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.

Throughout the developing nervous system, considerable synaptic re-organization takes place as postsynaptic neurons extend dendrites and incoming axons refine their synapses, strengthening some and eliminating others. It is well accepted that these processes rely on synaptic activity; however, the mechanisms that lead to this developmental reorganization are not fully understood. Here, we explore the regulation of cap-dependent translation, a mechanism known to play a role in synaptic growth and plasticity. Using sympathetic ganglia in α3 nicotinic acetylcholine receptor (nAChR)-knockout (KO) mice, we establish that electrophysiologically silent synapses between preganglionic axons and postsynaptic sympathetic neurons do not refine, and the growth of dendrites and the targeting of synapses on postsynaptic neurons are impaired. Remarkably, genetically removing 4E-BP, a suppressor of cap-dependent translation, from these α3 nAChR-KO mice largely restores these features. We conclude that synaptic connections can re-organize and refine without postsynaptic activity during post-natal development when 4E-BP-regulated cap-dependent translation is enhanced.

In vertebrates, synaptic activity exerts an important influence on the formation of neural circuits, yet our understanding of its role in directing presynaptic and postsynaptic differentiation during synaptogenesis is incomplete. This study investigates how activity influences synaptic differentiation as synapses mature during early postnatal life. Specifically, we ask what happens to presynaptic terminals when synapses develop without functional postsynaptic receptors and without fast synaptic transmission.To address this issue, we investigated cholinergic nicotinic synapses in sympathetic ganglia of mice with a null mutation for the ␣3 nicotinic ACh receptor gene. Disrupting the ␣3 gene completely eliminates fast excitatory synaptic potentials on postganglionic sympathetic neurons, establishing a crucial role for ␣3-containing postsynaptic receptors in synaptic transmission. Interestingly, the preganglionic nerve terminals form morphologically normal synapses with sympathetic neurons, and these synapses persist without activity in postnatal animals. Surprisingly, when stimulating the preganglionic nerve at physiological rates, we discovered a significant decrease in ACh output from the presynaptic terminals in these ␣3 Ϫ/Ϫ sympathetic ganglia. We show that this decrease in ACh output from the presynaptic terminals results, in part, from a lack of functional high-affinity choline transporters. We conclude the following: (1) fast synaptic transmission in mammalian SCG requires ␣3 expression; (2) in the absence of activity, the preganglionic nerve forms synapses that appear morphologically normal and persist for several weeks; and (3) to sustain transmitter release, developing presynaptic terminals require an activity-dependent retrograde signal.

Serotonin 5-HT 3 receptors (5-HT 3 Rs) are ligand-gated ion channels expressed by many peripheral neurons and are involved in several physiological processes. To learn more about the developmental regulation of 5-HT 3 R expression, we investigated rat sympathetic and vagal sensory neurons. We found that sympathetic and sensory neurons differ in their regulation of 5-HT 3 R expression during early postnatal life and as these neurons develop in culture. In SCG neurons 5-HT 3 R transcript levels are low at postnatal day 1 (P1) and increase 7.5-fold by P21; this increase occurs even after elimination of preganglionic innervation. In comparison, 5-HT 3 R mRNA levels in P1 nodose neurons are over 14-fold greater than in P1 SCG and change little by P21. We show that 5-HT 3 R transcript levels in nodose neurons depend on intact target innervation and drop by 60% after axotomy. When P1 SCG neurons develop in culture, we observed a significant increase in 5-HT 3 R expression: after 7 d in culture, transcript levels increase ninefold versus a threefold increase for neurons developing for 7 d in vivo. In contrast, 5-HT 3 R mRNA levels in cultured nodose neurons drop by 70% within 24 hr; however, this drop is transient. After 2 d, transcript levels begin to increase, and after 7 d, they are above initial values. We show that this delayed increase in 5-HT 3 R expression depends on neurotrophins. In both nodose and sympathetic neurons we found that the changes in 5-HT 3 R gene expression correlate directly with the appearance of 5-HTevoked current densities. Key words: 5-HT 3 receptor; ligand-gated ion channel; sympathetic; superior cervical ganglion; sensory; nodose; trigeminal; mRNA expression; neurotrophins; axotomyThe serotonin 5-HT 3 receptor (5-HT 3 R), a neurotransmittergated ion channel (Yakel and Jackson, 1988;Derkach et al., 1989;Maricq et al., 1991) present on many mammalian peripheral neurons, participates in several diverse physiological functions (Fozard, 1984;Jackson and Yakel, 1995). Activation of 5-HT 3 Rs located on peripheral vagal sensory nerve endings initiates reflexes affecting respiration, circulation, emesis, and swallowing (Douglas, 1975;Sanders-Bush and Mayer, 1996). 5-HT 3 Rs on spinal and vagal sensory neurons are involved in nociceptive signaling and nausea (Fozard, 1984). In the C NS 5-HT 3 Rs are implicated in anxiety, depression, and drug dependence (Apud, 1993;Greenshaw, 1993). In addition, 5-HT 3 Rs are expressed by sympathetic neurons; however, the role for these receptors in sympathetic f unction has not been f ully determined (Wallis and North, 1978;Yang et al., 1992).Many vagal afferent neurons expressing 5-HT 3 Rs are located in the nodose ganglion. These sensory neurons have typical unipolar polarities; their axons bif urcate into a peripheral branch that innervates much of the viscera, including heart, lungs, trachea, and gut, and a central branch that terminates mainly in the nucleus tractus solitarius (Andresen and Kunze, 1994). The physiological responses to serotonin elicited from ...

Mitochondria are the major energy providers in most mammalian cells. Mitochondrial dysfunction has been linked to multiple diseases and pathophysiologies, including a growing number of neurodegenerative disorders. Moreover, as key regulators of cell death, mitochondria are also prime targets for cancer research and at the center of drug development. The mitochondrial membrane potential is essential for ATP production and an important parameter to assess the functional state of these organelles. As MitoTracker® dyes concentrate in active mitochondria, they are ideal tools to evaluate mitochondrial function. We have developed a simple, fast and reliable protocol that measures MitoTracker® signals associated with mitochondria. Our protocol was successfully applied to assess changes in the mitochondrial membrane potential in diverse cell types from different organisms. The method developed by us provides a powerful tool for mitochondrial research and can be easily adapted for drug screening.