (APV), had little effect on either the monosynaptic e.p.s.p.s or responses to QA or KA at concentrations that abolished responses to L‐aspartate. Under voltage clamp, the peak synaptic current (e.p.s.c.) was linearly related to the membrane potential, increasing in amplitude with hyperpolarization and decreasing with depolarization from the resting potential. The decay of a somatic e.p.s.c. was well fitted by a single exponential function with a time constant of 0.6 ms at 25 degrees C. E.p.s.c.s which had proximal dendritic locations had decay time constants of 1‐2 ms. The decay time constant was voltage‐insensitive between ‐80 and +10 mV. We suggest that an acidic amino acid receptor other than that for NMDA mediates excitatory transmission at the s.c.‐s.c. synapse; and that the underlying conductance mechanism is voltage insensitive with an estimated mean channel lifetime of less than 1 ms.

Neuronal maturation in mammalian cell culture is dependent on spontaneous electrical activity.

Fetal mouse spinal cord (SC) and dorsal root ganglion (DRG) neurons undergo a process of maturation in cell culture lasting a month or more. We have investigated the role of electrical activity in this maturational process with the use of tetrodotoxin (TTX), the specific blocker of the voltage-sensitive sodium channel responsible for action potential generation. This agent completely eliminates the spikes and related synaptic activity which occur abundantly in untreated cultures. Such blockade of electrical activity in the cultures, when begun early (day 1 or day 8 in vitro), results in a 85-95% reduction in the number of large SC neurons, without affecting DRG neuron numbers. TTX treatment initiated when cultures are mature (day 70) has no significant effect on either DRG or SC neurons. Intermediate effects are obtained when treatment is initiated at day 35 in vitro. The activity of the nerve-specific enzyme choline acetyltransferase, is significantly decreased by early TTX treatment, while DNA and protein content of the cultures (primarily contributed by glial and fibroblastic cells) is not affected.