Daniel J. Bonthius

FGF-2, NGF and IGF-1, but not BDNF, utilize a nitric oxide pathway to signal neurotrophic and neuroprotective effects against alcohol toxicity in cerebellar granule cell cultures

Neuronal death is a prominent neuropathological component of fetal alcohol syndrome (FAS). Identification of molecular agents and pathways that can ameliorate alcohol-induced cell loss offers possible therapeutic strategies for FAS and potential insight into its pathogenesis. This study investigated the effects of growth factors on cellular survival in alcohol-exposed cerebellar granule cell (CGC) cultures and examined the role of the nitric oxide (NO)-cGMP-PKG (cGMP-dependent protein kinase) pathway in the cell survival-promoting effects of these growth factors. Primary CGC cultures were exposed to 0 or 400 mg/dl ethanol, accompanied by either no growth factor or 30 ng/ml fibroblast growth factor-2 (FGF-2), nerve growth factor (NGF), insulin-like growth factor-1 (IGF-1), brain-derived neurotrophic factor (BDNF) or epidermal growth factor (EGF). Viable neurons were quantified after 1 day of exposure. Two distinct types of cell survival-promoting effects of growth factors were detectable: (1) a neurotrophic effect, in which the growth factors diminished the background death of neurons that occurred in alcohol-free cultures; and (2) a neuroprotective effect, in which the growth factors diminished alcohol-induced cell death. The various growth factors differed markedly in their patterns of cell survival promotion. While BDNF and FGF-2 exerted both a neurotrophic and a neuroprotective effect, IGF-1 had only a neurotrophic effect and did not protect against alcohol toxicity, and NGF had only a neuroprotective effect and did not diminish background cell death. EGF had neither a neurotrophic nor a neuroprotective effect. In order to determine the role of the NO-cGMP-PKG pathway in the cell survival-promoting effects mediated by growth factors, cultures were exposed to one of several pharmacological inhibitors of the pathway, including NAME, LY83583 and PKG inhibitor. The cell survival-promoting effects of FGF-2, NGF and IGF-1 were all substantially reduced by each of the pathway inhibitors. In contrast, neither the neurotrophic nor the neuroprotective effects of BDNF were altered by any of the pathway inhibitors. Thus, growth factors differ in their patterns of neurotrophic and neuroprotective effects, and they differ in their reliance on the NO-cGMP-PKG pathway. While FGF-2, NGF and IGF-1 all signal their survival-promoting effects through the NO-cGMP-PKG pathway, BDNF does not rely upon this pathway for signal transduction in CGC cultures.

Altered GABAergic function accompanies hippocampal degeneration in mice lacking ClC-3 voltage-gated chloride channels

Mice lacking ClC-3 chloride channels, encoded by the Clcn3 gene, undergo neurodegeneration of the hippocampal formation and retina [Neuron, 29 (2001) 185-196; Genes Cells, 7 (2002) 597-605]. We independently created a mouse lacking the Clcn3 gene which demonstrated similar central nervous system abnormalities, including early postnatal degeneration of retinal photoreceptors. However, we observed a characteristic spatial-temporal sequence of hippocampal neurodegeneration that differs from the pattern previously reported. Anterior-to-posterior degeneration and astrogliosis of the dentate gyrus and hippocampus progressed over months. Sequential loss of hippocampal neuronal subpopulations began in the dentate gyrus and progressed to CA3, followed by CA1 neurons. Projection neurons of the entorhinal cortex degenerated, secondary to the loss of their synaptic targets within the hippocampal formation. Other characteristics of the Clcn3(-/-) mice included an abnormal gait, kyphosis, and absence of hindlimb escape extension upon tail elevation. Spontaneous seizures were observed in four adult Clcn3(-/-) mice, and one mouse died during the event. We hypothesized that neuronal injury may be related to recurrent seizures. Clcn3(-/-) mice had normal serum electrolytes and pH, and exhibited neither hyperglycemia nor rebound hypoglycemia following a glucose load. They displayed a greatly reduced susceptibility to pentylenetetrazole-induced seizures and an abnormally prolonged sedation to benzodiazepines. There was no change in vulnerability to kainic acid-induced seizures. Immunostaining revealed a progressive loss of GABA synthesizing cells in the dentate gyrus. The death of these cells was preceded by increased GABA(A) receptor immunoreactivity. These data suggest that GABA(A) inhibitory neurotransmission is altered in Clcn3(-/-) mice. The increase in GABA(A) receptor density may represent a compensatory response either to chronic excessive excitatory stimuli or reduced inhibitory input from local GABAergic interneurons within the dentate gyrus.

Congenital lymphocytic choriomeningitis virus infection : Spectrum of disease

Lymphocytic choriomeningitis virus (LCMV) is a human pathogen and an emerging neuroteratogen. When the infection occurs during pregnancy, the virus can target and damage the fetal brain and retina. We examined the spectrum of clinical presentations, neuroimaging findings, and clinical outcomes of children with congenital LCMV infection.

Induction of cortical spreading depression with potassium chloride upregulates levels of messenger RNA for glial fibrillary acidic protein in cortex and hippocampus: inhibition by MK-801.

The present study evaluates the time course and spatial extent of changes in GFAP mRNA expression following the induction of spreading depression. Spreading depression was elicited by applying filterpaper pledgets soaked in KCl (3 M) to exposed parietal cortex for ten minutes. Animals were killed 1.5, 3, 6, 12, 24, 48, 96 and 192 h post-KCl application, and the forebrains were prepared for quantitative in situ hybridization. The KCl treatment led to a many-fold increase in GFAP mRNA content in the ipsilateral hippocampus and neocortex and, to a lesser extent, in the contralateral hippocampus, but did not affect GFAP mRNA levels in the contralateral cortex or in the thalamus. The time course of increased expression of GFAP mRNA in the hippocampus differed markedly from that of the cortex. In the hippocampus, GFAP mRNA levels rose rapidly to a maximum at 24 h post-exposure, then fell rapidly. In the cortex, levels rose more slowly and did not reach a maximum until 4 days post-exposure. Analysis of GFAP mRNA levels by dot blot hybridization using samples from a separate set of animals killed at one and 4 days following the KCl exposure confirmed both the upregulation in GFAP mRNA levels and the regional time course differences. Intraperitoneal injection of MK-801, a non-competitive NMDA antagonist which prevents spreading depression, blocked the upregulation of GFAP mRNA in both the hippocampus and the cortex, as demonstrated by both in situ and dot blot hybridization. The results suggest that the physiological changes accompanying spreading depression have a powerful influence on glial cell gene expression.

Deficiency of neuronal nitric oxide synthase (nNOS) worsens alcohol-induced microencephaly and neuronal loss in developing mice.

Previous work conducted in vitro suggests that nitric oxide (NO) protects developing neurons against the toxic effects of alcohol. We tested the hypothesis that neonatal mice carrying a null mutation for neuronal nitric oxide synthase (nNOS), the enzyme which synthesizes NO in neurons, have increased vulnerability to alcohol-induced microencephaly and neuronal loss. Wild-type mice and mutant (nNOS(-/-)) mice received a single intraperitoneal injection of ethanol (0.0, 2.2, 3.3, or 4.4 g/kg) daily over postnatal days (PD) 4-9 and were sacrificed on PD 10. Peak blood alcohol concentrations were approximately 170, 280, and 385 mg/dl for the 2.2, 3.3 and 4.4 g/kg/day treatment groups, respectively, and did not differ significantly between wild-type and nNOS(-/-) strains. Exposure to alcohol induced dose-dependent reductions in total brain weight, forebrain weight and cerebellum weight in both strains of mice. However, the reductions in brain weight were significantly more severe in the nNOS(-/-) mice than in wild type. Quantification of cerebellar neurons revealed that alcohol-induced losses of Purkinje cells and granule cells were both significantly greater in the nNOS(-/-) mice than in wild type. The increased vulnerability of nNOS-deficient neurons to alcohol-induced cell death was confirmed in vitro. Cerebellar granule cell cultures derived from nNOS(-/-) and wild-type mice were exposed for 24 h to 0, 100, 200 or 400 mg/dl ethanol. At each alcohol concentration, the nNOS(-/-) neurons had a significantly greater cell loss than did the wild-type neurons. The results demonstrate that deficiency of nNOS decreases the ability of developing neurons to survive the toxic effects of alcohol. Because NO upregulates intracellular cGMP, which can activate cGMP-dependent protein kinase (PKG), we hypothesize that the NO-cGMP-PKG pathway has a neuroprotective role against alcohol toxicity within the developing brain.

Acute and long-term neuronal deficits in the rat olfactory bulb following alcohol exposure during the brain growth spurt.

This study demonstrates that there is a relative recovery in the number of olfactory bulb granule cells following an initial alcohol-induced deficit, while the number of mitral cells remains permanently and severely depressed. The importance of pattern of exposure in influencing the severity of alcohol-induced neuronal loss in the olfactory bulb is also demonstrated. Sprague-Dawley rat pups were reared artificially and were administered alcohol over postnatal days (PD) 4 through 9, a period of rapid brain growth comparable to part of the human third trimester. Two groups received a daily alcohol dose of 4.5 g/kg, administered either as a 5.1% or 10.2% solution. A third group received a higher daily alcohol dose of 6.6 g/kg administered continuously as a 2.5% solution. Pups were either sacrificed on PD 10 or were allowed to grow to adulthood and sacrificed on PD 90. The number of mitral cells and granule cells and the area of the subependymal zone were determined from single sections. On PD 10, immediately following the alcohol exposure, both the mitral cells and the granule cells were significantly reduced in number, relative to controls, in both of the groups receiving the concentrated (5.1% and 10.2%) alcohol treatments. On PD 90, however, only the mitral cell number remained significantly reduced in the groups receiving the concentrated solutions, while the number of granule cells no longer differed significantly from that of controls. The group receiving the higher daily dose (6.6 g/kg) in continuous fractions had no significant cell loss at 10 or 90 days of age.(ABSTRACT TRUNCATED AT 250 WORDS)

Maturation‐Dependent Alcohol Resistance in the Developing Mouse: Cerebellar Neuronal Loss and Gene Expression During Alcohol‐Vulnerable and ‐Resistant Periods

BACKGROUND Alcohol abuse during pregnancy injures the fetal brain. One of alcohols most important neuroteratogenic effects is neuronal loss. Rat models have shown that the cerebellum becomes less vulnerable to alcohol-induced neuronal death as it matures. We determined if maturation-dependent alcohol resistance occurs in mice and compared patterns of gene expression during the alcohol resistant and sensitive periods. METHODS Neonatal mice received alcohol daily over postnatal day (PD) 2 to 4 or PD8 to 10. Purkinje cells and granule cells were quantified on PD25. The temporal expression patterns of 4 neuro-developmental genes and 3 neuro-protective genes in the cerebellum were determined daily over PD0 to 15 to determine how gene expression changes as the cerebellum transitions from alcohol-vulnerable to alcohol-resistant. The effect of alcohol on expression of these genes was determined when the cerebellum is alcohol sensitive (PD4) and resistant (PD10). RESULTS Purkinje and granule cells were vulnerable to alcohol-induced death at PD2 to 4, but not at PD8 to 10. Acquisition of maturation-dependent alcohol resistance coincided with changes in the expression of neurodevelopmental genes. The vulnerability of cerebellar neurons to alcohol toxicity declined in parallel with decreasing levels of Math1 and Cyclin D2, markers of immature granule cells. Likewise, the rising resistance to alcohol toxicity paralleled increasing levels of GABA alpha-6 and Wnt-7a, markers of mature granule neurons. Expression of growth factors and genes with survival promoting function (IGF-1, BDNF, and cyclic AMP response element binding protein) did not rise as the cerebellum transitioned from alcohol-vulnerable to alcohol-resistant. All 3 were expressed at substantial levels during the vulnerable period and were not expressed at higher levels later. Acute alcohol exposure altered the expression of neurodevelopmental genes and growth factor genes when administered either during the alcohol vulnerable period or resistant period. However, the patterns in which gene expression changed varied among the genes and depended on timing of alcohol administration. CONCLUSIONS Mice have a temporal window of vulnerability in the first week of life, during which cerebellar neurons are more sensitive to alcohol toxicity than during the second week. Expression of genes governing neuronal maturation changes in synchrony with the acquisition of alcohol resistance. Growth factors do not rise as the cerebellum transitions from alcohol-vulnerable to alcohol-resistant. Thus, a process intrinsic to neuronal maturation, rather than rising levels of growth factors, likely underlies maturation-dependent alcohol resistance.

Stimulation of the cAMP pathway protects cultured cerebellar granule neurons against alcohol-induced cell death by activating the neuronal nitric oxide synthase (nNOS) gene.

Neuronal loss is a key component of fetal alcohol syndrome pathophysiology. Therefore, identification of molecules and signaling pathways that ameliorate alcohol-induced neuronal death is important. We have previously reported that neuronal nitric oxide synthase (nNOS) can protect developing cerebellar granule neurons (CGN) against alcohol-induced death both in vitro and in vivo. However, the upstream signal controlling nNOS expression in CGN is unknown. Activated cAMP response element binding protein (CREB) has been strongly linked to the survival of multiple cell types, including CGN. Furthermore, the promoter of the nNOS gene contains two cAMP response elements (CRE). Using cultures of CGN, we tested the hypothesis that cAMP mediates nNOS activation and the protective effect of nNOS against alcohol-induced cell death. Forskolin, an activator of the cAMP pathway, stimulated expression of a reporter gene under the control of the nNOS promoter, and this stimulation was substantially reduced when the two CREs were mutated. Forskolin increased nNOS mRNA levels several fold, increased production of nitric oxide, and abolished alcohols toxic effect in wild type CGN. Furthermore, forskolins protective effect was substantially reduced in CGN cultures genetically deficient for nNOS (from nNOS-/- mice). Delivery of nNOS cDNA using a replication-deficient adenoviral vector into nNOS-/- CGN abolished alcohol-induced neuronal death. In addition, overexpression of nNOS in wild type CGN ameliorated alcohol-induced cell death. These results indicate that the neuroprotective effect of the cAMP pathway is mediated, in part, by the pathways downstream target, the nNOS gene.

Lymphocytic choriomeningitis virus infection of the developing brain: critical role of host age

Lymphocytic choriomeningitis virus (LCMV) is a common human pathogen that causes substantial injury to the developing brain when the infection occurs during pregnancy. However, among children with congenital LCMV infection, there is considerable variability in the site, nature, and severity of neuropathology and in the clinical outcome. We hypothesize that the variability in neuropathology and outcome is due to differences in the gestational timing of LCMV infection.

Spreading depression and reverberatory seizures induce the upregulation of mRNA for glial fibrillary acidic protein

The present study evaluates the relative roles of seizure activity and spreading depression in upregulating glial fibrillary acidic protein (GFAP) mRNA expression. Stimulating electrodes were placed bilaterally in the angular bundle, and recording electrodes were placed bilaterally in the dentate gyrus of adult rats. Intense electrographic seizures were induced by delivering stimulus trains through one stimulating electrode. In some cases, spreading depression accompanied the seizures, while in other cases, the seizures occurred in the absence of spreading depression. Animals were killed 24 h following the last stimulus train, and the forebrains were prepared for quantitative in situ hybridization. Seizure activity and spreading depression led to significant increases in GFAP mRNA levels in the hippocampal formation. Seizure activity alone (without spreading depression) induced a 4-fold increase in GFAP mRNA levels in the hilus and molecular layer of the dentate gyrus and in stratum lacunosum-moleculare of the hippocampus. When seizure activity was accompanied by spreading depression, there was a 10-fold increase in GFAP mRNA levels in these same regions. Regional differences within the hippocampal formation in glial cell response were evident. While GFAP mRNA levels in stratum lacunosum-moleculare of the hippocampus were upregulated by seizure activity and spreading depression, levels in hippocampal stratum radiatum of the hippocampus remained unchanged. The results suggest that abnormal neuronal activity can influence glial cell gene expression and that spreading depression is a stronger signal than seizure activity in upregulating GFAP mRNA levels.