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Cellular and synaptic actions of acetylcholine in the lamprey spinal cord.

ABSTRACT: This study investigated cellular and synaptic mechanisms of cholinergic neuromodulation in the in vitro lamprey spinal cord. Most spinal neurons tested responded to local application of acetylcholine (ACh) with depolarization and decreased input resistance. The depolarization persisted in the presence of either tetrodotoxin or muscarinic antagonist scopolamine and was abolished with nicotinic antagonist mecamylamine, indicating a direct depolarization through nicotinic ACh receptors. Local application of muscarinic ACh agonists modulated synaptic strength in the spinal cord by decreasing the amplitude of unitary excitatory and inhibitory postsynaptic potentials. The postsynaptic response to direct application of glutamate was unchanged by muscarinic agonists, suggesting a presynaptic mechanism. Cholinergic feedback from motoneurons was assessed using stimulation of a ventral root in the quiescent spinal cord while recording intracellularly from spinal motoneurons or interneurons. Mainly depolarizing potentials were observed, a portion of which was insensitive to removal of extracellular Ca2+, indicating electrotonic coupling. Hyperpolarizing potentials were also observed and were attenuated by the glycinergic antagonist strychnine, whereas depolarizing responses were potentiated by strychnine. Mecamylamine also reduced hyperpolarizing responses. The pharmacology of these responses suggests a Renshaw-like feedback pathway in lamprey. Immunohistochemistry for choline acetyltransferase, performed in combination with retrograde filling of motoneurons, demonstrated a population of nonmotoneuron cholinergic cells in the lamprey spinal cord. Thus endogenous cholinergic modulation of the lamprey spinal locomotor network is likely produced by both motoneurons and cholinergic interneurons acting via combined postsynaptic and presynaptic actions.

SUBMITTER: Quinlan KA

PROVIDER: S-EPMC2525711 | biostudies-other | 2008 Aug

REPOSITORIES: biostudies-other

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Project description:Axon regrowth after spinal cord injury (SCI) is inhibited by several types of inhibitory extracellular molecules in the central nervous system (CNS), including chondroitin sulfate proteoglycans (CSPGs), which also are components of perineuronal nets (PNNs). The axons of lampreys regenerate following SCI, even though their spinal cords contain CSPGs, and their neurons are enwrapped by PNNs. Previously, we showed that by 2 weeks after spinal cord transection in the lamprey, expression of CSPGs increased in the lesion site, and thereafter, decreased to pre-injury levels by 10 weeks. Enzymatic digestion of CSPGs in the lesion site with chondroitinase ABC (ChABC) enhanced axonal regeneration after SCI and reduced retrograde neuronal death. Lecticans (aggrecan, versican, neurocan, and brevican) are the major CSPG family in the CNS. Previously, we cloned a cDNA fragment that lies in the most conserved link-domain of the lamprey lecticans and found that lectican mRNAs are expressed widely in lamprey glia and neurons. Because of the lack of strict one-to-one orthology with the jawed vertebrate lecticans, the four lamprey lecticans were named simply A, B, C, and D. Using probes that distinguish these four lecticans, we now show that they all are expressed in glia and neurons but at different levels. Expression levels are relatively high in embryonic and early larval stages, gradually decrease, and are upregulated again in adults. Reductions of lecticans B and D are greater than those of A and C. Levels of mRNAs for lecticans B and D increased dramatically after SCI. Lectican D remained upregulated for at least 10 weeks. Multiple cells, including glia, neurons, ependymal cells and microglia/macrophages, expressed lectican mRNAs in the peripheral zone and lesion center after SCI. Thus, as in mammals, lamprey lecticans may be involved in axon guidance and neuroplasticity early in development. Moreover, neurons, glia, ependymal cells, and microglia/macrophages, are responsible for the increase in CSPGs during the formation of the glial scar after SCI.

Project description:Previously, we reported that RhoA knockdown by morpholino antisense oligonucleotides (MOs), and enzymatic digestion of chondroitin sulfate proteoglycans (CSPGs) at the site of injury with chondroitinase ABC (ChABC), each can reduce retrograde neuronal apoptosis after spinal cord transection in the lamprey. To elucidate the mechanisms in neuronal survival and axon regeneration, we have investigated whether these two effects are additive in vivo. We used lampreys as a spinal cord injury model. MOs were used to knockdown RhoA and Chondroitinase ABC (ChABC) was used to digest CSPGs in vivo. Retrograde labeling, fluorochrome-labeled inhibitor of caspase activity (FLICA), immunohistochemistry, and western blots were performed to assess axonal regeneration, neuronal apoptotic signaling and Akt activation. Four treatment combinations were evaluated at 2-, 4-, and 10-weeks post-transection: (1) Control MO plus enzyme buffer (Ctrl); (2) control MO plus ChABC; (3) RhoA MO plus enzyme buffer (RhoA MO); and (4) RhoA MO plus ChABC (RhoA MO + ChABC). Consistent with our previous findings, at 4-weeks post-transection, there was less caspase activation in the ChABC and RhoA MO groups than in the Ctrl group. Moreover, the RhoA MO plus ChABC group had the best protective effect on identified reticulospinal (RS) neurons among the four treatment combinations. At 2 weeks post-transection, when axons have retracted maximally in the rostral stump and are beginning to regenerate back toward the lesion, the axon tips in the three treatment groups each were closer to the transection than those in the Ctr MO plus enzyme buffer group. Long-term axon regeneration also was evaluated for the large, individually identified RS neurons at 10 weeks post-transection by retrograde labeling. The percent regenerated axons in the RhoA MO plus ChABC group was greater than that in any of the other groups. Akt phosphorylation levels at threonine 308 was quantified in the identified RS neurons by western blots and immunofluorescence. The RhoA MO plus ChABC treatment enhanced pAkt-308 phosphorylation more than any of the other treatment groups. Although some of the effects of CSPGs are mediated through RhoA activation, some growth-inhibiting mechanisms of RhoA and CSPGs are independent of each other, so combinatorial therapies may be warranted.

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Cellular and synaptic actions of acetylcholine in the lamprey spinal cord.

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