LIM-homeodomain proteins Lhx3 and Lhx8 induce the cholinergic neurotransmitter identity in spinal motor neurons and forebrain cholinergic neurons through Isl1 and NLI hexamer complex transcriptional activity.

<p>LIM-homeodomain proteins Lhx3 and Lhx8 induce the cholinergic neurotransmitter identity in spinal motor neurons and forebrain cholinergic neurons through Isl1 and NLI hexamer complex transcriptional activity.</p

Expression of neurotransmitters and neurotransmitter receptors in the early motor circuit.

<p>A–H) In-situ detection of neurotransmitter receptor subunit mRNA (green) and <i>Isl1</i> mRNA (red) in adjacent sections reveals differential expression in medial LMC and lateral LMC motor neurons from HH St. 24–30. G) KCC2 (green) and Isl1 (red) protein detection. H–J) <i>GAD-1</i> mRNA expression in the whole spinal cord (dotted line) from HH St. 24–30. Coloured contours of spinal regions correspond to those summarizing neurotransmitter receptor expression in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093836#pone-0093836-t001" target="_blank">Table 1</a>. Scale bars represent 100 μm.</p

Pharmacological interrogation of motor neuron population activities.

<p>A) Co-electroporation of <i>Hb9-GFP</i> and <i>R-GECO</i> expression plasmids, followed by dissection and calcium imaging reveals patterns of activity in LMC motor neurons. B) Application of the nicotinic antagonist D-Tubocurarine (DTC, 10 μM) to HH St. 26 spinal cords blocks detectable activity. C) 10 μM DTC application blocks activity at HH St. 30. D–E) Application of the muscarinic antagonist Scopolamine (Scop., 10 μM) at HH St. 26 (D) and HH St. 30 (E) reduces the frequency of firing. F–G) Application of the GABA-A antagonist picrotoxin (PTX, 10 μM) at HH St. 26 (F) and HH St. 30 (G) reduces the frequency of firing. H) Cycle frequency at HH St. 26 in the presence or absence of blockers. I) Cycle length at HH St. 26 in the presence or absence of drugs. J) Cycle frequency at HH St. 30 in the presence or absence of blockers. K) Cycle length at HH St. 30 in the presence or absence of drugs. These comparisons are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093836#pone-0093836-t002" target="_blank">Tables 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093836#pone-0093836-t003" target="_blank">3</a>.</p

Electrical properties of Lim1 and Isl1-positive LMC neurons.

<p>A) Overall electrical activity profiles of Isl1⁺ and Lim1⁺ LMC subpopulations at HH St. 29/30 are similar; traces shown are recorded from two different preparations. B) Neurons filled with Alexa 568 dye are identified by post-recording immunostaining with Lhx1, Isl1 and Foxp1 antibodies. C) Comparison of membrane potential and membrane resistance (D) reveals both of these characteristics to be similar between cell types, whereas the membrane capacitance is higher in Isl1⁺ neurons (E). The magnitude of individual cycles is no different between cell types (F), though cycles are longer in Lim1⁺ cells (G) and there is a shorter interval between cycles in Lim1⁺ cells (H). The average number of cycles in an episode is not different (I), nor are episodes (J) or the intervals between episodes (K) longer in one cell type than another. Camera lucida tracing of filled neurons (L) allows counts of primary (black, M) and secondary (grey, N) processes, demonstrating no difference in branch characteristics of Isl1⁺ and Lim1⁺ neurons at the stages examined.</p

Summary of characteristics of lateral and medial LMC neurons.

<p>Summary of characteristics of lateral and medial LMC neurons.</p

Cycles occurring in active neurons through development.

<p>Cycles occurring in active neurons through development.</p

Calcium imaging reflects the electrical activity of spinal motor neurons.

<p>A) Co-electroporation of <i>RGECO</i> and <i>Hb9-GFP</i> expression plasmids labels LMC motor neurons which can be seen in an open book preparation at HH St. 30. Single motor neurons were simultaneously patch-clamped and imaged, showing calcium transients that correspond to voltage cycles. Peaks in voltage recordings are mirrored in calcium traces. B) The cycle detection routine transforms raw calcium traces into their first differential (1°∂). When the 1°∂ is >2 s.d from the mean, an cycle is detected that lasts until the first time the 1°Δ reaches 0 after passing a local minimum. Signal to noise ratio (SNR) is also calculated. C) Comparison of voltage and calcium recordings shows a very significant correlation between the two. Critically, our cycle detection routine (B) detects the onset of calcium cycles that accurately reflect cycles in the voltage trace. D) Imaging neurons with intact axons shows that activity patterns are similar in intact preparations (grey lines) compared to open-book preparations (black lines).</p

mRNA Enrichment in Spinal Neuron Populations.

<p>This table describes the enrichment of mRNAs in spinal cord regions defined in the Results section at various stages.</p

Synchronized activity develops over time.

<p>A) Example of a field of view from an HH St. 28 embryo containing fluo-4 labelled motor neurons (red dotted regions, scale bar = 100 μM). B) Examples of calcium traces from single cells through development; cycles become less variable over time, and regularly fire in episodes of multiple cycles. C–I) Raster plots and correlograms of activity in populations of cells between HH St. 23 and HH St. 30. Black bars indicate uncorrelated cycles, in which <60% of the motor neurons in the field of view participate, red bars indicate correlated cycles, in which >60% of motor neurons participate. The firing pattern of neurons is initially apparently random, becoming more synchronous with time such that by HH St. 28, very few cycles occur in an asynchronous fashion. Similarity of activity patterns between two given neurons is represented by black-framed correlograms that increase in brightness over time, representing the increase correlation between cells. J) Mean correlation coefficient of neurons over time increases from HH St. 23 to HH St. 30, as the firing of individual neurons in the circuit becomes more synchronous.</p

Pharmacological interrogation of individual motor neuron activity.

<p>30-clamp recordings were made from HH St. 30 motor neurons identified by Hb9-EGFP, during which time agonists or antagonists were applied. A) Inhibition of nicotinic transmission with 5 μM D-Tubocurarine (DTC) completely abolished episodic activity (black arrowheads). The activity seen at the end of the treatment does not fulfil the criteria for a cycle. B) Muscarinic inhibition with 5 μM scopolamine (Scop.) resulted in changes in episode patterns, with an abolition of doublet episodes (black arrowheads) and an increased frequency of singlet cycles (white arrowheads). C) Nicotinic stimulation (2 μM) completely abolished the pattern of activity; irregular singlets were seen throughout the drug application, and many more small depolarisations also occurred. Activity did not return after cessation of nicotine application. D) Stimulation with acetylcholine (5 μM) increased the frequency of both singlet cycles, and short depolarisations. E) GABA-A inhibition with Picrotoxin (PTX, 5 μM) abolished episodic activity; irregular singlet cycles remained, interspersed with many short depolarisations that do not fit the criteria of cycles. F) GABA application (5 μM) increased the frequency of episodic activity without causing significant changes to episode structure. G) Application of 5 μM Serotonin increased the frequency of episodic activity, and reduced the number of cycles in episodes. H) The shape of voltage traces for a typical individual cycle in baseline conditions compared to a typical cycle in the presence of PTX demonstrate the disruption to cycles in the absence of GABA-A signaling.</p