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Research ArticleNew Research, Neuronal Excitability

Serotonergic Suppression of Mouse Prefrontal Circuits Implicated in Task Attention

Michael K. Tian, Eric F. Schmidt and Evelyn K. Lambe
eNeuro 27 October 2016, 3 (5) ENEURO.0269-16.2016; DOI: https://doi.org/10.1523/ENEURO.0269-16.2016
Michael K. Tian
1Department of Physiology, University of Toronto, Toronto, ON, Canada
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Eric F. Schmidt
2Laboratory of Molecular Biology, Rockefeller University, New York, NY
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Evelyn K. Lambe
1Department of Physiology, University of Toronto, Toronto, ON, Canada
3Department of Obstetrics and Gynecology, University of Toronto, Toronto, ON, Canada
4Department of Psychiatry, University of Toronto, Toronto, ON, Canada
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  • Figure 1.
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    Figure 1.

    5-HT inhibits L6 pyramidal neurons of medial prefrontal cortex. Responses to 5-HT were probed in voltage-clamp and current-clamp by bath application of 5-HT. Representative voltage-clamp traces of the 5-HT response in L6 pyramidal neurons show that responses to 5-HT are stable and persist in the presence of synaptic blockers (A), are significantly suppressed by WAY100635 (B), and are similarly suppressed by a combination of WAY100635 and synaptic blockers (C). D, 5-HT elicits strong outward currents on L6 pyramidal neurons of mPFC (n = 28) that can be pharmacologically modulated (F(2,53) = 11.8, p < 0.0001, one-way ANOVA). Post hoc analyses show that these currents persist in the presence of synaptic blockers (q = 0.2, p > 0.05, n = 7, Dunnett’s multiple comparison test) but are significantly suppressed by WAY100635 (q = 4.6, p < 0.0001, n = 19). Responses to 5-HT were probed in current-clamp in L6 pyramidal neurons of medial prefrontal cortex with current injection to elicit steady firing (∼2–3 Hz) at baseline. Representative current-clamp traces show that responses to 5-HT are inhibitory, repeatable, and unaffected by synaptic blockers (E) and not fully blocked by WAY100635 (F) or by WAY100635 and synaptic blockers (G). H, L6 pyramidal neurons of mPFC are strongly hyperpolarized by 5-HT (n = 26). Post hoc analyses of pharmacological effects on this hyperpolarization (F(2,50) = 17, p < 0.0001, one-way ANOVA) show that this inhibition is unaffected by synaptic blockers (q = 0.8, p > 0.5, n = 6, Dunnett’s multiple comparison test) but greatly reduced by WAY100635 (q = 5.4, p < 0.0001, n = 21). I, Action potential firing was significantly affected by 5-HT (F(4,103) = 37, p < 0.0001, one-way ANOVA). Post hoc analyses reveal that baseline firing was strongly suppressed by 5-HT (q = 9, p < 0.0001, n = 17, Dunnett’s multiple comparison test) and remained suppressed by 5-HT in synaptic blockers (q = 6.1, p < 0.0001, n = 6). The suppression was not blocked by WAY100635 (q = 8.8, p < 0.0001, n = 16) and returned to baseline levels after washout of 5-HT (q = 2.3, p > 0.05, n = 36).

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    Figure 2.

    Combined activation of serotonergic 5-HT1A and 5-HT2A receptors mediate inhibition of L6 neuronal excitability at suprathreshold potentials. Incremental current steps were injected into patched L6 pyramidal neurons, and their output in firing frequency was measured. Shown are representative recordings of the response to a 150-pA current step in single L6 pyramidal neurons (left) and the response to the same 150-pA current step in the presence of 5-HT (middle). The input–output relationship for each group is plotted (right). A, The input–output relationship of L6 pyramidal neurons is significantly right-shifted by 5-HT (F(1,168) = 31, p < 0.0001, repeated-measures two-way ANOVA). Post hoc analysis showed significantly fewer elicited action potentials at every input step (p < 0.05, Sidak’s multiple comparisons test). B, L6 excitability is significantly suppressed by 5-HT in the presence of WAY100635 (p < 0.0001, F(1,72) = 72, repeated-measures two-way ANOVA), an effect especially prominent at higher input steps (125- to 200-pA steps, p < 0.05, Sidak’s multiple comparisons test). C, L6 suppression by 5-HT is fully blocked by simultaneous blockade of both 5-HT1A and 5-HT2A receptors by specific antagonists WAY100635 and MDL100907 (F(1,32) = 0.8, p = 0.4, repeated-measures two-way ANOVA). D, TCB-2, a selective 5-HT2A receptor agonist, inhibits L6 neuronal firing (F(1,80) = 24, p < 0.0001, repeated-measures two-way ANOVA), also more prominently at higher input steps (125- to 200-pA steps, p < 0.05, Sidak’s multiple comparisons test).

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    Figure 3.

    Characterization of L6 neurons in medial prefrontal cortex (mPFC) expressing synaptotagmin 6 and epiphycan. A, Neurons expressing eGFP driven by the synaptotagmin-6 (Syt6) BAC promoter are localized to L6 pyramidal neurons in the prelimbic region of mPFC. B, EGFP is also seen in L6 pyramidal neurons in prelimbic mPFC by anti-EGFP immunohistochemistry in Epyc-Cre mice crossed to a Cre-dependent eGFP reporter. Image is adapted from www.gensat.org. C, Quantification (mean ± SEM) by qRT-PCR of the expression of selected genes in mPFC Epyc-vTRAP IP samples compared with whole PFC input. Positive values indicate enrichment in the IP, and negative values indicate depletion. Dotted lines indicate a twofold difference in either direction. Green bars, genes that are >2-fold enriched in the Epyc cells; red bars, genes that are >2-fold depleted; gray bars, genes expressed at levels similar to the rest of PFC. Of note, mRNAs for serotonin receptors Htr1a and Htr2a were expressed, but not enriched, in Epyc cells, a not-unexpected finding given the expression of 5-HT1A and 5-HT2A in other populations of neurons in mouse mPFC beyond L6. *p < 0.05; **p < 0.01 by Student’s t test.

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    Figure 4.

    Optogenetic activation of L6 pyramidal neurons of medial prefrontal cortex excites L5 interneurons. A, Schematic representation of light activation of L6 pyramidal neurons of medial prefrontal cortex in Epyc-ChR2 mice with axons projecting to L5 interneurons. B, Channelrhodopsin-expressing pyramidal L6 neurons were robustly excited by targeted light stimulation over L6. The effects of increasing L6 light power are shown for one example L6 pyramidal neuron. C, Light activation of L6 robustly excited L5 interneurons. The effects of increasing L6 light power are shown for three different L5 interneurons. D, Top, close-up of the initial light-evoked action potential in L6 to show the timing from onset of light (blue dotted line) to peak of the spike (black dotted line). Scale bar: 20 mV, 1 ms. Note: In L6 the onset of depolarization from light is <1 ms. Bottom, voltage-clamp recording showing the light-evoked postsynaptic response in a L5 interneuron to demonstrate response latency. Scale bar: 40 pA, 1 ms. Postsynaptic responses in L5 interneurons were initiated 1.1 ± 0.3 ms after initial spike of L6 pyramidal neurons, as indicated by an arrow.

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    Figure 5.

    Two distinct groups of interneurons are found in L5 and are activated by light stimulation of L6 pyramidal neurons. A, FS interneurons of L5 characterized by injection of current steps. B, Representative trace of an L5 FS interneuron activated by L6. Note the rapidly depressing response to L6 activation. C, Activation of FS interneurons by L6 elicited action potential firing primarily during the initial phase of activation that rapidly depressed over the duration of the stimulation (number of elicited action potentials in first half of stimulation vs. second half: t9 = 7.2, p < 0.0001, unpaired t test). D, nFS interneurons of L5 characterized by injection of current steps. E, Representative trace of a L5 nFS interneuron activated by L6, demonstrating a more regular firing pattern. F, L5 nFS interneurons were activated by L6 and fired in a regular pattern over the course of the stimulation (number of elicited action potentials in first half of stimulation vs. second half: t7 = 1.9, p = 0.1, unpaired t test).

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    Figure 6.

    L6 activation of L5 interneurons in medial prefrontal cortex is stable over time but suppressed by 5-HT. A, Excitatory effects on L5 interneurons by optogenetic activation of L6 were stable and repeatable over time. Shown here are the postsynaptic responses in a L5 interneuron to L6 light-stimulation repeated over 15 min. The number of spikes elicited initially and upon repetition in L5 interneurons is plotted on the bar graph at the right (mean ± SEM). There was no significant difference (t11 = 0.8, p = 0.4, paired t test), showing that the postsynaptic effect in L5 interneurons does not decrease over time under baseline conditions. B, L6 activation of L5 FS interneurons was significantly suppressed by 5-HT (t8 = 3.8, p = 0.005, paired t test, n = 9). Shown here are repeated recordings (baseline, 5-HT, washout) from three different L5 interneurons. The number of spikes elicited at baseline and in the presence of 5-HT in L5 interneurons are illustrated in the bar graph on the right (mean ± SEM). C, Antagonists of serotonergic 5-HT1A and 5-HT2A receptors blocked the inhibitory effects of 5-HT on L6 activation of L5 interneurons (t15 = 0.9, p = 0.4, paired t test, n = 16). Shown here is one representative L5 interneuron excited by L6 stimulation, which is suppressed by 5-HT applied alone, and no longer suppressed by 5-HT in the presence of WAY100635 and MDL100907. The results are plotted on the bar graph at the right (mean ± SEM).

Tables

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    Table 1.

    Intrinsic electrophysiological properties of three groups of neurons recorded: pyramidal neurons in L6, FS interneurons in L5, and nFS interneurons in L5.

    RMP (mV)Input resistance (MΩ)Spike amplitude (mV)Peak firing frequency (Hz)n
    Layer 6 pyramidal neurons–90.2 ± 0.6128.5 ± 3.983.1 ± 0.622.4 ± 0.8122
    Layer 5 FS interneurons–85.2 ± 1.2114.3 ± 9.859.5 ± 2.2109.8 ± 23.319
    Layer 5 nFS interneurons–82.7 ± 1.6235.9 ± 30.0*79.7 ± 1.9**34.1 ± 3.0**22
    • Neuronal properties shown are resting membrane potential (RMP), input resistance, spike amplitude, and peak firing frequency upon injection of a maximal suprathreshold current. Data are shown as mean ± SEM. Comparisons between L5 FS and nFS interneurons: *p < 0.05, **p < 0.001, unpaired t tests.

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    Table 2.

    TaqMan gene expression assay.

    SymbolGene nameAssayDye
    Aldh1l1Aldehyde dehydrogenase 1 family, member L1Mm03048957_m1FAM
    CnpCyclic nucleotide phosphodiesterase 1Mm01306640_m1FAM
    Foxp2Forkhead box P2Mm00475030_m1FAM
    Gad1Glutamic acid decarboxylase 1Mm00725661_s1FAM
    GapdhGlyceraldehyde-3-phosphate dehydrogenaseMm99999915_g1FAM
    Htr1a5-Hydroxytryptamine (serotonin) receptor 1AMm00434106_s1FAM
    Htr2a5-Hydroxytryptamine (serotonin) receptor 2AMm00555764_m1FAM
    Ntsr1Neurotensin receptor 1Mm00444459_m1FAM
    Slc17a7Vesicular glutamate transporter 1Mm00812886_m1FAM
    Syt6Synaptotagmin VIMm01308768_m1FAM
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Serotonergic Suppression of Mouse Prefrontal Circuits Implicated in Task Attention
Michael K. Tian, Eric F. Schmidt, Evelyn K. Lambe
eNeuro 27 October 2016, 3 (5) ENEURO.0269-16.2016; DOI: 10.1523/ENEURO.0269-16.2016

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Serotonergic Suppression of Mouse Prefrontal Circuits Implicated in Task Attention
Michael K. Tian, Eric F. Schmidt, Evelyn K. Lambe
eNeuro 27 October 2016, 3 (5) ENEURO.0269-16.2016; DOI: 10.1523/ENEURO.0269-16.2016
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Keywords

  • corticothalamic neurons
  • interneurons
  • medial prefrontal cortex
  • optogenetics
  • serotonin (5-HT)

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