Darrell A. Jackson

Prenatal availability of choline modifies development of the hippocampal cholinergic system

Choline supplementation during fetal development [embryonic days (E) 11–17] permanently enhances memory performance in rats. To characterize the neurochemical mechanisms that may mediate this effect, we investigated the development of indices of the cholinergic system in the hippocampus: choline acetyltransferase (ChAT), acetylcholinesterase (AChE), synthesis of acetylcholine (ACh) from choline transported by high‐affinity choline uptake (HACU), and potassium‐evoked ACh release. During E11–E17, Sprague‐Dawley pregnant rats consumed 0 [choline‐deficient (ChD)], 1.3 [control (ChC)], and 4.6 [choline‐supplemented (ChS)] mmol/(kg • day) of choline, respectively. On postnatal days 17 and 27, hippocampi of the ChD animals had the highest AChE and ChAT activities, and increased synthesis of ACh from choline transported by HACU, concomitant with reductions of tissue ACh content relative to the ChC and ChS rats and an inability to sustain depolarization‐evoked ACh release relative to the ChS animals. In contrast, AChE and ChAT activities, and ACh synthesized from choline transported by HACU, were lowest in ChS rats whereas depolarization‐evoked ACh release was the highest. This pattern of changes suggests that the hippocampus of the ChD animals is characterized by fast ACh recycling and efficient choline reutilization for ACh synthesis, presumably to maintain adequate ACh release despite the decrease of the ACh pool, whereas in the ChS animals ACh turnover and choline recycling is slower while the evoked release of ACh is high. Together, the data show a complex adaptive response of the hippocampal cholinergic system to prenatal choline availability and provide a novel example of developmental plasticity in the nervous system governed by the supply of a single nutrient.—Cermak, J. M., Holler, T., Jackson, D. A., Blusztajn, J. K. Prenatal availability of choline modifies development of the hippocampal cholinergic system. FASEB J. 12, 349–357 (1998)

Receptor subtypes mediating facilitation by serotonin of excitability of spinal motoneurons

Serotonin receptor ligands, with differential affinity for subtypes of serotonin (5-HT) receptors, were administered intravenously or iontophoretically to urethane-anesthetized rats and the effects of these compounds on glutamate-evoked firing of spinal motoneurons were tested. The excitability of spinal motoneurons was markedly enhanced after intravenous administration of the selective 5-HT1A ligand 8-hydroxy-2-(di-n-propylamino) tetralin (DPAT) in rats with acute spinal transections at C1. However, local application of DPAT, directly into the ventral horn by microiontophoresis, inhibited the glutamate-evoked firing of motoneurons in direct contrast to the facilitatory effects of iontophoretically applied 5-HT. The DPAT-induced inhibition may have been nonspecific, since it was not antagonized by methysergide. Other 5-HT agonists, with relatively selective affinity for 5-HT1B, 5-HT1C and 5-HT2 receptors, increased the excitability of spinal motoneurons when applied iontophoretically or intravenously. The excitatory effect of iontophoretically applied 5-HT was antagonized by the nonselective 5-HT antagonist, methysergide and by ketanserin and ritanserin, which have relatively selective affinity for 5-HT1C and 5-HT2 receptors. These results indicate that 5-HT1A receptors do not mediate facilitation of excitability of motoneurons produced by local application of 5-HT directly into the vicinity of the motoneurons. However, the marked increase in firing of motoneurons that was caused by intravenous administration of DPAT in spinal transected rats, suggests that 5-HT1A receptors in the spinal cord may participate in 5-HT-induced enhancement of somatomotor outflow, at sites presynaptic to the motoneurons. The iontophoretic results suggest that 5-HT1B, 5-HT1C and 5-HT2 receptors may all play a role in facilitation of the excitability of spinal motoneurons by locally applied 5-HT. Differentiation between these subtypes of receptor awaits the development of more completely selective agonists and antagonists.

Imprinting of hippocampal metabolism of choline by its availability during gestation: Implications for cholinergic neurotransmission

Choline supplementation during the second half of the gestational period in rats permanently improves visuospatial memory. Choline availability during this period also alters the turnover of choline and acetylcholine in the hippocampus in 3-4 week-old animals in a complex pattern consistent with the notion that cholinergic neurotransmission is enhanced by prenatal choline supplementation.

Molecular analysis of the regulation of muscarinic receptor expression and function

We have investigated the molecular mechanisms involved in the regulation of muscarinic acetylcholine receptor gene expression and localization and generated knockout mice to study the role of the M1 muscarinic receptor in vivo. We have used the MDCK cell system to demonstrate that different subtypes of mAChR can be targeted to different regions of polarized cells. We have also examined the developmental regulation of mAChR expression in the chick retina. Early in development, the M4 receptor is the predominant mAChR while the levels of the M2 and M3 receptors increase later in development. The level of M2 receptor is also initially very low in retinal cultures and undergoes a dramatic increase over several days in vitro. The level of M2 receptor can be increased by a potentially novel, developmentally regulated, secreted factor produced by retinal cells. The promoter for the chick M2 receptor gene has been isolated and shown to contain a site for GATA-family transcription factors which is required for high level cardiac expression. The M2 promoter also contains sites which mediate induction of transcription in neural cells by neurally active cytokines. We have generated knockout mice lacking the M1 receptor and shown that these mice do not exhibit pilocarpine-induced seizures and muscarinic agonist-induced suppression of the M-current potassium channel in sympathetic neurons.

Thyrotropin‐Releasing Hormone (TRH) Effects on Spinal Cord Neuronal Excitability

TRH is found in terminals in the dorsal, lateral, and ventral horns of the spinal cord and apparently has at least a weak facilitatory effect on excitability of neurons in all these locations. These findings suggest that TRH may facilitate transmission in somatosensory pathways, enhance sympathetic outflow from the spinal cord, and facilitate somatic motoneuron excitability, at least transiently. All studies that have examined TRH effects on spinal neuronal excitability have used exogenously administered TRH. Virtually nothing is known about how spinal neuronal functioning might be affected by TRH released from terminals after activation of TRH-containing cell bodies. The acquisition of this knowledge awaits the development of specific TRH antagonists. Preliminary experiments suggest that TRH may have prolonged facilitatory effects on the excitability of developing or damaged spinal cord neurons. Further studies are necessary to determine how TRH interacts with other neuroactive peptides and monoamines to affect excitability of neurons in the developing, damaged, and normal adult spinal cord.

Thyrotropin releasing hormone (TRH) modifies excitability of spinal cord dorsal horn cells

Thyrotropin releasing hormone (TRH) has been identified recently in fibers and cell bodies in the dorsal horn of the spinal cord, but its function in the dorsal horn is not known. The present study investigated the effects of TRH applied by iontophoresis on the excitability of dorsal horn cells that were responsive to mechanical stimulation of the ipsilateral hindlimb. TRH facilitated glutamate-induced firing of these cells without directly driving the cells in the absence of glutamate. These results suggest that TRH may modulate transmission of somatosensory information in the spinal cord.

Subtype-specific regulation of muscarinic receptor expression and function by heterologous receptor activation.

Incubation of cultured embryonic chicken heart cells with the β-adrenergic agonist isoproterenol resulted in a dose-dependent increase in the number of mAChR on the surface of intact cells. The isoproterenol-mediated increase in mAChR number was time dependent and reached a maximum by 48 h. Chick heart cells treated with isoproterenol exhibited a greater than 6-fold increase in the sensitivity for carbachol-mediated inhibition of adenylyl cyclase activity as compared to control. Stimulation of cultured heart cells for 24 h with isoproterenol resulted in a 25-35% increase in cm2 mRNA levels as compared to control cm2 mRNA levels. In contrast, the level of cm4 mRNA was not significantly affected by isoproterenol treatment. cm2 mRNA levels were maximally elevated by 15 h following isoproterenol stimulation and remained elevated for up to 72 h. Incubation of cells with isoproterenol in the presence of Rp-cAMP, an inhibitor of cAMP-dependent protein kinase, blocked the increase in the level of cm2 mRNA. Thus, prolonged activation of β-adrenergic receptors results in an increase in mAChR number and muscarinic responsiveness in chick heart cells due to a cAMP-dependent protein kinase mediated increase in cm2 mRNA levels.

Differential role of beta-arrestin ubiquitination in agonist-promoted down-regulation of M 1 vs M 2 muscarinic acetylcholine receptors

Background Sustained agonist-promoted ubiquitination of β-arrestin has been correlated with increased stability of the GPCR – β-arrestin complex. Moreover, abrogation of β-arrestin ubiquitination has been reported to inhibit receptor internalization with minimal effects on receptor degradation. Results Herein we report that agonist activation of M1 mAChRs produces a sustained β-arrestin ubiquitination but no stable co-localization with β-arrestin. In contrast, sustained ubiquitination of β-arrestin by activation of M2 mAChRs does result in stable co-localization between the M2 mAChR and β-arrestin. Internalization of receptors was unaffected by proteasome inhibitors, but down-regulation was significantly reduced, suggesting a role for the ubiquitination machinery in promoting down-regulation of the receptors. Given the ubiquitination status of β-arrestin following agonist treatment, we sought to determine the effects of β-arrestin ubiquitination on M1 and M2 mAChR down-regulation. A constitutively ubiquitinated β-arrestin 2 chimera in which ubiquitin is fused to the C-terminus of β-arrestin 2 (YFP-β-arrestin 2-Ub) significantly increased agonist-promoted down-regulation of both M1 and M2 mAChRs, with the effect substantially higher on the M2 mAChR. Based on this observation, we were interested in examining the effects of disruption of potential ubiquitination sites in the β-arrestin sequence on receptor down-regulation. Agonist-promoted internalization of the M2 mAChR was not affected by expression of β-arrestin lysine mutants lacking putative ubiquitination sites, β-arrestin 2K18R, K107R, K108R, K207R, K296R, while down-regulation and stable co-localiztion of the receptor with this β-arrestin lysine mutant were significantly reduced. Interestingly, expression of β-arrestin 2K18R, K107R, K108R, K207R, K296R increased the agonist-promoted down-regulation of the M1 mAChR but did not result in a stable co-localiztion of the receptor with this β-arrestin lysine mutant. Conclusion These findings indicate that ubiquitination of β-arrestin has a distinct role in the differential trafficking and degradation of M1 and M2 mAChRs.

NADPH oxidase mediates the oxygen-glucose deprivation/reperfusion-induced increase in the tyrosine phosphorylation of the N -methyl-D-aspartate receptor NR2A subunit in retinoic acid differentiated SH-SY5Y Cells

Background Evidence exists that oxidative stress promotes the tyrosine phosphorylation of N-methyl-D-aspartate receptor (NMDAR) subunits during post-ischemic reperfusion of brain tissue. Increased tyrosine phosphorylation of NMDAR NR2A subunits has been reported to potentiate receptor function and exacerbate NMDAR-induced excitotoxicity. Though the effect of ischemia on tyrosine phosphorylation of NMDAR subunits has been well documented, the oxidative stress signaling cascades mediating the enhanced tyrosine phosphorylation of NR2A subunits remain unclear. Results We report that the reactive oxygen species (ROS) generator NADPH oxidase mediates an oxidative stress-signaling cascade involved in the increased tyrosine phosphorylation of the NR2A subunit in post-ischemic differentiated SH-SY5Y neuroblastoma cells. Inhibition of NADPH oxidase attenuated the increased tyrosine phosphorylation of the NMDAR NR2A subunit, while inhibition of ROS production from mitochondrial or xanthine oxidase sources failed to dampen the post-ischemic increase in tyrosine phosphorylation of the NR2A subunit. Additionally, inhibition of NADPH oxidase blunted the interaction of activated Src Family Kinases (SFKs) with PSD-95 induced by ischemia/reperfusion. Lastly, inhibition of NADPH oxidase also markedly reduced cell death in post-ischemic SH-SY5Y cells stimulated by NMDA. Conclusions These data indicate that NADPH oxidase has a key role in facilitating NMDAR NR2A tyrosine phosphorylation via SFK activation during post-ischemic reperfusion.

Agonist mediated internalization of M 2 mAChR is β-arrestin-dependent

Background Muscarinic acetylcholine receptors (mAChRs) undergo agonist-promoted internalization, but evidence suggesting that the mechanism of internalization is β-arrestin dependent has been contradictory and unclear. Previous studies using heterologous over-expression of wild type or dominant-negative forms of β-arrestins have reported that agonist-promoted internalization of M2 mAChRs is a β-arrestin- and clathrin-independent phenomenon. In order to circumvent the complications associated with the presence of endogenous β-arrestin that may have existed in these earlier studies, we examined agonist-promoted internalization of the M2 mAChR in mouse embryonic fibroblasts (MEFs) derived from β-arrestin knockout mice that lack expression of either one or both isoforms of β-arrestin (β-arrestin 1 and 2). Results In wild type MEF cells transiently expressing M2 mAChRs, 40% of surface M2 mAChRs underwent internalization and sorted into intracellular compartments following agonist stimulation. In contrast, M2 mAChRs failed to undergo internalization and sorting into intracellular compartments in MEF β-arrestin double knockout cells following agonist stimulation. In double knockout cells, expression of either β-arrestin 1 or 2 isoforms resulted in rescue of agonist-promoted internalization. Stimulation of M2 mAChRs led to a stable co-localization with GFP-tagged β-arrestin within endocytic structures in multiple cell lines; the compartment to which β-arrestin localized was determined to be the early endosome. Agonist-promoted internalization of M2 mAChRs was moderately rescued in MEF β-arrestin 1 and 2 double knockout cells expressing exogenous arrestin mutants that were selectively defective in interactions with clathrin (β-arrestin 2 ΔLIELD), AP-2 (β-arrestin 2-F391A), or both clathrin/AP-2. Expression of a truncated carboxy-terminal region of β-arrestin 1 (319–418) completely abrogated agonist-promoted internalization of M2 mAChRs in wild type MEF cells. Conclusion In summary, this study demonstrates that agonist-promoted internalization of M2 mAChRs is β-arrestin- and clathrin-dependent, and that the receptor stably co-localizes with β-arrestin in early endosomal vesicles.