Journal: Scientific Reports

Article Title: Neurotrophic factors and target-specific retrograde signaling interactions define the specificity of classical and neuropeptide cotransmitter release at identified Lymnaea synapses

doi: 10.1038/s41598-020-70322-5

Figure Lengend Snippet: Synaptic transmission via classical and peptide cotransmitters is target-specific. ( A ). Schematic of the Lymnaea CNS, depicting in situ locations of the neurons used in this study. Dorsal surface of the ganglia: L/R, left and right; Ce, cerebral; Pe, pedal; Pl, pleural; P, parietal; V, visceral. Adapted from . ( B ). Summary diagram of the neurons and synaptic connections that form the respiratory central pattern generator (rCPG) circuit, and the associated motor neurons that drive opening (expiration) and closing (inspiration) of the pneumostome. ( C ). Summary diagram of the cardioregulatory network, and the motor neurons implicated in modulating the rate and amplitude of auricular and ventricular contractions. ( D ). Simultaneous intracellular current clamp recordings of endogenous synapses from an isolated intact CNS preparation, performed in control conditions ( i ; Lymnaea normal saline), or with AChR antagonists ( ii ; 5 μM MLA, 10 μM TC, 20 μM TEA) to isolate peptidergic transmission. N = 11. Stimulation of VD4 elicits primarily cholinergic inhibition of RPeD1 (i ACh in i , antagonized in ii ) and primarily peptidergic excitation of VF (e FMRF in i and ii ). Lucifer yellow dye injections illustrate the morphology of identified cardiorespiratory neurons and their projections ( iii ). N ≥ 3 each. Scale bar, 1 mm. ( E ). Summary data, mean synaptic response of VF is unchanged by addition of AChR antagonists, indicating primarily peptidergic transmission (excitation of VF measured as number of action potentials induced in response to VD4 burst). P = 0.813 (Paired Samples T-test). ( F ). Summary data, mean synaptic response of RPeD1 is attenuated by AChR antagonists, indicating primarily cholinergic transmission (inhibition of RPeD1 measured as duration of hyperpolarization and firing cessation (s) induced in response to VD4 burst). ** P = 0.003 (Wilcoxon Signed Ranks test). Error bars, SEM. ( G ). Summary diagram illustrating that synaptic transmission at distinct postsynaptic targets is differentially mediated by classical and peptide neurotransmitters.

Article Snippet: Signals were amplified with a Neuro data dual channel intracellular recording amplifier (Neuron Data Instruments), digitized (Digidata 1440; Molecular Devices), and recorded with Axoscope 10.2 (Molecular Devices).

Techniques: Transmission Assay, In Situ, Isolation, Control, Saline, Inhibition

Journal: Scientific Reports

Article Title: Neurotrophic factors and target-specific retrograde signaling interactions define the specificity of classical and neuropeptide cotransmitter release at identified Lymnaea synapses

doi: 10.1038/s41598-020-70322-5

Figure Lengend Snippet: Target-specific use of cotransmitters is modulated by extrinsic neurotrophic factor signaling. ( A – B ). Characterization of cotransmission and isolated FMRF transmission by simultaneous intracellular current clamp recordings in soma-soma paired neurons (image inserts; scale bar, 50 μm), performed in CM or DM control ( Ai , iii and Bi , iii ), or with AChR antagonists ( Aii , iv and Bii , iv ; 5 μM MLA, 10 μM TC, 20 μM TEA) to isolate peptidergic transmission. Inserts show ACh-PSPs ( i , iii ; − 100 mV holding potential), inhibited by AChR antagonists ( ii , iv ). Mixed cholinergic-peptidergic cotransmission is observed at VD4-VF synapses in CM, with biphasic cholinergic and excitatory peptidergic responses ( Ai , ii ; e ACh , i ACh , e FMRF ), and at VD4-RPeD1 synapses in DM, with biphasic cholinergic and inhibitory peptidergic responses ( Biii , iv ; e ACh , i ACh , i FMRF ). Primarily cholinergic transmission is observed at VD4-RPeD1 synapses in CM ( Aiii , iv ; e ACh , i ACh , absence of i FMRF ), and VD4-VF synapses in DM ( Bi , ii ; i ACh , absence of e FMRF ), demonstrating that peptide cotransmitter specificity is determined by postsynaptic target identity and extrinsic NTF signaling. ( C ). Summary data, mean peptidergic excitation of VF (number of action potentials induced) is unchanged by AChR antagonists, and influenced by NTFs. N ≥ 9. ** P ≤ 0.002 (Independent Samples Kruskal–Wallis test). ( D ). Summary data, mean inhibition of RPeD1 (duration in s, reflects ACh + FMRF cotransmission or isolated FMRF transmission) is reduced by AChR antagonists in CM but not DM, indicating differential peptidergic inhibition influenced by NTFs. N ≥ 10. * P ≤ 0.010; *** P < 0.001 (Independent Samples Kruskal–Wallis test). ( E ). Summary data, mean amplitudes of ACh-PSPs are unchanged in CM or DM control. N ≥ 9. P = 0.490 (Independent Samples Kruskal–Wallis test). ( F ). Summary data, mean cholinergic excitation (number of action potentials induced), primarily biphasic cholinergic responses are observed in CM, and primarily inhibitory cholinergic responses are observed in DM. N ≥ 9. ** P = 0.001; *** P < 0.001 (Independent Samples Kruskal–Wallis test). Error bars, SEM.

Article Snippet: Signals were amplified with a Neuro data dual channel intracellular recording amplifier (Neuron Data Instruments), digitized (Digidata 1440; Molecular Devices), and recorded with Axoscope 10.2 (Molecular Devices).

Techniques: Isolation, Transmission Assay, Control, Inhibition

Journal: Scientific Reports

Article Title: Neurotrophic factors and target-specific retrograde signaling interactions define the specificity of classical and neuropeptide cotransmitter release at identified Lymnaea synapses

doi: 10.1038/s41598-020-70322-5

Figure Lengend Snippet: Target-specific use of classical and peptide cotransmitters is established by distinct mechanisms. ( A–B ). Characterization of cotransmission and isolated FMRF transmission by simultaneous intracellular current clamp recordings in neurons plated in a triple-soma configuration (image inserts; scale bar, 20 μm), performed in CM or DM control ( Ai and Bi ), or with AChR antagonists ( Aii and Bii ; 5 μM MLA, 10 μM TC, 20 μM TEA) to isolate peptidergic transmission. Inserts show ACh-PSPs ( i ; − 100 mV holding potential), inhibited by AChR antagonists ( ii ). Mixed cholinergic-peptidergic cotransmission is observed at VD4-VF synapses in CM, with inhibitory cholinergic and excitatory peptidergic responses ( Ai , ii ; i ACh , e FMRF ), and at VD4-RPeD1 synapses in DM, with inhibitory cholinergic and peptidergic responses ( Bi , ii ; i ACh , i FMRF ). Primarily cholinergic transmission is observed at VD4-RPeD1 synapses in CM ( Ai , ii ; e ACh , i ACh , absence of i FMRF ), and VD4-VF synapses in DM ( Bi , ii ; i ACh , absence of e FMRF ), ( C ). Summary data, mean peptidergic excitation of VF (number of action potentials induced) is unchanged by AChR antagonists, and influenced by NTFs. N ≥ 9. ** P ≤ 0.002 (Independent Samples Kruskal–Wallis test). ( D ). Summary data, mean inhibition of RPeD1 (duration in s, reflects ACh + FMRF cotransmission or isolated FMRF transmission) is reduced by AChR antagonists in CM but not DM, indicating differential peptidergic inhibition influenced by NTFs. N ≥ 9. ** P ≤ 0.009 (Independent Samples Kruskal–Wallis test). ( E ). Summary data, mean amplitudes of ACh-PSPs are unchanged in CM or DM control, but are larger at VD4-RPeD1 synapses than VD4-VF synapses, indicating that the use of classical and peptide cotransmitters in synaptic transmission are tuned by distinct synapse-specific mechanisms. N ≥ 9. * P ≤ 0.012 (Independent Samples Kruskal–Wallis test). ( F ). Summary data, mean cholinergic excitation (number of action potentials induced), primarily biphasic cholinergic responses are observed in CM, and primarily inhibitory cholinergic responses are observed in DM. N ≥ 9. * P = 0.023 (Independent Samples Kruskal–Wallis test). Error bars, SEM.

Article Snippet: Signals were amplified with a Neuro data dual channel intracellular recording amplifier (Neuron Data Instruments), digitized (Digidata 1440; Molecular Devices), and recorded with Axoscope 10.2 (Molecular Devices).

Techniques: Isolation, Transmission Assay, Control, Inhibition

Journal: Scientific Reports

Article Title: Neurotrophic factors and target-specific retrograde signaling interactions define the specificity of classical and neuropeptide cotransmitter release at identified Lymnaea synapses

doi: 10.1038/s41598-020-70322-5

Figure Lengend Snippet: Reciprocal inhibitory transmission between VD4-RPeD1 is attenuated by FMRF neuropeptide transmission from VD4. ( A ). Simultaneous intracellular current clamp recordings in soma-soma paired VD4-RPeD1 neurons. Stimulation of RPeD1 induces DA-mediated inhibition in VD4 when pairs are cultured in CM ( i ; i DA ), but not in DM ( ii ; absence of i DA ), indicating that NTF-dependent regulation of FMRF peptidergic transmission (see also Figs. , ) tunes the function of the reciprocal inhibitory synapse between VD4-RPeD1, which underlies respiratory central pattern generator (rCPG) activity and breathing behaviour in Lymnaea (see also Fig. ). ( B ). Summary data, correlation of the mean dopaminergic synaptic response at RPeD1-VD4 synapses (y axis; VD4 inhibition) with the mean peptidergic synaptic response at VD4-RPeD1 synapses measured in the presence of AChR antagonists (x axis; RPeD1 inhibition; see also Figs. , ). ** P = 0.001; R = − 0.534; R = 0.2849 (Pearson correlation).

Article Snippet: Signals were amplified with a Neuro data dual channel intracellular recording amplifier (Neuron Data Instruments), digitized (Digidata 1440; Molecular Devices), and recorded with Axoscope 10.2 (Molecular Devices).

Techniques: Transmission Assay, Inhibition, Cell Culture, Activity Assay

Journal: iScience

Article Title: Serotonergic neurons control cortical neuronal intracellular energy dynamics by modulating astrocyte-neuron lactate shuttle

doi: 10.1016/j.isci.2022.105830

Figure Lengend Snippet: Response of cortical neuronal intracellular ATP signals to optogenetic activation of raphe serotonergic neurons (A) Schematic illustration of the fiber photometric recording of intracellular ATP levels in cortical pyramidal neurons under photostimulation of serotonergic neurons in the raphe. CTX, cortex; DRN, dorsal raphe nucleus. (B) Representative traces of cortical neuronal intracellular ATP signals (Thy1-ATeam), EEG, and EMG signals with vigilance states of animals under serotonergic photostimulation. One-second blue light illumination for the opening of ChR2(C128S) in serotonergic neurons was followed by 1-s yellow light illumination for the closing of ChR2(C128S), with a 30-s interval between illuminations. Vertical blue and yellow lines indicate the 1-s illumination of each light color. (C) Traces of averaged Thy1-ATeam signals with state probabilities, EEG power density spectrum, and EMG activity under serotonergic photostimulation during wake, NREM sleep, and REM sleep states, respectively. Traces of Thy1-ATeam signals represent mean ± SEM (n = 5 sessions from 5 mice). (D) Area under the curve (AUC) of Thy1-ATeam signal responses to the optogenetic activation of serotonergic neurons. ∗p< 0.05 versus Control (Ctrl); two-sided Wilcoxon signed-rank test (n = 5 sessions from 5 mice). In the control condition, 1-s yellow light illumination was used instead of blue light. (E) Comparison of the AUC, peak value, and the peak time of Thy1-ATeam signal responses to serotonergic activation across the states for the data in (C). ∗p< 0.05 versus Wake; Friedman test with post hoc Steel-Dwass test (n = 5 sessions from 5 mice). (F) Comparison of fluctuations in Thy1-ATeam signals under serotonergic photostimulation-induced awakening and spontaneous awakening from NREM sleep (left) and REM sleep state (right), respectively (n = 5 sessions from 5 mice). (G) Comparison of the peak values and peak times of Thy1-ATeam signals under serotonergic photostimulation-induced and spontaneous awakening for the data in (F). ∗p< 0.05 versus Sponta; two-sided Wilcoxon signed-rank test (n = 5 sessions from 5 mice). (H) The effect of fluoxetine on the AUC of Thy1-ATeam signal responses to serotonergic photostimulation. ∗p< 0.05 versus Pre (before fluoxetine treatment); two-sided Wilcoxon signed-rank test (n = 5 sessions from 5 mice). (I) Effect of serotonin receptor subtype-selective antagonists on the AUC of Thy1-ATeam signal responses to serotonergic photostimulation. ∗p< 0.05 versus Pre (before each drug administration); two-sided Wilcoxon signed-rank test (n = 5 sessions from 5 mice). (J) The effect of selective noradrenergic lesions by DSP-4 (left) and muscarinic and nicotinic cholinergic receptor antagonists (scopolamine (Sco) and mecamylamine hydrochloride/methyllycaconitine citrate (M/M), respectively; right) on the AUC of Thy1-ATeam signal responses to serotonergic photostimulation. p < 0.05 versus Pre (before each drug administration); two-sided Wilcoxon signed-rank test (n = 5 sessions from 5 mice). Data are expressed as the mean ± SEM. See also Figures S1–S4 .

Article Snippet: Alteration of state-dependent cortical neuronal intracellular ATP signal dynamics by chemogenic inhibition of dorsal raphe serotonergic neurons (A) Schematic illustration of the fiber photometric recording of intracellular ATP levels in cortical pyramidal neurons in Sert-Cre::Thy1-ATeam mice injected with AAV-hSyn-DIO-hM4D(Gi)-mCherry into the dorsal raphe nucleus.

Techniques: Activation Assay, Activity Assay, Control, Comparison

Journal: iScience

Article Title: Serotonergic neurons control cortical neuronal intracellular energy dynamics by modulating astrocyte-neuron lactate shuttle

doi: 10.1016/j.isci.2022.105830

Figure Lengend Snippet: Lactate mediates the increase in intracellular ATP levels in cortical neurons by activating raphe serotonergic neurons (A) Representative trace of diminished cortical neuronal intracellular ATP (Thy1-ATeam) signal responses to serotonergic photostimulation by topical administration of 4-CIN in comparison with Pre (before drug administration) in the same mouse. The blue line above the data represents the timing of serotonergic photostimulation for 30 s. (B) Effect of 4-CIN on cortical neuronal intracellular ATP dynamics under serotonergic photostimulation is shown as averaged traces of Thy1-ATeam signals before (Pre) and after the administration (n = 5 sessions from 5 mice). Vertical blue and yellow lines indicate the 1-s illumination of each light color. (C) Alteration of AUC and peak value of Thy1-ATeam signal responses to serotonergic activation by 4-CIN administration. ∗p< 0.05 versus Pre (before drug administration); two-sided Wilcoxon signed-rank test (n = 5 sessions from 5 mice). (D) Representative trace of blunted Thy1-ATeam signal responses to serotonergic photostimulation by topical administration of DAB in comparison with Pre (before drug administration) in the same mouse. (E) Effect of DAB on neuronal intracellular ATP dynamics under serotonergic photostimulation is shown as averaged traces of Thy1-ATeam signals (n = 5 sessions from 5 mice). (F) Alteration of AUC and peak value of Thy1-ATeam signal responses to serotonergic activation by treatment with DAB for the data in (D and E). ∗p< 0.05 versus Pre; two-sided Wilcoxon signed-rank test (n = 5 sessions from 5 mice). (G) Representative trace of sharpened Thy1-ATeam signal responses to serotonergic photostimulation by topical administration of L-lactate in comparison with Pre (before drug administration) in the same mouse. (H) Effect of L-lactate on neuronal intracellular ATP dynamics under serotonergic photostimulation. is shown as averaged traces of Thy1-ATeam signals (n = 5 sessions from 5 mice). (I) Alteration of AUC and peak value of Thy1-ATeam signal responses to serotonergic activation by treatment with L-lactate for the data in (G and H). ∗p< 0.05 versus Pre; two-sided Wilcoxon signed-rank test (n = 5 sessions from 5 mice). Data are expressed as the mean ± SEM. See also Figure S4 .

Article Snippet: Alteration of state-dependent cortical neuronal intracellular ATP signal dynamics by chemogenic inhibition of dorsal raphe serotonergic neurons (A) Schematic illustration of the fiber photometric recording of intracellular ATP levels in cortical pyramidal neurons in Sert-Cre::Thy1-ATeam mice injected with AAV-hSyn-DIO-hM4D(Gi)-mCherry into the dorsal raphe nucleus.

Techniques: Comparison, Activation Assay

Journal: iScience

Article Title: Serotonergic neurons control cortical neuronal intracellular energy dynamics by modulating astrocyte-neuron lactate shuttle

doi: 10.1016/j.isci.2022.105830

Figure Lengend Snippet: Alteration of state-dependent cortical neuronal intracellular ATP signal dynamics by chemogenic inhibition of dorsal raphe serotonergic neurons (A) Schematic illustration of the fiber photometric recording of intracellular ATP levels in cortical pyramidal neurons in Sert-Cre::Thy1-ATeam mice injected with AAV-hSyn-DIO-hM4D(Gi)-mCherry into the dorsal raphe nucleus. CTX, cortex; DRN, dorsal raphe nucleus. (B) Representative traces of cortical neuronal intracellular ATP signals (Thy1-ATeam), EEG, and EMG signals with vigilance states of mice under saline or CNO administration. (C) Traces of averaged Thy1-ATeam signal in the cortex during the transition from NREM sleep to wake state under saline (control) or CNO administration (n = 6 sessions from 4 mice). (D) AUC (left), peak value (middle), and peak timing (right) of the Thy1-ATeam signal immediately after the transition from NREM sleep to wake state under saline or CNO administration. Two-sided Wilcoxon signed-rank test (n = 6 sessions from 4 mice). (E) Traces of averaged Thy1-ATeam signal during the transition from REM sleep to wake state under saline or CNO administration (n = 5 sessions from 3 mice). (F) AUC (left), peak value (middle), and peak timing (right) of the Thy1-ATeam signal immediately after the transition from REM sleep to wake state under saline or CNO administration. ∗p< 0.05 versus saline; two-sided Wilcoxon signed-rank test (n = 5 sessions from 3 mice). (G) Traces of averaged Thy1-ATeam signal during the transition from the quiet-awake to active-awake substate during the wake state under saline or CNO administration (n = 6 sessions from 4 mice). (H) AUC (left), peak value (middle), and peak timing (right) of the Thy1-ATeam signal immediately after the transition from the quiet-awake to active-awake substate under saline or CNO administration. Two-sided Wilcoxon signed-rank test (n = 6 sessions from 4 mice). (G) Traces of averaged Thy1-ATeam signal at the micro-awakening events during the NREM sleep under saline or CNO administration (n = 6 sessions from 4 mice). (J) AUC (left), peak value (middle), and peak timing (right) of the Thy1-ATeam signal at the micro-awakening events during the NREM sleep under saline or CNO administration. Two-sided Wilcoxon signed-rank test (n = 6 sessions from 4 mice). Bar graphs show mean ± SEM.

Article Snippet: Alteration of state-dependent cortical neuronal intracellular ATP signal dynamics by chemogenic inhibition of dorsal raphe serotonergic neurons (A) Schematic illustration of the fiber photometric recording of intracellular ATP levels in cortical pyramidal neurons in Sert-Cre::Thy1-ATeam mice injected with AAV-hSyn-DIO-hM4D(Gi)-mCherry into the dorsal raphe nucleus.

Techniques: Inhibition, Injection, Saline, Control

Journal: iScience

Article Title: Serotonergic neurons control cortical neuronal intracellular energy dynamics by modulating astrocyte-neuron lactate shuttle

doi: 10.1016/j.isci.2022.105830

Figure Lengend Snippet: Serotonergic control of ANLS and neuronal energy levels Serotonin increases astrocytic cAMP and Ca 2+ signals, triggering glycogenolysis for lactate production and its extracellular release. In turn, neurons increase intracellular ATP levels partly via lactate uptake from the extracellular space. CTX, cortex; DRN, dorsal raphe nucleus.

Article Snippet: Alteration of state-dependent cortical neuronal intracellular ATP signal dynamics by chemogenic inhibition of dorsal raphe serotonergic neurons (A) Schematic illustration of the fiber photometric recording of intracellular ATP levels in cortical pyramidal neurons in Sert-Cre::Thy1-ATeam mice injected with AAV-hSyn-DIO-hM4D(Gi)-mCherry into the dorsal raphe nucleus.

Techniques: Control