Roderic L. Smith

Alteration of GABAA Receptor Function Following Gene Transfer of the CLC-2 Chloride Channel

The effect of GABAA receptor activation varies from inhibition to excitation depending on the state of the transmembrane anionic concentration gradient (delta anion). delta anion was genetically altered in cultured dorsal root ganglion neurons via adenoviral vector-mediated expression of ClC-2, a Cl- channel postulated to regulate the Cl- concentration in neurons in which GABAA receptor activation is predominantly inhibitory. ClC-2 expression was verified by the presence of the appropriate mRNA, protein, and membrane conductance. CIC-2 expression resulted in a large negative shift in the Cl- equilibrium potential (ECl) that attenuated the GABA-mediated membrane depolarization and prevented GABAA receptor-mediated action potentials. These results establish that gene transfer of transmembrane ion channels to neurons can be used to demonstrate their physiological function, and that delta anion can be genetically manipulated to alter the function of neuronal GABAA receptors in situ.

Ontogeny of cation–Cl− cotransporter expression in rat neocortex

Neuronal precursors and immature cortical neurons actively accumulate Cl- and as a consequence depolarize in response to GABAA receptor activation. With maturity, intracellular Cl- decreases resulting in a shift towards GABAA inhibition. These observations suggest that changes in expression of cation-Cl- cotransporters may have a significant role in the ontogeny of neuronal Cl- homeostasis. Using ribonuclease protection analysis and in situ hybridization we examined the developmental expression of all presently known members of the cation-Cl- cotransporter gene family in rat brain. Of the inwardly directed cotransporters, NKCC-1, NKCC-2, and NCC-1, only NKCC-1 was detected at significant levels in brain. NKCC-1 was expressed in neurons, appearing first in cortical plate but not in ventricular or subventricular zone. Expression levels peaked by the third postnatal week and were maintained into adulthood. The outwardly directed cotransporters, KCC-1 and KCC-2, demonstrated significantly different levels and time courses of expression. KCC-1 was expressed prenatally at very low levels which increased little over the course of development. In contrast, KCC-2 expression appeared perinatally and increased dramatically after the first week of postnatal life. Differential changes in expression of this gene family occurred during periods of critical shifts in chloride homeostasis and GABA response suggestive of a role in these processes. Furthermore the absence of expression of known inwardly directed cotransporters in Cl- accumulating neuroepithelia and lack of evidence for glial expression suggests that as yet unidentified members of this gene family may be involved in chloride homeostasis in immature neuronal precursors and neuroglia.

Activation of second-messenger pathways reactivates latent herpes simplex virus in neuronal cultures

Herpes simplex virus type 1 (HSV-1) establishes latent infections in neurons of sympathetic and sensory ganglia in humans, and reactivation of latent virus results in recurrent disease. Previously, we reported establishment of latent HSV-1 infections in neuronal cultures derived from rats, monkeys, and humans; reactivation occurs following nerve growth factor (NGF) deprivation. The processes controlling HSV latency are not understood. Using the in vitro neuronal latency system, we have shown that latent HSV-1 reactivated in response to stimulation of at least two second-messenger pathways. Stimulation of cAMP-dependent pathways by several mechanisms or activation of protein kinase C by phorbol myristate acetate (PMA) resulted in reactivation of latent HSV-1. The reactivation kinetics following treatment with activators of protein kinase A and C were accelerated compared with those following NGF deprivation. 2-Aminopurine, which inhibits NGF-stimulated protein kinases and other classes of protein kinases, but does not effect protein kinase A or C, blocked reactivation produced by NGF deprivation or treatment with a cAMP analog, but not reactivation by PMA treatment. These results demonstrate that latent HSV-1 reactivates in neurons in vitro in response to activation of second-messenger pathways.

A new form of feedback at the GABAA receptor

GABA is inhibitory in adults, but it is excitatory in young animals. A recent study shows that activation of the GABAA receptor itself may promote this developmental switch.

DEVELOPMENTAL EXPRESSION OF CLC-2 IN THE RAT NERVOUS SYSTEM

Abstract Regulation of expression of the voltage-gated chloride channel, ClC-2, was investigated during development and adult life in rat brain. RNase protection assays demonstrated a marked increase in levels of expression of ClC-2 in brain during early postnatal development which was also detected in adult brain. In situ hybridization of E15 and E18 rat brains demonstrated ClC-2 expression in deep brain nuclei and scattered cells within the neuroepithelial layers, but not in the regions of subventricular zone that primarily give rise to glial populations. By E18 all neurons within the emerging cortical plate and its equivalent in other areas of the CNS were heavily labeled. During the first postnatal week, ClC-2 was highly expressed in most neurons. By P7 a pattern of differential expression emerged with evidence of decreased expression of ClC-2 mRNA in many neuronal populations. In adult rat brain, ClC-2 was expressed at highest levels in large neurons as found within layer V of cortex, Ammons Horn of hippocampus, or mitral cells of the olfactory bulb and Purkinje cells within the cerebellum. Many smaller neurons within the diencephalon maintained significant levels of expression. A functional conductance was readily detected in hippocampal neurons during the first postnatal week, which had the same characteristic properties as the conductance observed in adult neurons. The observed expression and functional presence of ClC-2 suggest a widespread role in neuronal chloride homeostasis in early postnatal life, and demonstrated that cell specific shut-down resulted in the adult pattern of expression.

Expression of mRNA for the elav-like neural-specific RNA binding protein, HuD, during nervous system development

The expression of mRNA for the neuronal antigen HuD (Elavl4) associated with paraneoplastic encephalomyelitis and sensory neuronopathy was evaluated in the developing and adult rat nervous system. Using RNase protection assay and non-radioactive in situ hybridization histochemistry HuD expression was shown to be expressed at high levels at the earliest time point observed (E15), but declined significantly during the first postnatal week to levels which were maintained into adulthood. In the adult, HuD expression became restricted primarily to large pyramidal-like neurons. Exceptions of note were many smaller neurons within a variety of thalamic nuclei. Expression of HuD was observed to be coincident with terminal differentiation of all neuronal structures evaluated regardless of the timing of their development, providing correlative evidence for a role in neuronal differentiation or the maintenance of neuronal phenotype. The marked restriction of HuD mRNA expression with maturity suggests that its functional role in adult neurons varies significantly throughout the CNS.

Characterization of Promoter Function and Cell-Type-Specific Expression from Viral Vectors in the Nervous System

ABSTRACT Viral vectors have become important tools to effectively transfer genes into terminally differentiated cells, including neurons. However, the rational for selection of the promoter for use in viral vectors remains poorly understood. Comparison of promoters has been complicated by the use of different viral backgrounds, transgenes, and target tissues. Adenoviral vectors were constructed in the same vector background to directly compare three viral promoters, the human cytomegalovirus (CMV) immediate-early promoter, the Rous sarcoma virus (RSV) long terminal repeat, and the adenoviral E1A promoter, driving expression of the Escherichia coli lacZ gene or the gene for the enhanced green fluorescent protein. The temporal patterns, levels of expression, and cytotoxicity from the vectors were analyzed. In sensory neuronal cultures, the CMV promoter produced the highest levels of expression, the RSV promoter produced lower levels, and the E1A promoter produced limited expression. There was no evidence of cytotoxicity produced by the viral vectors. In vivo analyses following stereotaxic injection of the vector into the rat hippocampus demonstrated differences in the cell-type-specific expression from the CMV promoter versus the RSV promoter. In acutely prepared hippocampal brain slices, marked differences in the cell type specificity of expression from the promoters were confirmed. The CMV promoter produced expression in hilar regions and pyramidal neurons, with minimal expression in the dentate gyrus. The RSV promoter produced expression in dentate gyrus neurons. These results demonstrate that the selection of the promoter is critical for the success of the viral vector to express a transgene in specific cell types.

Inducible Cyclic AMP Early Repressor Produces Reactivation of Latent Herpes Simplex Virus Type 1 in Neurons In Vitro

ABSTRACT Herpes simplex virus type 1 (HSV-1) establishes a latent infection in neurons of the peripheral nervous system. During latent HSV-1 infection, viral gene expression is limited to latency-associated transcripts (LAT). HSV-1 remains latent until an unknown mechanism induces reactivation. The ability of the latent virus to periodically reactivate and be shed is essential to the transmission of disease. In vivo, the stimuli that induce reactivation of latent HSV-1 include stress, fever, and UV damage to the skin at the site of initial infection. In vitro, in primary neurons harboring latent HSV-1, nerve growth factor (NGF) deprivation or forskolin treatment induces reactivation. However, the mechanism involved in the induction of reactivation remains poorly understood. An in vitro neuronal model of HSV-1 latency was used to investigate potential mechanisms involved in the induction of reactivation of latent HSV-1. In situ hybridization analysis of neuronal cultures harboring latent HSV-1 showed a marked, rapid decrease in the percentage of LAT-positive neurons following induction of reactivation by NGF deprivation or forskolin treatment. Western blot analysis showed a corresponding increase in expression of the cellular transcription factor inducible cyclic AMP early repressor (ICER) during reactivation. In transient-transfection assays, ICER downregulated LAT promoter activity. Expression of ICER from a recombinant adenoviral vector induced reactivation and decreased the percentage of LAT-positive neurons in neuronal cultures harboring latent HSV-1. These results indicate that ICER represses LAT expression and induces reactivation of latent HSV-1.