Respiratory motoneuron properties during the transition from gill to lung breathing in the American bullfrog

Amphibian respiratory development involves a dramatic metamorphic transition from gill to lung breathing and coordination of distinct motor outputs. To determine whether the emergence of adult respiratory motor patterns was associated with similarly dramatic changes in motoneuron (MN) properties, we characterized the intrinsic electrical properties of American bullfrog trigeminal MNs innervating respiratory muscles comprising the buccal pump. In premetamorphic tadpoles (TK stages IX-XVIII) and adult frogs, morphometric analyses and whole cell recordings were performed in trigeminal MNs identified by fluorescent retrograde labeling. Based on the amplitude of the depolarizing sag induced by hyperpolarizing voltage steps, two MN subtypes (I and II) were identified in tadpoles and adults. Compared with type II MNs, type I MNs had larger sag amplitudes (suggesting a larger hyperpolarization-activated inward current), greater input resistance, lower rheobase, hyperpolarized action potential threshold, steeper frequency-current relationships, and fast firing rates and received fewer excitatory postsynaptic currents. Postmetamorphosis, type I MNs exhibited similar sag, enhanced postinhibitory rebound, and increased action potential amplitude with a smaller-magnitude fast afterhyperpolarization. Compared with tadpoles, type II MNs from frogs received higher-frequency, larger-amplitude excitatory postsynaptic currents. Input resistance decreased and rheobase increased postmetamorphosis in all MNs, concurrent with increased soma area and hyperpolarized action potential threshold. We suggest that type I MNs are likely recruited in response to smaller, buccal-related synaptic inputs as well as larger lung-related inputs, whereas type II MNs are likely recruited in response to stronger synaptic inputs associated with larger buccal breaths, lung breaths, or nonrespiratory behaviors involving powerful muscle contractions

Menin: a tumor suppressor that mediates postsynaptic receptor expression and synaptogenesis between central neurons of Lymnaea stagnalis.

Neurotrophic factors (NTFs) support neuronal survival, differentiation, and even synaptic plasticity both during development and throughout the life of an organism. However, their precise roles in central synapse formation remain unknown. Previously, we demonstrated that excitatory synapse formation in Lymnaea stagnalis requires a source of extrinsic NTFs and receptor tyrosine kinase (RTK) activation. Here we show that NTFs such as Lymnaea epidermal growth factor (L-EGF) act through RTKs to trigger a specific subset of intracellular signalling events in the postsynaptic neuron, which lead to the activation of the tumor suppressor menin, encoded by Lymnaea MEN1 (L-MEN1) and the expression of excitatory nicotinic acetylcholine receptors (nAChRs). We provide direct evidence that the activation of the MAPK/ERK cascade is required for the expression of nAChRs, and subsequent synapse formation between pairs of neurons in vitro. Furthermore, we show that L-menin activation is sufficient for the expression of postsynaptic excitatory nAChRs and subsequent synapse formation in media devoid of NTFs. By extending our findings in situ, we reveal the necessity of EGFRs in mediating synapse formation between a single transplanted neuron and its intact presynaptic partner. Moreover, deficits in excitatory synapse formation following EGFR knock-down can be rescued by injecting synthetic L-MEN1 mRNA in the intact central nervous system. Taken together, this study provides the first direct evidence that NTFs functioning via RTKs activate the MEN1 gene, which appears sufficient to regulate synapse formation between central neurons. Our study also offers a novel developmental role for menin beyond tumour suppression in adult humans

Etonogestrel Administration Reduces the Expression of PHOX2B and Its Target Genes in the Solitary Tract Nucleus

Heterozygous mutations of the transcription factor PHOX2B are responsible for Congenital Central Hypoventilation Syndrome, a neurological disorder characterized by inadequate respiratory response to hypercapnia and life-threatening hypoventilation during sleep. Although no cure is currently available, it was suggested that a potent progestin drug provides partial recovery of chemoreflex response. Previous in vitro data show a direct molecular link between progestins and PHOX2B expression. However, the mechanism through which these drugs ameliorate breathing in vivo remains unknown. Here, we investigated the effects of chronic administration of the potent progestin drug Etonogestrel (ETO) on respiratory function and transcriptional activity in adult female rats. We assessed respiratory function with whole-body plethysmography and measured genomic changes in brain regions important for respiratory control. Our results show that ETO reduced metabolic activity, leading to an enhanced chemoreflex response and concurrent increased breathing cycle variability at rest. Furthermore, ETO-treated brains showed reduced mRNA and protein expression of PHOX2B and its target genes selectively in the dorsal vagal complex, while other areas were unaffected. Histological analysis suggests that changes occurred in the solitary tract nucleus (NTS). Thus, we propose that the NTS, rich in both progesterone receptors and PHOX2B, is a good candidate for ETO-induced respiratory modulation

Calcium influx through L-type VGCCs and MAPK/ERK are required for the expression of excitatory nAChRs.

<p>(<b>a</b>) Single LPeD1 neurons were cultured in CM and impaled with intracellular electrodes. Exogenous application of ACh (10⁻⁶ M) triggered an excitatory response in the postsynaptic neuron, LPeD1 (n = 30). (<b>ai</b>) Exogenous application of ACh to LPeD1 somata induced an inhibitory response in the presence the L-type VGCC blocker nifedipine (n = 9) (10 µM). (<b>aii</b>) The expression of excitatory nAChRs was also prevented by the MEK1/2 inhibitor, U0126 (40 µM) n = 9. (<b>b</b>) Summary of the percentage of cells that functionally exhibited excitatory nAChRs with a significance of <i>P</i><0.05, as determined using Pearson's Chi-squared test.</p

Menin expression is upregulated by neurotrophic factors, and following microinjections of synthetic <i>L-MEN1</i> mRNA.

<p>Summary of the relative gene expression of <i>L-MEN1</i>, quantified via qPCR, with a significance of <i>P</i>(H1)<0.001 as determined using the REST randomization test (n = 3 for all conditions).</p

Synthetic <i>L-MEN1</i> rescues deficits in synapse formation induced by L-EGFR knockdown <i>in situ</i>.

<p>(<b>a</b>) Intact VD4 and LPeD1 neurons were simultaneously impaled with intracellular electrodes to confirm whether or not a synapse was present. Trains of action potentials triggered in VD4 induced a corresponding excitatory response in LPeD1 (n = 7). (<b>ai</b>) Control, LPeD1 transplants re-formed excitatory, cholinergic synapses with intact, host VD4 neurons (n = 17). (<b>aii</b>) LPeD1 neurons pre-treated with L-AChBP dsRNA (negative control; 200 ng/ml) prior to transplantation established excitatory synapses with host VD4 neurons (n = 9). (<b>aiii</b>) Incubation of LPeD1 neurons with L-EGFR dsRNA (200 ng/ml) prior to transplantation prevented excitatory synapse formation with host VD4 neurons (n = 17). (<b>b</b>) Whole CRG were pre-treated with L-EGFR dsRNA prior to isolation of the LPeD1 transplants. 4–6 hours later, LPeD1 was injected with <i>L-MEN1</i> mRNA. 2–3 hours following injection, the LPeD1 neurons were isolated and transplanted into host CRG, where they re-formed excitatory synapses with VD4 (n = 13). (<b>c–d</b>) Summary of the percentage of pairs that formed an excitatory synapse with a significance of <i>P</i><0.01, as determined using Pearson's Chi-squared test. (<b>e</b>) Summary of mean EPSP amplitude (mV) with a significance of <i>P</i><0.05, as determined using a univariate ANOVA with Tukey's <i>post hoc</i> test. Error bars represent standard error of the mean (SEM).</p

RT-PCR Gene Specific Primers (GSPs).

<p>RT-PCR Gene Specific Primers (GSPs).</p

Lucifer yellow images illustrating the single cell transplantation procedure.

<p>Arrows point to the somata of intact or transplanted neurons. Scale bars represent 100 µm. (<b>a</b>) The presynaptic neuron, VD4, is located in the visceral ganglia, and is characterized by two axons that span the entire central ring ganglia (CRG; 4× magnification). (<b>b</b>) The postsynaptic neuron, LPeD1, is located in the pedal ganglia, and has one axon which extends downwards toward the visceral ganglia, where it makes a synaptic connection with VD4 as well as many other neurons. In addition, LPeD1 projects axonal branches through peripheral nerves to innervate various organs (marked by asterisks; 4× magnification). (<b>c</b>) The native LPeD1 neuron is first ablated in the host ganglia via pronase injection (5% solution in <i>Lymnaea</i> saline) and is completely fragmented within 1 hr (10× magnification). (<b>d</b>) An LPeD1 neuron from a donor animal is then transplanted into the original location of the ablated LPeD1 in the host CRG, where it exhibits significant regeneration within 12 hours (4× magnification). (<b>di</b>) Regeneration 24 hours post-transplantation (10× magnification). Inset shows magnified details of neurite outgrowth extending down through the left parietal and pleural ganglia. (<b>e</b>) Schematic of the <i>Lymnaea</i> CRG, indicating the relative size and position of LPeD1 (1) and VD4 (4).</p

L-EGF is sufficient to induce the functional expression of excitatory nAChRs.

<p>(<b>a</b>) Single LPeD1 neurons were cultured in CM and impaled with intracellular electrodes. Exogenous application of ACh (10⁻⁶ M) triggered an excitatory response in the postsynaptic neuron, LPeD1 (n = 33). (<b>ai</b>) The EGFR inhibitor PD153035 (200 nM) prevented the CM-induced expression of excitatory nAChRs (n = 16). (<b>b</b>) Single LPeD1 neurons cultured in the absence of trophic factors (DM) exhibited an inappropriate (does not exist <i>in vivo</i>), inhibitory response to exogenous ACh application by ‘default’ (n = 9). (<b>bi</b>) L-EGF (400 ng/ml) was sufficient to rescue the expression of excitatory nAChRs in LPeD1 neurons cultured in DM (n = 5). (<b>c</b>) Experimental preparation. Phase contrast image of a single LPeD1 neuron impaled by a sharp intracellular electrode at 10× magnification. Another electrode with a tip diameter of 1–5 µm was used to pressure apply ACh onto the cell body of LPeD1, at a distance that was sufficient not to cause a mechanical stimulation artifact. (<b>d</b>) Summary of the percentage of cells that functionally exhibited excitatory nAChRs with a significance of <i>P</i><0.001, as determined using Pearson's Chi-squared test.</p