MPTP-Parkinsonism is accompanied by persistent expression of a delta-FosB-like protein in dopaminergic pathways.

Parkinson's disease (PD) is characterized by the relatively selective and progressive loss of dopaminergic neurons in the substantia nigra. During the early stages of PD, there are marked compensatory changes in the dopaminergic system, although little is known of how these responses are orchestrated. Since the induction of cellular immediate-early genes (cIEG) has been linked to adaptive responses in the nervous system, we examined their expression in the N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) murine model of PD. MPTP elicited an induction of c-fos, fosB, Delta-fosB and c-jun mRNAs in the striatum that persisted for 24 h. There was a parallel increase in AP-1-like DNA binding activity for up to 7 days post-treatment. At 7 days, AP-1 complexes were specifically supershifted with antisera to FosB and JunD. Immunoblotting of MPTP-treated striata with a FosB-specific antiserum revealed elevated levels of approximately 35 and approximately 46 kDa cross-reactive proteins. Only the 35 kDa protein was increased at 7 days. Thus, the persistent AP-1 complex seen in the MPTP-treated striatum is composed of JunD and a 35 kDa FosB-related protein, possibly Delta-FosB. In situ hybridization revealed elevated expression of fosB and Delta-fosB in the MPTP-treated brain. Expression of both transcripts was highest in ventral striatum, nucleus accumbens and other terminal fields of the mesolimbic system, such as the olfactory tubercle and Islands of Calleja. Thus, the increased fosB expression accompanying MPTP treatment was predominantly associated with dopaminergic pathways. Since FosB was expressed in both vulnerable and spared neuronal populations, we suggest that Delta-FosB-JunD heterodimers play a role in the adaptive response to MPTP neurotoxicity.

Synthesis of willardiine and 6-azawillardiine analogs: pharmacological characterization on cloned homomeric human AMPA and kainate receptor subtypes.

Both willardiine and azawillardiine analogs (18-28) have been reported to be potent and selective agonists for either AMPA or kainate receptors. We report here the novel synthesis and pharmacological characterization of a range of willardiine (18-23) and 6-azawillardiine (24-28) analogs on cells individually expressing human homomeric hGluR1, hGluR2, hGluR4, or hGluR5 receptors. Reaction of the sodium salts of substituted uracils (7-12) or 6-azauracils (13-16) with (S)-3-[(tert-butoxycarbonyl)amino]oxetan-2-one (17) in dry DMF, subsequent deprotection in TFA, and purification by ion-exchange chromatography gave mainly the willardiine analog in which alkylation took place on N1 of the uracil ring. We have investigated the subtype selectivity of these compounds by examining their binding affinity for homomeric hGluR1, -2, -4, or -5 (and hGluR6 in the case of 5-iodowillardiine (22)). From this study we have demonstrated that 22 has high affinity for hGluR5 and, compared to kainate, displays excellent selectivity for this receptor over both the AMPA receptor subtypes and the homomeric kainate receptor, hGluR6. 5-Fluorowillardiine (19) has higher affinity than AMPA for both homomeric hGluR1 and hGluR2 and compared to AMPA displays greater selectivity for AMPA receptor subtypes over the kainate receptor, hGluR5. Some structural features required for optimal activity at homomeric AMPA or kainate receptor subtypes have also been identified. It would appear that quite large lipophilic substituents at the 5-position of the uracil ring not only are accommodated by hGluR5 receptors but also lead to enhanced affinity for these receptors. In contrast to this, for optimal binding affinity to hGluR1, -2, or -4, smaller, electron-withdrawing substituents are required. For optimal activity at hGluR4 receptors a 6-aza-substituted willardiine is favored. The subtype-selective compounds described here are likely to be useful tools to probe the distribution and the physiological roles of the various glutamate receptor subunits in the central nervous system.

A hippocampal GluR5 kainate receptor regulating inhibitory synaptic transmission.

The principal excitatory neurotransmitter in the vertebrate central nervous system, L-glutamate, acts on three classes of ionotripic glutamate receptors, named after the agonists AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxalole-4-propionic acid), NMDA (N-methyl-D-aspartate) and kainate. The development of selective pharmacological agents has led to a detailed understanding of the physiological and pathological roles of AMPA and NMDA receptors. In contrast, the lack of selective kainate receptor ligands has greatly hindered progress in understanding the roles of kainate receptors. Here we describe the effects of a potent and selective agonist, ATPA ((RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid) and a selective antagonist, LY294486 ((3SR, 4aRS, 6SR, 8aRS)-6-((((1H-tetrazol-5-yl) methyl)oxy)methyl)-1, 2, 3, 4, 4a, 5, 6, 7, 8, 8a-decahydroisoquinoline-3-carboxylic acid), of the GluR5 subtype of kainate receptor. We have used these agents to show that kainate receptors, comprised of or containing GluR5 subunits, regulate synaptic inhibition in the hippocampus, an action that could contribute to the epileptogenic effects of kainate.

Absence of a persistently elevated 37 kDa fos-related antigen and AP-1-like DNA-binding activity in the brains of kainic acid-treated fosB null mice.

Chronic stimulation of the nervous system or acute administration of kainic acid results in a persistent increase in AP-1-like DNA-binding activity in the brain. However, the composition and function of these AP-1 complexes remain controversial. By comparing wild-type and fosB-null mice treated with kainic acid, we establish that the complexes comprise JunD in association with an approximately 37 kDa Delta-FosB species. Delta-FosB was expressed persistently in neurons in many areas of the CNS, even though fosB mRNA only increased transiently. This implies that the 37 kDa protein is very stable. fosB-/- mice are predisposed to seizures. Therefore, the chronic expression of Delta-FosB elicited by kainic acid seizures may be indicative of a compensatory/protective role in the pathophysiology of epilepsy.

Agonist and toxin sensitivities of ACh-evoked currents on neurons expressing multiple nicotinic ACh receptor subunits.

1. We have investigated the pharmacological properties of functional nicotinic acetylcholine receptors (nAChRs) on neonatal rat sympathetic neurons from the superior cervical ganglion (SCG) to learn more about the subunit composition of these receptors. These neurons express five nAChR transcripts: alpha 3, alpha 5, alpha 7, beta 2, and beta 4; this finding suggests that SCG neurons may express several different, physiologically distinct, subtypes of nAChRs. 2. To identify potential subtypes, we have characterized currents evoked by different nicotinic agonists and determined their sensitivity to blockade by alpha-bungarotoxin (alpha-BTX) and by neuronal bungarotoxin (n-BTX). From dose-response curves, we find that the ED50 for both cytisine and dimethylphenylpiperazinium (DMPP) is 20 microM and for ACh is 52 microM. n-BTX blocks the ACh-gated currents rapidly, but the kinetics for n-BTX removal is dependent on the duration of the application: brief applications were quickly reversible, whereas prolonged applications took orders of magnitude longer to reverse. 3. Using fast (ms) agonist application, we observed no rapidly desensitizing currents despite the high levels of alpha 7 in these neurons, nor did we observe any currents that could be blocked by alpha-BTX. 4. Using Xenopus oocytes expressing alpha 7 receptors, we show that choline evokes a significant current that is blocked by alpha-BTX. In contrast, choline is much less potent on alpha 3 beta 4 receptors expressed in Xenopus oocytes. Choline can also act as a weak agonist for nAChRs on rat SCG neurons, but its evoked current is not blocked by alpha-BTX. 5. Our results indicate that, when measured at the macroscopic level, most functional nAChRs on SCG neurons behave as a uniform population of receptors, at least with respect to agonist activation and toxin blockade. In comparison with known receptors expressed in heterologous systems, the physiological properties of ACh-evoked currents on SCG neurons are most similar to receptors that have coassembled with both beta 2 and beta 4.

Kainic acid-induced neuronal death is associated with DNA damage and a unique immediate-early gene response in c-fos-lacZ transgenic rats.

Previously, we established that persistent upregulation of c-fos expression preceded kainic acid (KA)-induced neuronal death in mice. To discriminate between events that are products of the seizures elicited by KA and those that are specifically associated with its neurotoxic actions, we have examined the expression of cellular immediate-early genes (cIEGs) following KA or pentylenetetrazol (PTZ) treatment in c-fos-lacZ transgenic rats. While both chemoconvulsants elicit seizures, only KA causes selective neuronal death. Following treatment of transgenic rats with KA there was a protracted expression of Fos-lacZ that lasted for 2-3 d. In contrast, PTZ elicited a transient increase in the transgene product that lasted about 6 hr. Normally, Fos and Fos-lacZ were detected only in neuronal nuclei. However, 6 hr following kainic acid (but not PTZ) administration, beta-galactosidase activity appeared in the cytoplasm of neurons within vulnerable regions (as determined by the terminal transferase biotinylated-UTP nick end labeling (TUNEL) procedure). Like c-fos, transcripts for other cIEGs were elevated for longer periods in the KA-treated rat hippocampus. In addition, fra-1 and fra-2 were only induced in the KA-treated rat. These changes in mRNA levels were paralleled by a sustained increase in AP-1 DNA binding activity. Thus, quantitative and qualitative changes in AP-1 DNA binding complexes accompany neurotoxic cell death that are not observed following seizures.

The developmental increase in ACh current densities on rat sympathetic neurons correlates with changes in nicotinic ACh receptor alpha-subunit gene expression and occurs independent of innervation.

Determining factors that control the expression of neurotransmitter receptors and the mechanisms by which these factors operate is essential to understand how synapses form during development and how receptor numbers change in the adult. In this study, we have investigated one such factor, the influence of innervation, on the developmental expression of nicotinic ACh receptors (nAChRs) on neonatal rat sympathetic neurons, both in terms of ACh current densities, and in terms of mRNA levels for the transcripts that encode these receptors. To date, nine genes have been cloned that encode neuronal nAChRs subunits in mammals. We demonstrate that mRNA encoding five nAChR subunits, alpha 3, alpha 5, alpha 7, beta 2, and beta 4, are present in neonatal rat sympathetic neurons. We show that mRNA levels for alpha 3 and alpha 7 subunits increase by more than threefold over the first 2 postnatal weeks, a period when most synapses are forming on the neurons; however, we observed no significant change in mRNA levels for alpha 5, beta 2, or beta 4. Using whole-cell voltage-clamp techniques, we show that the increase in alpha-subunit mRNA correlates with increases in ACh current densities, which double over the same period. To investigate the role of innervation, we cut the preganglionic nerve at birth and measured subunit mRNA levels and ACh current densities in denervated neurons 1-2 weeks later. Our results indicate that the preganglionic nerve differentially affects the mRNA level for the five nAChR transcripts, yet it has little influence on the developmental increase in ACh current densities.

The expression of nicotinic acetylcholine receptors by PC12 cells treated with NGF.

The expression of neuronal nicotinic ACh receptors (nAChRs) and the subunits that compose these receptors by PC12 cells exposed to NGF has been studied. The analysis of total RNA reveals that the neuronal nAChR subunits alpha 3, alpha S, beta 2, beta 3, and beta 4, but not alpha 2 and alpha 4, are expressed in our PC12 cells. Within 48 hr of adding NGF to cultures, the RNA corresponding to alpha 3, alpha 5, beta 3, and beta 4 is decreased, but the beta 2 RNA increases for up to 6 d after NGF treatment. To determine the influence of NGF treatment on subunit protein expression, subunit-specific antisera were prepared. Immunocytochemistry detected antigen for alpha 3, alpha 5, beta 2, beta 3, and beta 4 (but not alpha 2 and alpha 4) in both NGF-treated and nontreated PC12 cells. The expression of nAChR subunit proteins, as measured by direct binding of antibodies to PC12 cells, does not change subsequent to 6 d of treatment with NGF. Whole-cell recording of PC12 cells shows that both the individual cell current density and response to the agonist cytisine were not altered after 5-7 d in NGF. However, the number of cells exhibiting detectable ACh-induced currents doubled. These results indicate that NGF increases the number of PC12 cells expressing ACh-sensitive nAChR currents but the activation is not the result of altering the amounts of individual nAChR subunit proteins. These data, taken together with the decrease in most nAChR subunit RNAs (except beta 2), suggest that NGF regulation of nAChRs may be through a posttranscriptional mechanism.

Effects of ganglionic satellite cells and NGF on the expression of nicotinic acetylcholine currents by rat sensory neurons.

1. We have investigated two factors that affect the expression of nicotinic acetylcholine (ACh) currents on neonatal rat sensory neurons: an influence derived from ganglionic satellite cells, and nerve growth factor (NGF). 2. With the use of whole-cell patch-clamp techniques on rat nodose neurons, we have measured the proportion of neurons sensitive to ACh and have quantified their ACh current densities. The majority (60%) of nodose neurons from neonatal animals do not express nicotinic acetylcholine receptors (nAChRs); the remaining 40% had ACh current densities that ranged from 0.4 to 93 pA/pF. Furthermore, neither the proportion nor the ACh current densities change over the first two postnatal weeks in vivo. 3. The expression of ACh currents by these neurons in vivo is controlled, in part, by an influence from the ganglionic satellite cells: culturing neurons in the absence of other cell types results in an increase in the proportion of ACh-sensitive neurons, whereas coculturing neurons with their satellite cells maintains functional nAChR expression in its in vivo state. Furthermore, satellite cells are not required continually, as a brief exposure to this influence, either in vivo or in culture, is sufficient to exert its effect on functional nAChR expression. 4. On removal of this satellite cell influence, the neurons respond to NGF treatment by increasing their ACh current densities: the median ACh current density for neurons grown for 2-3 wk with NGF was 32.5 pA/pF, whereas, the median ACh current density for neurons cultured without NGF for the same time was 4.5 pA/pF.(ABSTRACT TRUNCATED AT 250 WORDS)

Nerve growth factor induces functional nicotinic acetylcholine receptors on rat sensory neurons in culture.

Neonatal sensory neurons from rat nodose ganglia express nicotinic acetylcholine receptors when grown in tissue culture without other cell types. The present study investigates the role of nerve growth factor in inducing these receptors. Nerve growth factor has little effect on the growth and survival of nodose neurons in culture, although most neurons were found by quantitative radioautography to have high-affinity nerve growth factor receptors. Nerve growth factor strongly influenced the expression of nicotinic receptors on these neurons: the proportion of acetylcholine-sensitive neurons was approximately 60% in cultures with nerve growth factor compared with 15% in cultures grown without nerve growth factor. The proportion of acetylcholine-sensitive neurons increased over the first week, plateaued by day 12 and remained high for at least three weeks. In contrast, without NGF, the proportion of acetylcholine-sensitive neurons was low throughout the three-week period. The results indicate that nerve growth factor is an important factor in promoting nicotinic receptors on these neurons in culture.