Kevin J. Anderson

An essential role for the H218/AGR16/Edg‐5/LPB2 sphingosine 1‐phosphate receptor in neuronal excitability

A wealth of indirect data suggest that the H218/AGR16/Edg‐5/LPB2 sphingosine 1‐phosphate (S1P) receptor plays important roles in development. In vitro, it activates several forms of development‐related signal transduction and regulates cellular proliferation, differentiation and survival. It is expressed during embryogenesis, and mutation of an H218‐like gene in zebrafish leads to profound defects in embryonic development. Nevertheless, the in vivo functions served by H218 signalling have not been directly investigated. We report here that mice in which the H218 gene has been disrupted are unexpectedly born with no apparent anatomical or physiological defects. In addition, no abnormalities were observed in general neurological development, peripheral axon growth or brain structure. However, between 3 and 7 weeks of age, H218–/– mice have seizures which are spontaneous, sporadic and occasionally lethal. Electroencephalographic abnormalities were identified both during and between the seizures. At a cellular level, whole‐cell patch‐clamp recordings revealed that the loss of H218 leads to a large increase in the excitability of neocortical pyramidal neurons. Therefore, H218 plays an essential, unanticipated and functionally important role in the proper development and/or mediation of neuronal excitability.

The Phosphorylated Axonal Form of the Neurofilament Subunit NF-H (pNF-H) as a Blood Biomarker of Traumatic Brain Injury

The detection of neuron-specific proteins in blood might allow quantification of the degree of neuropathology in experimental and clinical contexts. We have been studying a novel blood biomarker of axonal injury, the heavily phosphorylated axonal form of the high molecular weight neurofilament subunit NF-H (pNF-H). We hypothesized that this protein would be released from damaged and degenerating neurons following experimental traumatic brain injury (TBI) in amounts large enough to allow its detection in blood and that the levels detected would reflect the degree of injury severity. An enzyme-linked immunosorbent assay (ELISA) capture assay capable of detecting nanogram amounts of pNF-H was used to test blood of rats subjected to experimental TBI using a controlled cortical impact (CCI) device. Animals were subjected to a mild (1.0 mm), moderate (1.5 mm), or severe (2.0 mm) cortical contusion, and blood samples were taken at defined times post-injury. The assay detected the presence of pNF-H as early as 6 h post-injury; levels peaked at 24-48 h, and then slowly decreased to baseline over several days post-injury. No signal above baseline was detectable in control animals. Analysis of variance (ANOVA) showed a significant effect of lesion severity, and post hoc analysis revealed that animals given a moderate and severe contusion showed higher levels of blood pNF-H than controls. In addition, the peak levels of pNF-H detected at both 24 and 48 h post-injury correlated with the degree of injury as determined by volumetric analysis of spared cortical tissue. Relative amounts of pNF-H were also determined in different areas of the central nervous system (CNS) and were found to be highest in regions containing large-diameter axons, including spinal cord and brainstem, and lowest in the cerebral cortex and hippocampus. These findings suggest that the measurement of blood levels of pNF-H is a convenient method for assessing neuropathology following TBI.

Regional distribution of Fluoro-Jade B staining in the hippocampus following traumatic brain injury

Fluoro-Jade B (FJB) is an anionic fluorescein derivative that has been reported to specifically stain degenerating neurons. We were interested in applying FJB staining in a well-characterized model of traumatic brain injury (TBI) in order to estimate the total number of neurons in different regions of the hippocampus that die after a mild or moderate injury. Rats were subjected to a mild or moderate unilateral cortical contusion (1.0- or 1.5-mm displacement from the cortical surface) with a controlled cortical impacting device. Animals were allowed to survive for 1, 2, or 7 days and the total number of FJB-positive neurons in hippocampal areas CA1, CA3, and the dentate gyrus granule layer was estimated using sterological methods. The region that had the highest number of FJP-positive neurons after TBI was the dentate gyrus. In both 1- and 1.5-mm injuries, FJB-positive granule cells were observed throughout the rostro-caudal extent of the dentate. In contrast, labeled pyramidal neurons of area CA3 were most numerous after the 1.5-mm injury. The area that had the fewest number of FJB-labeled cells was area CA1 with only scattered neurons seen in the 1.5-mm group. In both injury groups and in all hippocampal regions, more FJB-positive neurons were seen at the earlier times post injury (1 and 2 days) than at 7 days. FJB appears to be a reliable marker for neuronal vulnerability following TBI.

PATHOBIOLOGY OF DYNORPHINS IN TRAUMA AND DISEASE

Dynorphins, endogenous opioid neuropeptides derived from the prodynorphin gene, are involved in a variety of normative physiologic functions including antinociception and neuroendocrine signaling, and may be protective to neurons and oligodendroglia via their opioid receptor-mediated effects. However, under experimental or pathophysiological conditions in which dynorphin levels are substantially elevated, these peptides are excitotoxic largely through actions at glutamate receptors. Because the excitotoxic actions of dynorphins require supraphysiological concentrations or prolonged tissue exposure, there has likely been little evolutionary pressure to ameliorate the maladaptive, non-opioid receptor mediated consequences of dynorphins. Thus, dynorphins can have protective and/or proapoptotic actions in neurons and glia, and the net effect may depend upon the distribution of receptors in a particular region and the amount of dynorphin released. Increased prodynorphin gene expression is observed in several disease states and disruptions in dynorphin processing can accompany pathophysiological situations. Aberrant processing may contribute to the net negative effects of dysregulated dynorphin production by tilting the balance towards dynorphin derivatives that are toxic to neurons and/or oligodendroglia. Evidence outlined in this review suggests that a variety of CNS pathologies alter dynorphin biogenesis. Such alterations are likely maladaptive and contribute to secondary injury and the pathogenesis of disease.

Distribution of Heterotopic Neurons in Normal Hemispheric White Matter: A Morphometric Analysis

One of the frequent abnormalities described in the context of surgically resected temporal lobe (TL) specimens is the presence of heterotopic neurons within white matter (WM). We have attempted to morphometrically define the distribution of these heterotopic neurons in normal subjects, comparing the incidence of heterotopic neurons in TL WM with that in occipital (OL) and frontal lobe (FL) sections. Using a combination of routine and special stains combined with immunohistochemical confirmation 20 adult autopsy cases were examined. WM from TL, FL, and OL sections was outlined and the area was measured by image analysis. Using defined criteria, heterotopic neurons within these areas were counted. Results confirm our hypothesis that normal adult TL WM contains a significantly higher population of residual/heterotopic neurons than OL and FL WM groups. It is felt that these neurons represent interstitial remnants of the subplate which have failed to undergo programmed cell death. The significance of these findings with regard to assessment of similar findings in temporal lobectomy specimens is addressed. A second intriguing association of this TL WM heterotopia concerns its possible relationship to the more frequent occurrence of “malformative neoplasms” with neuronal elements (such as ganglioglioma and dysembryoplastic neuroepithelial tumor) in the temporal lobe.

Quantitative autoradiographic analysis of excitatory amino acid receptors in the cat spinal cord

Using quantitative autoradiography, we have studied the density and distribution of N-methyl-D-aspartate (NMDA), kainate and AMPA receptors and the binding site for the sodium-dependent EAA transporter in sections from the cat spinal cord. NMDA, kainate and AMPA receptors were found in highest concentrations in laminae I and II of the dorsal horn. Lower levels of all receptors were seen in other regions of the spinal cord grey matter. The distribution of the sodium-dependent transporter was unlike that of any of the receptor populations with highest levels found in the ventral horn with slightly lower levels in other regions of grey matter. The pattern of binding sites was consistent throughout all levels of the spinal cord.

Autoradiographic characterization of putative excitatory amino acid transport sites

Removal of excitatory amino acids from the extracellular space is now postulated to occur through at least two distinct transport systems that are distinguished by their ionic dependency. Thus, both sodium-dependent and chloride-dependent systems have been described in the mammalian central nervous system. In this report we attempt to characterize these sites by autoradiography, using D-[3H]aspartate and L-[3H]glutamate as ligands. Previous studies have shown that sequestration of radioligand into membrane vesicles can be a potential artifact when examining transport sites. We have found that sequestration can be alleviated by incubation of tissue sections in xylenes prior to incubation with radioligand. Using in vitro autoradiography we have characterized the two binding sites with respect to their distribution, kinetics and pharmacology. Both appeared to have a single, saturable binding site with Kds in the low micromolar range. Sodium-dependent D-aspartate binding predominated, having a Bmax that was five times greater than chloride-dependent L-glutamate binding in whole brain. The levels of binding to the two sites varied between brain regions. Sodium-dependent D-aspartate binding was highest in the cerebellar molecular layer greater than dentate gyrus molecular layer greater than entorhinal cortex. Chloride-dependent L-glutamate binding was highest in the outer layers of cerebral cortex greater than dentate gyrus molecular layer greater than entorhinal cortex greater than striatum. Pharmacological characterization of these sites also showed major differences. Sodium-dependent D-aspartate binding was most potently inhibited by L-aspartate greater than threo-beta-hydroxyaspartate greater than L-cysteine sulfinic acid greater than L-cysteic acid. Chloride-dependent glutamate binding was most potently inhibited by L-glutamate greater than L-alpha-amino adipic acid greater than quisqualate greater than L-serine-o-sulfate. The differences in distribution, ligand binding properties and pharmacology of these sites suggest that a significant variable in excitatory amino acid circuitry may include heterogeneity in transporters associated with excitatory pathways.

Astrocyte hypertrophy in the Alzheimer's disease hippocampal formation

In Alzheimers disease (AD), neuritic plaques are often found in the hippocampal dentate gyrus along the boundary between inner and outer molecular layers. The dentate outer molecular layer in AD also exhibits axon sprouting in response to an early loss of entorhinal neurons. The relationship between the laminar arrangement of plaques and the sprouting remains unclear. In experimental entorhinal lesions in the rat, the denervated dentate outer molecular layer demonstrates hypertrophic astrocytes which may provide trophic support for the sprouting response. It is not known whether an equivalent astrocyte response occurs in AD or whether this response is related to the distribution of plaques. We used immunohistochemical staining for glial fibrillary acidic protein (GFAP) to demonstrate reactive astrocytes in the hippocampus in AD patients and age-matched controls. These results were compared to the astrocyte response to an experimental entorhinal lesion in the rat. Quantitative and qualitative analyses demonstrated a significant increase in GFAP-positive hypertrophic astrocytes in the dentate outer molecular layer in AD compared to controls. These astrocytes were randomly distributed within the outer layer and did not parallel the distribution of neuritic plaques. In the entorhinal-lesioned rat, reactive hypertrophied astrocytes also showed a selective distribution within the denervated outer molecular layer. Our results further support the similarity of the hippocampal response in AD and experimental entorhinal lesion but do not explain the laminar distribution of neuritic plaques along the denervated zone.

Fluoro-jade B stains quiescent and reactive astrocytes in the rodent spinal cord.

In an attempt to label dying neurons in the injured spinal cord, we used the novel fluorescein derivative Fluoro-Jade B, which has been reported to specifically label dead or dying neurons in the brain. Rats and mice were subjected to a moderate level of spinal cord injury using an IH impact device and sacrificed at 1, 2, 4, 7, 14, and 21 days post injury. Spinal cord tissue was processed for Fluoro-Jade B histochemistry and included sections throughout the injured region of the cord. No Fluoro-Jade positive neurons were observed in sections from any time point postinjury at any level of the spinal cord. Instead, Fluoro-Jade labeled astrocytes in uninjured control animals and injured animals. The specificity of astrocytic staining was confirmed by co-localizaton of Fluoro-Jade with glial fibrillary acidic protein. We also subjected a group of rats to a sequential cortical contusion injury and spinal cord injury. Sections from these animals showed numerous Fluoro-Jade positive neurons in the hippocampal formation and thalamus underlying the cortical contusion; however, the staining pattern in the spinal cord was identical to those animals that had received spinal cord injury alone.

Age-Related Changes in [3H]MK-801 Binding in the Fischer 344 Rat Brain

In this study we tested the hypothesis that the efficacy of L-glutamate to stimulate [3H]MK-801 binding to the NMDA receptor/channel complex is altered as a function of aging. L-Glutamate, or related excitatory amino acid (EAA), is the endogenous neurotransmitter of the NMDA receptor/channel complex. These studies examined the efficacy and potency with which L-glutamate produces receptor activation, channel opening and subsequent MK-801 binding as a function of increasing age by comparing dose-response curves (EC50 and Emax) from 6-, 12-, and 24-month-old F-344 rats. The number of NMDA receptors, as determined by [3H]MK-801 binding in the presence of a saturating concentration of L-glutamate, was reduced in the inner frontal cortex, entorhinal cortex and the lateral striatum in aged rats when compared with young adults. When a range of L-glutamate concentrations were used, differences in Emax were noted in the same brain regions in addition to several others in aged and middle-aged animals when compared with young-adult animals. No changes in EC50 values were noted in any of the brain regions at either age when compared with young-adults.