Invited review: Intermittent hypoxia and respiratory plasticity.

Intermittent hypoxia elicits long-term facilitation (LTF), a persistent augmentation (hours) of respiratory motor output. Considerable recent progress has been made toward an understanding of the mechanisms and manifestations of this potentially important model of respiratory plasticity. LTF is elicited by intermittent but not sustained hypoxia, indicating profound pattern sensitivity in its underlying mechanism. During intermittent hypoxia, episodic spinal serotonin receptor activation initiates cell signaling events, increasing spinal protein synthesis. One associated protein is brain-derived neurotrophic factor, a neurotrophin implicated in several forms of synaptic plasticity. Our working hypothesis is that increased brain-derived neurotrophic factor enhances glutamatergic synaptic currents in phrenic motoneurons, increasing their responsiveness to bulbospinal inspiratory inputs. LTF is heterogeneous among respiratory outputs, differs among experimental preparations, and is influenced by age, gender, and genetics. Furthermore, LTF is enhanced following chronic intermittent hypoxia, indicating a degree of metaplasticity. Although the physiological relevance of LTF remains unclear, it may reflect a general mechanism whereby intermittent serotonin receptor activation elicits respiratory plasticity, adapting system performance to the ever-changing requirements of life.

Leigh syndrome in an infant resulting from mitochondrial DNA depletion.

Leigh syndrome is an encephalomyelopathy that results from a heterogeneous group of mitochondrial disorders characterized by symmetric brainstem spongioform lesions. An infant born with hypotonia and lactic acidosis was found to have symmetric brainstem lesions on T(2)-weighted magnetic resonance imaging consistent with Leigh syndrome. Muscle biopsy failed to reveal ragged-red fibers or cells devoid of cytochrome C oxidase or succinate dehyrogenase. Southern blot analysis of mitochondrial DNA isolated from the patient's quadriceps muscle indicated severe mitochondrial DNA depletion, which was suggested as the cause for the Leigh syndrome seen in this patient. Consideration of mitochondrial DNA depletion as an etiology when evaluating the patient with Leigh syndrome is encouraged.

Sensory stimulation increases cortical expression of the fragile X mental retardation protein in vivo.

Fragile X syndrome is a common cause of mental retardation that results from the absence of the fragile X mental retardation protein (FMRP), an RNA binding protein whose function remains unclear. Recent in vitro work has demonstrated that the protein is translated near the synapse in an activity dependent manner [33]. We therefore asked whether expression of FMRP might be altered by neuronal activity in vivo. Using immunoblots of different sub-cellular fractions of the rat somatosensory cortex, we show that the levels of FMRP increase significantly following unilateral whisker stimulation, a model of experience dependent plasticity. This increase is greatest between 2 and 8 h after the stimulus and is seen in both a synaptosomal fraction as well as a sub-cellular fraction enriched for polyribosomal complexes. In contrast, detectable levels of FMRP within the somatosensory cortex show either a decrease or no change after a kainic acid induced seizure compared to water treated controls. Our findings demonstrate that FMRP expression levels are modulated in vivo in response to neuronal activity and suggest a role for FMRP in activity dependent plasticity.

Seizures and sensory stimulation result in different patterns of brain derived neurotrophic factor protein expression in the barrel cortex and hippocampus.

Brain-derived neurotrophic factor (BDNF) is important for the development and trophic support of neurons, and may be involved in controlling axonal sprouting and synaptic plasticity. In order to investigate the activity-dependent regulation of the BDNF gene, BDNF expression was examined within the rat somatosensory cortex (SSC) and hippocampus following vibrissae stimulation, kainic acid induced seizure, and pentylenetetrazol (PTZ) induced seizure. The specific goals of this study were to determine the time course and magnitude of BDNF's activity-dependent expression, and to compare the expression patterns of three commonly used neuronal activation paradigms. Our results demonstrate three novel observations. First, the patterns of BDNF protein expression are dependent upon the neuronal stimulation model used. Both unilateral whisker stimulation (a model of experience dependent plasticity) and kainic acid induced seizure were able to increase the levels of BDNF protein within the SSC and hippocampus. In contrast, PTZ induced seizure did not increase BDNF protein levels in either tissue. Second, there is a dissociation between BDNF mRNA and protein levels following PTZ induced seizure. PTZ seizures resulted in strong increases of BDNF mRNA levels without corresponding increases of the protein. Finally, whisker stimulation resulted in an unexpected increase in BDNF mRNA and protein levels within the hippocampus. These results suggest specific types of neuronal activity can regulate gene expression differently. Furthermore, temporal and spatial differences between the expression of BDNF protein and mRNA levels suggest that the BDNF gene is regulated at the level of translation as well as transcription.

Multiple promoters direct stimulus and temporal specific expression of brain-derived neurotrophic factor in the somatosensory cortex.

Neuronal activity rapidly induces expression of brain-derived neurotrophic factor (BDNF) in the adult rat cortex. The rat BDNF gene has four differentially regulated promoters, each of which produce an mRNA containing a unique 5' exon (I-IV) and a common 3' exon (V) that encodes the mature BDNF protein. The present study used an exon-specific RT-PCR analysis to determine the time course of the induction from both seizures and whisker stimulation. Our data show that specific promoters are utilized at different stages of the activity-dependent induction of the BDNF gene. Furthermore, the data show a differential utilization of the four promoters following a specific stimulus.

Cerebrospinal fluid free choline in movement disorders of paediatric onset.

We measured free choline in cerebrospinal fluid (CSF) of 78 patients with movement disorders of paediatric onset and various controls as a putative index of central phospholipid metabolism. Most of the disorders studied were myoclonic disorders, such as progressive myoclonus epilepsy, the opsoclonus-myoclonus syndrome, and essential myoclonus, but other movement disorders, interictal seizure disorders, and different neurological and nonneurological disorders were also included. There were no significant differences in CSF choline concentrations in myoclonic disorders or other movement disorders compared with controls. The CSF choline levels were lowest in children with seizure disorders including progressive myoclonus epilepsy. In progressive myoclonus epilepsy, the CSF choline values resembled other epileptic disorders rather than other myoclonic disorders. When all the data were analysed collectively, no significant relation of CSF choline was found to patient age, gender, aliquot of CSF measured, or the length of time the sample was stored at -70 degrees C. Separate analyses of data from children and adults showed a trend toward a biphasic relation between patient age and CSF choline which could be pursued in developmental studies of normal subjects. Reduced CSF choline may indicate increased choline incorporation into brain phospholipids, disturbances of choline metabolism, decreased choline release, or non-neural factors.

Cortical NMDAR-1 gene expression is rapidly upregulated after seizure.

The promoter region of the NMDAR-1 receptor has a cis-regulatory element that is capable of binding to the NGFI-A family of transcription factors. Based on this observation, we hypothesized that situations that cause a change in NGFI-A levels would result in a change in NMDAR-1 expression. In these studies, we have demonstrated that a seizure results in a rapid significant increase in NMDAR-1 mRNA and protein expression, at a time when NGFI-A protein levels are expected to be elevated. Our results indicate that control of NMDAR-1 expression is stimulus, time and tissue dependent.

Child neurology and the Internet.

The Internet has become available as a tool to disseminate and gain access to information as well as to provide an ease of communication. The current question before us is how will we, as child neurologists, take advantage of this opportunity? This article delineates examples of current useful resources, investigates the potential of the Internet for neurology, and suggests a "vision" for the future of neurology on the Internet.

NGFI-C expression is affected by physiological stimulation and seizures in the somatosensory cortex.

NGFI-C is an early response gene which encodes a Cys2/His2 zinc finger protein. NGFI-C has previously been demonstrated to be inducible in PC12 cells after NGF stimulation. This study sought to localize this gene in somatosensory cortex, and investigate its possible induction by physiological and seizure stimuli. To determine if NGFI-C message levels are affected by stimulation, RT-PCR was performed on mRNA extracts from somatosensory cortex. NGFI-C mRNA levels were increased to levels four-fold over baseline after a seizure. In a paradigm used as a model of experience-dependent plasticity, vibrissae stimulation also increased the level of NGFI-C expression in the contralateral barrel cortex to 180% of control levels. In situ analysis using digoxigenin-labelled cRNA probes demonstrated NGFI-C containing neurons throughout layers 2 through 6 in somatosensory cortex. A higher cell density was seen after stimulation. Qualitatively, staining was more intense in post-seizure and post-stimulus cortex than in control cortex. Analysis of related zinc finger expression in serial sections revealed that NGFI-C is expressed in a distinct but overlapping cell populations relative to NGFI-A, Krox 20, and Egr-3. These studies demonstrate the inducible nature of NGFI-C message in response to a physiological vibrissae stimulus, as well as to seizures. However, the levels and pattern of expression differ between these two stimuli.

Transcription factor expression is induced by axonal stimulation and glutamate in the glia of the developing optic nerve.

Recent experiments have demonstrated that stimulation of the developing optic nerve affects several glial cell characteristics, such as ionic fluxes and cell proliferation. This investigation asked if transcription factor expression may be another stimulation-dependent process in the glia of the developing optic nerve. In unstimulated optic nerves, an antibody to c-fos-related antigens demonstrated positive cell body staining at postnatal days (P) 2, 7, 14, and 60. This nuclear staining was most prominent at early postnatal ages, although young adult (P60) optic nerves showed occasional positive cells. To demonstrate the inducibility of transcription factor antigens, optic nerves from P7 animals received intermittent 15-20 Hz electrical stimulation for 5-15 min. Two hours after this stimulation, an increased number of immunoreactive cells for c-fos-related antigens, c-jun, and NGFI-A was demonstrated. Additionally, optic nerves were exposed for 5-30 min to a solution of 300 microM glutamate, latter maintained in a glutamate-free solution for 2 h, and then quickly frozen. Glutamate-treated nerves showed an increased expression of c-fos-related antigens compared to control nerves. No c-fos increase was seen in the absence of calcium. Expression of c-fos or NGFI-A occurred in cells that were S-100 positive, and most likely represented type 1 astrocytes. These studies indicate that developing (P7) optic nerves show a baseline expression of c-fos-related antigens, c-jun and NGFI-A. Stimulation through electrical nerve stimulation or glutamate results in an increased expression of these transcription factors.

krox 20 messenger RNA and protein expression in the adult central nervous system.

krox 20 is an inducible immediate early response gene. To determine if krox 20 has a physiological role in the adult central nervous system (CNS), this study sought to demonstrate the presence of krox 20 in adult rat brain. RNA analysis showed the presence of krox 20 transcripts in the CNS, including the cortex. Polyclonal antibodies to a Krox 20 fusion protein demonstrated 79 and 55 kDa antigens in nuclear CNS homogenates. Neither RNA nor protein analysis was able to demonstrate an induction of krox 20 by a seizure at times when other immediate early response genes are known to be induced. Immunohistochemical analysis revealed staining at several levels throughout the nervous system. This staining was predominantly nuclear, consistent with the role of krox 20 as a transcription factor. These data show that krox 20 is present in the adult CNS, yet differs in response to stimuli as compared to other related transcription factors with a zinc finger motif, such as NGFI-A and NGFI-C.

Induction of transcription factors in somatosensory cortex after tactile stimulation.

Immediate early response genes have been shown to be inducible in the central nervous system after a variety of stimuli. Induction of these transcription factors in cerebral cortex by a physiological stimulus had not previously been demonstrated. In this study, tactile stimuli induced multiple transcription factors in the somatosensory cortex. Adult male rats were lightly anesthetized with urethane. Tactile stimuli was delivered by a paint brush gently stroking an animals whiskers on one side of its face for a 15 min period. Two h later, the animals were sacrificed. Cortex contralateral to the stimulation was compared with ipsilateral cortex using antibodies raised against immediate early response gene products NGFI-A, NGFI-B, and c-fos. The different transcription factors showed slightly different patterns of response to the tactile stimulus. However, the induction of immunohistochemical staining was most prominent in layer 4 with all antibodies under study. This increase in the number of cell bodies stained was less robust than that seen in the somatosensory cortex after a seizure, and showed more of a predominance in layer 4 cells. These data demonstrate that physiologic stimulation can induce immediate early response genes in cortical cells, and that multiple immediate early response genes react to a stimulus.

The mouse dopamine D2A receptor gene: sequence homology with the rat and human genes and expression of alternative transcripts.

To understand the possible involvement of dopamine receptors in the pathogenesis of various neurological disorders, we have cloned and sequenced a dopamine D2A receptor gene from the mouse. A mouse genomic library was screened with probes derived from the published sequence of a rat D2A receptor cDNA. Using restriction endonuclease mapping, Southern blotting, and DNA sequencing, we have determined the cDNA sequence and genomic organization of the mouse D2A receptor gene. Unlike other guanine nucleotide-binding protein-coupled receptors, but similar to its rat and human counterparts, the mouse D2A receptor gene has seven introns and spans at least 30 kb of genomic DNA. The mouse D2A sequence shows 99% amino acid homology with the rat and 95% amino acid homology with the human sequence. As would be predicted, sequence differences are significantly more frequent outside of the hypothesized transmembrane spanning domain regions of the protein. Using the polymerase chain reaction with primers made from neighboring exons, we have identified two alternatively spliced D2A transcripts in the mouse. However, in contrast to the other species studied, the mouse expresses primarily the mRNA representing the larger, 444-amino-acid form of the receptor. Mouse pituitary expresses only the mRNA of the 444-amino-acid form of the D2A receptor. Hence, the mouse may offer the best model to study the in vivo physiology of the long form of the D2A receptor.

Relationship between the neural dysgenesis and increased production of class I MHC H-2Kk mRNA and protein in neurons of murine trisomy 16 fetuses.

Neuronal cells from murine trisomy 16 fetuses have increased levels of class I MHC H-2Kk. To determine whether this increased level of H-2Kk protein product resulted from an increased synthesis of mRNA, a 33 base antisense cDNA probe complementary to a region in exon 2 of the H-2Kk sequence (nucleotide 392-424) was synthesized. This probe was used to examine, by in situ hybridization and immunohistochemistry, the neural distribution of H-2Kk mRNA and protein product. A marked elevation of the H-2Kk mRNA and protein were localized in mts16 neuronal populations that were susceptible to dysgenesis. The results implicate the expression of the H-2Kk in the neuropathology of mts16 and its human counterpart, Down syndrome.

Differential expression of dopaminergic D2 receptor messenger RNAs during development.

To define the possible roles of multiple types of dopamine D2 receptors, mRNA levels for two forms of D2 receptor were determined during a variety of developmental stages. Transcripts encoding the 444 amino acid form appear as early as embryonic day 14. In contrast, the mRNA encoding the 415 amino acid form of the receptor does not appear until embryonic day 17, and remains a minor form throughout prenatal development. The adult levels of the mRNAs of these two D2 receptor forms are not attained until long after birth.

Organization and expression of the rat D2A receptor gene: identification of alternative transcripts and a variant donor splice site.

We have recently reported the creation of a cell line expressing D2 receptors encoded by a gene distinct from that described by Bunzow et al. [Bunzow, J. R., Van Tol, H. H. M., Grandy, D. K., Albert, P., Salon, J., Christie, M., Machida, C. A., Neve, K., & Civelli, O. (1988) Nature 336, 783-787]. To provide a framework for understanding structural differences between these and other G-protein-coupled receptors, the structure of the rat gene coding for the Bunzow et al. cDNA (called D2A here) was delineated. The D2A gene contains eight exons and spans at least 50 kb. Sets of oligonucleotide primers were used in combination with the polymerase chain reaction (PCR) to determine the presence of alternative transcripts within the introns. In contrast to other G-protein-coupled receptors, the D2A gene undergoes alternative RNA processing within intron 5, resulting in an insertion of 29 amino acids to the predicted 415 amino acid sequence of the D2A protein. By use of the PCR assay the relative abundance and tissue distribution of the alternative D2A transcripts (herein termed D2A415 and D2A444) were determined. A variant donor splice site was also identified at the end of exon 4, a GC dinucleotide instead of the canonical GT. The variant dinucleotide was also present in the mouse but not in the human D2A gene.