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| <?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN" "JATS-archivearticle1.dtd"><article article-type="research-article" dtd-version="1.1d1" xmlns:xlink="http://www.w3.org/1999/xlink"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">02726</article-id><article-id pub-id-type="doi">10.7554/eLife.02726</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Short report</subject></subj-group><subj-group subj-group-type="heading"><subject>Neuroscience</subject></subj-group></article-categories><title-group><article-title>Phasic activation of ventral tegmental neurons increases response and pattern similarity in prefrontal cortex neurons</article-title></title-group><contrib-group><contrib contrib-type="author" corresp="yes" id="author-12031"><name><surname>Iwashita</surname><given-names>Motoko</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/><aff id="aff1"><institution content-type="dept">National Institute of Mental Health</institution>, <institution>National Institutes of Health</institution>, <addr-line><named-content content-type="city">Bethesda</named-content></addr-line>, <country>United States</country></aff></contrib></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Häusser</surname><given-names>Michael</given-names></name><role>Reviewing editor</role><aff><institution>University College London</institution>, <country>United Kingdom</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>moko0927@gmail.com</email></corresp></author-notes><pub-date date-type="pub" publication-format="electronic"><day>30</day><month>09</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e02726</elocation-id><history><date date-type="received"><day>06</day><month>03</month><year>2014</year></date><date date-type="accepted"><day>28</day><month>09</month><year>2014</year></date></history><permissions><license xlink:href="http://creativecommons.org/publicdomain/zero/1.0/"><license-p>This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/publicdomain/zero/1.0/">Creative Commons CC0 public domain dedication</ext-link>.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife02726.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.02726.001</object-id><p>Dopamine is critical for higher neural processes and modifying the activity of the prefrontal cortex (PFC). However, the mechanism of dopamine contribution to the modification of neural representation is unclear. Using in vivo two-photon population Ca<sup>2+</sup> imaging in awake mice, this study investigated how neural representation of visual input to PFC neurons is regulated by dopamine. Phasic stimulation of dopaminergic neurons in the ventral tegmental area (VTA) evoked prolonged Ca<sup>2+</sup> transients, lasting ∼30 s in layer 2/3 neurons of the PFC, which are regulated by a dopamine D1 receptor-dependent pathway. Furthermore, only a conditioning protocol with visual sensory input applied 0.5 s before the VTA dopaminergic input could evoke enhanced Ca<sup>2+</sup> transients and increased pattern similarity (or establish a neural representation) of PFC neurons to the same sensory input. By increasing both the level of neuronal response and pattern similarity, dopaminergic input may establish robust and reliable cortical representation.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02726.001">http://dx.doi.org/10.7554/eLife.02726.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.02726.002</object-id><title>eLife digest</title><p>Around 120 years ago, Ivan Pavlov unintentionally sparked a new field of psychology research. He did so by noting that his dogs had learned to associate the sound of the bell that he rang before feeding them with the food itself, such that they would salivate upon hearing the bell even when there was no food present. This form of learning—now known as associative learning—has since been demonstrated in species from honeybees to humans.</p><p>For the brain to associate two events, such as the sound of a bell and the delivery of food, it must encode the first event and keep that information available or ‘on-line’ until the occurrence of the second event, at which point the two can be linked together. This process takes place in part of the brain called the prefrontal cortex, but the mechanism by which it occurs is largely unclear.</p><p>Now, Iwashita has obtained new insights into the molecular basis of associative learning by studying how the activity of the prefrontal cortex is affected by the activity of a second region of the brain. This second region, called the ventral tegmental area, is part of the brain's reward circuit: it becomes active whenever an animal experiences a desirable event, such as receiving food, and supplies a neurotransmitter called dopamine to its target areas, which include the prefrontal cortex.</p><p>Electrodes were used to mimic the changes in brain activity that occur when a mouse learns to associate a visual stimulus with a reward: this involved repeatedly activating the visual cortex in a conscious mouse, followed by activation of the ventral tegmental area. Short-lived increases in calcium levels were seen in the prefrontal cortex, raising the possibility that these ‘calcium transients’ are the signal that enables the brain to link two events. Moreover, blocking proteins called dopamine D1 receptors in the prefrontal cortex reduced the calcium transients, which is consistent with existing evidence that dopamine from the ventral tegmental area is required for associative learning.</p><p>Intriguingly, the calcium transients lasted for roughly 30 s, which is also the maximum length of time by which a stimulus and a reward can be separated and still be associated. Given that the calcium transients could not be detected in anesthetized mice, a full understanding of the mechanisms underlying associative learning may require studies of the conscious brain.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02726.002">http://dx.doi.org/10.7554/eLife.02726.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>dopamine</kwd><kwd>prefrontal cortex</kwd><kwd>neuronal representation</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>mouse</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000025</institution-id><institution>National Institute of Mental Health</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Iwashita</surname><given-names>Motoko</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100001691</institution-id><institution>Japan Society for the Promotion of Science</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Iwashita</surname><given-names>Motoko</given-names></name></principal-award-recipient></award-group><funding-statement>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>Transient increases in calcium release in prefrontal cortex neurons, observable only in awake animals, may form the basis of associative learning.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>The prefrontal cortex (PFC) plays an important role in adaptive behavior such as associative learning (<xref ref-type="bibr" rid="bib5">Duncan, 2001</xref>). Dopaminergic input from the ventral tegmental area (VTA) is crucial for PFC function (<xref ref-type="bibr" rid="bib21">Schultz, 2007</xref>). In primates, sensory cues, which are used in associative learning tasks, create specific temporal activity patterns in PFC neurons and a neural representation of the sensory cue (<xref ref-type="bibr" rid="bib13">Jacob et al., 2013</xref>). However, how dopamine contributes to form neural representations at the neuronal network level is largely unknown. One potential mechanism is that dopaminergic (DA) neurons target dopamine over a number of inhibitory and excitatory neurons via their widespread axonal arborizations (<xref ref-type="bibr" rid="bib15">Matsuda et al., 2009</xref>). Thus, investigating modification of neuronal activity at the population level will reveal the role of dopamine signaling in the formation of neural representation. Therefore, utilizing in vivo two-photon Ca<sup>2+</sup> imaging in awake mice, this study investigated how neural representation in PFC neurons is developed under regulation by dopamine, in response to visual sensory input.</p></sec><sec id="s2" sec-type="results|discussion"><title>Results and discussion</title><p>PFC neuronal activity was recorded through a cranial window at the secondary motor cortex (M2). The M2 is categorized as the dorsomedial PFC in rodents in some publications, based on both its anatomical features, including thalamocortical and cortical–basal ganglia connections, and its functional role in motor decision (<xref ref-type="bibr" rid="bib26">Sul et al., 2011</xref>; <xref ref-type="bibr" rid="bib29">Uylings et al., 2003</xref>; <xref ref-type="bibr" rid="bib11">Hoover and Vertes, 2007</xref>). The M2 receives inputs from both the VTA and the secondary visual cortex lateral area (V2L) (<xref ref-type="fig" rid="fig1">Figure 1A,B</xref>). Microelectrodes were implanted in these two brain areas to supply electrical stimulation (<xref ref-type="fig" rid="fig1">Figure 1C,D</xref>).<fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.02726.003</object-id><label>Figure 1.</label><caption><title>Ca<sup>2+</sup> imaging setup and calcium transients in response to VTA stimulation with and without anesthesia.</title><p>(<bold>A</bold>) Retrograde tracing. Fluorogold was injected into the prefrontal cortex (PFC). Cells in the secondary visual cortex lateral area (V2L) were labeled. Scale bar, 250 μm. (<bold>B</bold>) Anterograde tracing. FITC-dextran was injected into the V2L. Labeled fibers were observed in the PFC. Scale bar, 100 μm. (<bold>C</bold>) Experimental setup for calcium imaging. A cranial window was opened over M2 (PFC). Two electrodes were implanted in the ventral tegmental area (VTA) and the V2L. (<bold>D</bold>) Two-photon image of calcium indicator (OGB-1)-labeled layer 2/3 cells in M2 (neurons, green; sulforhodamine-101 counterstained astrocytes, red to orange). Approximately 50–80 cells were analyzed in each animal. Scale bar, 30 μm. (<bold>E</bold>) The Ca<sup>2+</sup> transients evoked in an awake animal and in an animal under anesthesia. Population average of Ca<sup>2+</sup> transients (dF/F) in response to 10 pulses at 50 Hz VTA stimulation (n = 4 animals). VTA electrical stimulation was applied at the 30 s time point (red arrowhead). In contrast to a clear response in awake mice (left), the long-lasting Ca<sup>2+</sup> transients were not detected in mice under anesthesia (right, 4% isoflurane). The same animals were used for the ‘Awake’ and ‘Under anesthesia’ experiments. (<bold>F</bold>) The effect of isoflurane on Ca<sup>2+</sup> transients. The summed values of Ca<sup>2+</sup> transients from the 30 to 50 s time points were compared with the ‘Awake’ value (changing ratio). Paired t test, *p < 0.05. Error bars represent SEM.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02726.003">http://dx.doi.org/10.7554/eLife.02726.003</ext-link></p></caption><graphic xlink:href="elife02726f001"/></fig></p><p>To determine the single input response, the PFC neuronal response to VTA stimulation was first measured by recording Ca<sup>2+</sup> transients. Most DA neurons are spontaneously active with firing patterns that range from regular tonic firing (1–5 Hz) to phasic firing (40–50 Hz) (<xref ref-type="bibr" rid="bib19">Robinson et al., 2004</xref>; <xref ref-type="bibr" rid="bib31">Grace and Bunney, 1984a</xref>; <xref ref-type="bibr" rid="bib32">Grace and Bunney, 1984b</xref>). In addition, salient events such as experiencing a novel environment or receiving a reward-associated signal, evoke phasic firing in the VTA (<xref ref-type="bibr" rid="bib21">Schultz, 2007</xref>; <xref ref-type="bibr" rid="bib12">Horvitz, 2000</xref>). The PFC activity evoked by VTA stimulation in these physiological ranges was therefore examined.</p><p>When low frequency VTA microstimulation was used (1 Hz–10 Hz, tonic range), Ca<sup>2+</sup> transients in the PFC neurons were evoked and decayed within 5 s. This steep increase and short decay is comparable with the reported response to sensory input observed in cortical neurons of anesthetized animals (<xref ref-type="bibr" rid="bib18">Ohki et al., 2005</xref>). In contrast, high frequency VTA stimulation (40–50 Hz, phasic range) evoked substantially elongated Ca<sup>2+</sup> transients that lasted 20–30 s and then returned to the original baseline (<xref ref-type="fig" rid="fig2">Figure 2</xref>). In addition, these Ca<sup>2+</sup> transients of the PFC neurons reached a peak relatively slowly, 6–7 s after stimulation. Furthermore, high frequency VTA stimulation robustly evoked Ca<sup>2+</sup> transients in all tested animals, whereas low frequency VTA stimulation did not as only about half of the animals tested showed detectable Ca<sup>2+</sup> transients in the PFC. Importantly, these long-lasting Ca<sup>2+</sup> transients were only detected in awake and not in anesthetized mice (<xref ref-type="fig" rid="fig1">Figure 1E,F</xref>), possibly because of the potential inhibition of voltage-gated calcium channels by isoflurane (<xref ref-type="bibr" rid="bib9">Herring et al., 2009</xref>; <xref ref-type="bibr" rid="bib25">Study, 1994</xref>). This result highlights the advantage of a system using awake animals to elucidate dopamine regulation of neuronal responses in the PFC.<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.02726.004</object-id><label>Figure 2.</label><caption><title>Ca<sup>2+</sup> transients in response to ventral tegmental area (VTA) stimulation.</title><p>(<bold>A</bold>) The left panel shows the population average of Ca<sup>2+</sup> transients across the cells of a single animal. The right panel shows the Ca<sup>2+</sup> transients of each single neuron using a color map according to its dF/F value. A train of 10 pulses was applied at each frequency of VTA stimulation (1, 5, 10, 20, 30, 40, and 50 Hz). (<bold>B</bold>) Population average of Ca<sup>2+</sup> transients across eight animals. VTA electrical stimulation was applied at the 30 s time point (red arrowhead). Error bars represent SEM.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02726.004">http://dx.doi.org/10.7554/eLife.02726.004</ext-link></p></caption><graphic xlink:href="elife02726f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02726.005</object-id><label>Figure 2—figure supplement 1.</label><caption><title>Ca<sup>2+</sup> transients in response to secondary visual cortex lateral area (V2L) stimulation.</title><p>(<bold>A</bold>) The left panel shows the population average of Ca<sup>2+</sup> transients across the cells of a single animal. The right panel shows the Ca<sup>2+</sup> transients of each single neuron indicated in a color map according to its dF/F value. A train of 10 pulses was applied at each frequency of V2L stimulation (1, 5, 10, 20, 30, 40, and 50 Hz). (<bold>B</bold>) Population average of Ca<sup>2+</sup> transients across eight animals. V2L electrical stimulation was applied at a 30 s time point (red arrowhead). Each stimulation frequency evoked a Ca<sup>2+</sup> response with a short decay time of 5 s. Error bars represent SEM.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02726.005">http://dx.doi.org/10.7554/eLife.02726.005</ext-link></p></caption><graphic xlink:href="elife02726fs001"/></fig></fig-group></p><p>To test whether long-lasing Ca<sup>2+</sup> transients can be evoked by other neural inputs, the same microstimulation protocol used in the VTA (1–50 Hz) was applied to a region of the visual cortex, the V2L. Both stimulation frequencies only evoked short Ca<sup>2+</sup> transients in PFC neurons (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>). Input from the VTA, especially through high frequency, phasic stimulation can therefore induce a specific signal required to evoke long-lasting Ca<sup>2+</sup> transients in PFC neurons. Phasic stimulation of the VTA is known to facilitate DA release from its terminals (<xref ref-type="bibr" rid="bib28">Tsai et al., 2009</xref>; <xref ref-type="bibr" rid="bib14">Lavin et al., 2005</xref>), therefore, it was hypothesized that DA receptors are involved in induction of the long-lasting Ca<sup>2+</sup> transients. DA receptors are members of the G-protein coupled receptor family and are composed of two groups: D1 and D2, which have opposing effects on intracellular signaling (<xref ref-type="bibr" rid="bib22">Seamans and Yang, 2004</xref>; <xref ref-type="bibr" rid="bib23">Soltani et al., 2013</xref>). Selective DA receptor antagonists for each family were administered by intraperitoneal injection (i.p.). Treatment with the D1 antagonist SCH23390 (1 mg/kg) reduced the long-lasting Ca<sup>2+</sup> transients by 50% and the decay time constant by 60%. In contrast, no detectable attenuation was observed in mice treated with the D2 antagonist eticlopride (0.5 mg/kg) (<xref ref-type="fig" rid="fig3">Figure 3A,B</xref>). In addition, the short Ca<sup>2+</sup> response evoked by a 5 Hz VTA stimulation was not affected by the D1 or D2 antagonist (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1A,B</xref>). However, this short Ca<sup>2+</sup> response was reduced by NMDA and AMPA antagonists (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1C,D</xref>). In the VTA, up to 65% of the neurons are dopaminergic and the others are GABAergic or glutamatergic (<xref ref-type="bibr" rid="bib16">Nair-Roberts et al., 2008</xref>; <xref ref-type="bibr" rid="bib6">Gorelova et al., 2012</xref>), and some DA neurons co-release glutamate (<xref ref-type="bibr" rid="bib10">Hnasko et al., 2010</xref>). However, glutamatergic terminals are dominant in the mesocortical pathway (<xref ref-type="bibr" rid="bib6">Gorelova et al., 2012</xref>). Inhibition of the short Ca<sup>2+</sup> response by glutamate receptor antagonists suggests that glutamatergic neurons contribute to the 5 Hz responses. Initial experiments implicated the D1 receptors for the long-lasting Ca<sup>2+</sup> transient. To exclude a potential role of glutamatergic neurons in initiating the long calcium transients, a cocktail of NMDA and AMPA receptor antagonists was used. However, the long-lasting Ca<sup>2+</sup> transients were not affected by NMDA and AMPA antagonists (<xref ref-type="fig" rid="fig3">Figure 3C,D</xref>). This result further suggests that the long-sustained increase in intracellular Ca<sup>2+</sup> concentration is not mediated by glutamatergic local recurrent networks. Over all, pharmacological experiments clearly suggest D1 receptors are mainly involved in the long-lasting Ca<sup>2+</sup> response in PFC neurons.<fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.02726.006</object-id><label>Figure 3.</label><caption><title>Long-lasting Ca<sup>2+</sup> transients depend on D1 receptors.</title><p>(<bold>A</bold> and <bold>B</bold>) The effect of the D1 antagonist SCH23390 (1 mg/kg) (upper panel), the D2 antagonist eticlopride (0.5 mg/kg) (middle panel), and H<sub>2</sub>O, used as a control (bottom panel), on the long-lasting Ca<sup>2+</sup> transients evoked by 10 pulses at 50 Hz stimulation of the ventral tegmental area (VTA) (n = 6 animals in each experimental group). (<bold>C</bold> and <bold>D</bold>) The effect of a cocktail of NMDA and AMPA antagonists (CPP: 3 mg/kg, and CNQX: 10 mg/kg, i.p.) on long-lasting Ca<sup>2+</sup> transients (n = 4 animals). (<bold>A</bold> and <bold>C</bold>) Population average of Ca<sup>2+</sup> transients across animals in each group. VTA electrical stimulation was applied at the 30 s time point (red arrowhead). (<bold>B</bold> and <bold>D</bold>) The changing ratio of the summed values of Ca<sup>2+</sup> transients between the 30 and 50 s time points compared with the ‘Before’ value (left panels), the peak of dF/F (middle panels), and the decay time constant (right panels). Paired t test with Holm's adjustment, *p < 0.05. Error bars represent SEM.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02726.006">http://dx.doi.org/10.7554/eLife.02726.006</ext-link></p></caption><graphic xlink:href="elife02726f003"/></fig><fig id="fig3s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02726.007</object-id><label>Figure 3—figure supplement 1.</label><caption><title>Short Ca<sup>2+</sup> transients do not depend on D1 or D2 receptors but on glutamate receptors.</title><p>(<bold>A</bold> and <bold>B</bold>) The effect of D1 antagonist SCH23390 (1 mg/kg, upper panel), D2 antagonist eticlopride (0.5 mg/kg, middle panel) and H<sub>2</sub>O, used as a control (bottom panel), on the short Ca<sup>2+</sup> transients evoked by 10 pulses at 5 Hz for ventral tegmental area (VTA) stimulation (n = 6 animals in each experimental group). (<bold>C</bold> and <bold>D</bold>) The effect of a cocktail of NMDA and AMPA antagonists (CPP: 3 mg/kg, and CNQX: 10 mg/kg, i.p.) on short Ca<sup>2+</sup> transients (n = 4 animals). (<bold>A</bold> and <bold>C</bold>) Population average of Ca<sup>2+</sup> transients across animals in each group. VTA electrical stimulation was applied at a 30 s time point (red arrowhead). (<bold>B</bold> and <bold>D</bold>) To evaluate the effect of the drugs, the summed values of Ca<sup>2+</sup> transients from the 30 to 35 s time points were compared with the ‘Before’ value (changing ratio). Paired t test with Holm's adjustment, *p < 0.05. Error bars represent SEM.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02726.007">http://dx.doi.org/10.7554/eLife.02726.007</ext-link></p></caption><graphic xlink:href="elife02726fs002"/></fig></fig-group></p><p>To resolve the potential role of dopamine-induced, long-lasting Ca<sup>2+</sup> responses for PFC circuitry modification, the PFC neuronal response to sensory input from the V2L after combined repetitive stimulation of the V2L and VTA was investigated. An experimental paradigm composed of a pre-conditioning phase, conditioning phase, and test phase was used (<xref ref-type="fig" rid="fig4">Figure 4A</xref>). In the pre-conditioning phase, V2L stimulations (20 Hz, five pulses) were given three times to acquire the base response of PFC neurons to V2L inputs. In the conditioning phase, the VTA and V2L were simultaneously stimulated every minute for 30 min. After this conditioning phase, repetitive V2L stimulation (20 Hz, five pulses) was applied at three time points: right after, 1 hr after, and 2 hr after completion of the conditioning phase (test phase, <xref ref-type="fig" rid="fig4">Figure 4A</xref>), to determine whether or not the responses of the PFC neurons were modified.<fig-group><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.02726.008</object-id><label>Figure 4.</label><caption><title>Combined repetitive stimulation of the secondary visual cortex lateral area (V2L) and the ventral tegmental area (VTA) causes a modification of prefrontal cortex (PFC) neuronal response.</title><p>(<bold>A</bold>) Experimental design for stimulation of the V2L and VTA composed of three phases: pre-conditioning, conditioning, and test. Five pulses at 5 Hz and 10–15 pulses at 50 Hz were used for V2L and VTA electrical stimulation, respectively. T1: Timing1; T2: Timing2. (<bold>B</bold>) Shift in population average of dF/F value. Post hoc tests revealed that dF/F values were significantly different in many of the comparisons between the time points in each conditioning paradigm (Ryan's test). (<bold>C</bold>) Changing dF/F value calculated by subtracting the ‘Before’ value from the ‘2 hr after’ value (dF/F<sub>(2 hr-before)</sub>) to simplify the results of (<bold>B</bold>). The T1 conditioning paradigm showed a significantly larger temporal change than the others. (<bold>D</bold>) Difference in pattern similarity. Pattern similarity calculated from cosine similarity revealed that only T1 conditioning significantly increased the value of ‘2 hr after’ when compared with the ‘Before’ value. n = 8 animals in each experimental group. Ryan's post hoc test: *p < 0.05, **p < 0.01, *p < 0.001. Error bars represent SEM.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02726.008">http://dx.doi.org/10.7554/eLife.02726.008</ext-link></p></caption><graphic xlink:href="elife02726f004"/></fig><fig id="fig4s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02726.009</object-id><label>Figure 4—figure supplement 1.</label><caption><title>Percentage distributions of neurons by dF/F in each conditioning group and reliability of calcium transients occurrence following three repetitive secondary visual cortex lateral area (V2L) stimulations.</title><p>(<bold>A</bold>) Percentage distributions of neurons, pooled from eight animals in each conditioning group, are shown. The number of neurons showing a dF/F value above 0.2 increased 2 hr after stimulation (right panel) compared with ‘Before’ (left panel), especially in T1 at ‘2 hr after’. Note, the dF/F probability distribution in T1 conditioning at ‘1 hr after’ (far right) is comparable to the ventral tegmental area (VTA) only and T2 at ‘2 hr after’. (<bold>B</bold>) The reliability of calcium transients occurrence across three repetitive V2L stimulations was calculated by Cronbach's alpha. Error bars represent 95% confidence intervals. None of the conditioning group, including T1, showed a significant shift in the value of Cronbach's alpha, suggesting no reliability shift in either group. T1: Timing1; T2: Timing2.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02726.009">http://dx.doi.org/10.7554/eLife.02726.009</ext-link></p></caption><graphic xlink:href="elife02726fs003"/></fig><fig id="fig4s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02726.010</object-id><label>Figure 4—figure supplement 2.</label><caption><title>No correlation between Ca<sup>2+</sup> influx evoked by ventral tegmental area (VTA) stimulation and neuronal activity increase in response to secondary visual cortex lateral area (V2L) stimulation.</title><p>Scatter plots of the Ca<sup>2+</sup> transients evoked by VTA phasic stimulation from the 30 to 50 s time points against the increased value of dF/F, calculated by subtracting the dF/F value of ‘Before’ from ‘2 hr after’ conditioning (dF/F<sub>(2 hr−Before)</sub>) of Timing1 conditioning. Each plot represents one animal and each dot represents a different single neuron of the animal. There is no significant correlation between the amount of Ca<sup>2+</sup> transients evoked by VTA stimulation and the increased value of dF/F. This indicates that the higher amount of intracellular Ca<sup>2+</sup> is not the main factor modifying the response to input from the sensory cortex.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02726.010">http://dx.doi.org/10.7554/eLife.02726.010</ext-link></p></caption><graphic xlink:href="elife02726fs004"/></fig></fig-group></p><p>For the conditioning phase, the effect of timing on V2L and VTA stimulation was also tested by using two different time intervals, Timing1 (T1) and Timing2 (T2) (<xref ref-type="fig" rid="fig4">Figure 4A</xref>). In T1, V2L stimulation was applied half a second before VTA phasic stimulation. T1 simultaneously simulates a visual experience and VTA activation. For T2, V2L stimulation was applied 45 s after VTA phasic stimulation, which is 15 s before the next VTA stimulation, when no Ca<sup>2+</sup> transients were observed (<xref ref-type="fig" rid="fig4">Figure 4A</xref>). Therefore, T2 is used as a control, at a point when visual and VTA inputs are temporally separated. As additional controls, the V2L and the VTA were stimulated separately during the conditioning phase.</p><p>Two-way repeated measures ANOVA with temporal change (before, right after, 1 hr after, and 2 hr after conditioning) and conditioning paradigm (V2L only, VTA only, Timing1, and Timing2) as factors, revealed that population averages of dF/F values of Ca<sup>2+</sup> transients signal showed significant differences in temporal change (<italic>F</italic>(3, 84) = 48.486, p < 0.0001) and the conditioning paradigm × temporal change interaction (<italic>F</italic>(9,84) = 3.173, p = 0.0024) (<xref ref-type="fig" rid="fig4">Figure 4B</xref>). T1 conditioning significantly increased the dF/F value more than other conditioning paradigms, indicating that T1 conditioning has the largest modification effect on the PFC response to a sensory input (one-way ANOVA: <italic>F</italic>(3,28) = 6.504, p = 0.0018; post hoc Ryan's test: p < 0.01; <xref ref-type="fig" rid="fig4">Figure 4C</xref>). This increase in value is due to an increased number of neurons showing high dF/F values (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1A</xref>). In addition, there was no significant correlation between the Ca<sup>2+</sup> level in response to VTA stimulation (at conditioning) and the increased Ca<sup>2+</sup> level in response to V2L stimulation ‘2 hr after’ T1 conditioning (test phase) (<xref ref-type="fig" rid="fig4s2">Figure 4—figure supplement 2</xref>), suggesting that the increased neuronal response with V2L stimulation is independent of how well the neuron responds to the DA input. This relatively unexpected conclusion may be explained by the fact that the network dynamics of the PFC microcircuit are composed of inhibitory and excitatory neurons, both of which express DA receptors.</p><p>Finally, the neuronal population dynamics were investigated through analysis of the pattern similarity (cosine similarity, see the ‘Materials and methods’ section) of neuronal activity in the PFC across three repetitions of V2L stimuli, conducted at four time points: ‘before’, ‘right after’, ‘1 hr after’, and ‘2 hr after’ after the conditioning phase. Two-way repeated measures ANOVA on the temporal changes of the mean cosine similarities in each conditioning paradigm revealed significant differences in the temporal factor (<italic>F</italic>(3, 84) = 7.676, p = 0.0001) and the time × condition paradigm interaction (<italic>F</italic>(9, 84) = 2.483, p = 0.0145; <xref ref-type="fig" rid="fig4">Figure 4D</xref>). Post hoc tests also showed that the cosine similarity in T1 was significantly increased in ‘2 hr after’ compared with ‘before’ (Ryan's test, p < 0.01; <xref ref-type="fig" rid="fig4">Figure 4D</xref>). This increase in cosine similarity in T1 conditioning is not simply due to an increased number of high dF/F neurons (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1A</xref>), because ‘1 hr after’ in T1 conditioning had a significantly increased cosine similarity (<xref ref-type="fig" rid="fig4">Figure 4D</xref>) but its population probability distribution was not significantly different from that of VTA only (2 hr after) or T2 (2 hr after) (two-sample Kolmogorov–Smirnov test, p = 0.5128 and p = 0.1418, respectively) (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1A</xref>). This suggests that the increase in cosine similarity in T1 conditioning was not due to an increased number of high dF/F neurons. In addition, the increased cosine similarity was not due to increased reliability of Ca<sup>2+</sup> transients occurrence in response to three repetitive V2L stimulations, because the reliability calculated by Cronbach's alpha did not change between ‘before’ and ‘2 hr after’ in any of the conditioning groups, including T1 (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1B</xref>). These results indicate that only T1 conditioning improved the pattern similarity in PFC neurons, and the increase in pattern similarity could be the result of circuit (network) modification, and not simply the result of increased reliability of Ca<sup>2+</sup> transients occurrence or an increased number of high responding dF/F neurons.</p><p>This study revealed that only coincident visual sensory input with long-lasting Ca<sup>2+</sup> transients increased neuronal activity and induced robust repeatable neuronal responses, at the population level, to the same sensory input. By increasing both dF/F and pattern similarity, DA input may enhance PFC activity and establish cortical representation (<xref ref-type="bibr" rid="bib30">Xue et al., 2010</xref>). In fact, it is known that DA release plays a major role in dynamic cortical remodeling in the auditory cortex (<xref ref-type="bibr" rid="bib1">Bao et al., 2001</xref>), suggesting that the phenomenon observed here in the T1 conditioning paradigm may represent the neurophysiological basis for dynamic neural events. Besides network modification in PFC, it has also been demonstrated that PFC neurons show long-lasting Ca<sup>2+</sup> transients that depend on the D1 receptor pathway. This newly identified physiological phenomenon might have an important function in Ca<sup>2+</sup> regulation of neuronal processes (<xref ref-type="bibr" rid="bib3">Berridge, 1998</xref>; <xref ref-type="bibr" rid="bib4">Brenowitz et al., 2006</xref>; <xref ref-type="bibr" rid="bib20">Roussel et al., 2006</xref>; <xref ref-type="bibr" rid="bib2">Bardo et al., 2006</xref>). For example, association learning is only successful if the cue signal is less than 20–30 s before the reward signal. Long-lasting Ca<sup>2+</sup> transients may be tightly associated with this time window in association learning. Taken together, the long-lasting Ca<sup>2+</sup> transients reported here could be a key phenomenon required to explain the dynamics of the dopaminergic neural network and its role in PFC cognitive functions.</p></sec><sec id="s3" sec-type="materials|methods"><title>Materials and methods</title><sec id="s3-1"><title>Neuronal tracing</title><p>Neuronal retrograde tracer Fluorogold (4%; Fluorochrome, Denver, CO) or FITC-conjugated dextran (Life Technologies, Carlsbad, CA) were injected into the M2 or V2L, respectively, and 48 hr later, mice were fixed with 4% formalin in phosphate buffer. Brains were sliced using a vibratome and Fluorogold or FITC was identified by using fluorescence microscopy.</p></sec><sec id="s3-2"><title>Animal preparation</title><p>All procedures were conducted according to the animal welfare guidelines of the NIH and approved by the NIH Animal Care and Use Committee.</p><p>C57BL/6 mice ranging in age from 2 to 4 months were used. Throughout all procedures, body temperature was maintained at 37°C using a heating pad. Anesthesia was induced with Avertin (2.2.2-tribromoethanol; Sigma-Aldrich, St. Louis, MO), and mice were placed in a stereotaxic device.</p><p>Stainless bipolar stimulating electrodes were implanted in the V2L (2.5 mm lateral and 2.5 mm posterior from bregma, 0.5 mm below the cortical surface) and VTA (0.5 mm lateral and 3.1 mm posterior from bregma, 4.5 mm below the cortical surface).</p><p>A 1.5 mm craniotomy (with the dura carefully removed) was opened over M2 (0.5 mm lateral and 1.0 mm anterior from bregma).</p><p>Multi-cell bolus loading of neocortical cells with the calcium indicator Oregon-BAPTA Green 1-AM (OGB-1-AM; Life Technologies) and astrocyte marker sulforhodamine 101 (SR101; Life Technologies) was performed as previously described (<xref ref-type="bibr" rid="bib24">Stosiek et al., 2003</xref>; <xref ref-type="bibr" rid="bib17">Nimmerjahn et al., 2004</xref>). This multi-cell bolus loading was performed in superficial layer 2/3 (L2/3). The craniotomy was then covered with silicone (Kwik-Sil Adhesive; World Precision Instruments, Sarasota, FL) and sealed with a glass coverslip. A metal bar was glued directly on the skull with dental acrylic for future attachment to an imaging frame. About ∼2 hr after surgery, when the mouse had completely recovered from the anesthesia, cortical activity, measured by evoked Ca<sup>2+</sup> transients, was imaged in the awake, head-fixed mouse.</p></sec><sec id="s3-3"><title>Two-photon imaging</title><p>The mouse was placed under the microscope. Cortical activity was imaged using a two-photon microscope (Olympus Fluoview; Olympus, Japan) equipped with a 25 × (1.05 NA) water-immersion objective (Olympus). Excitation wavelength was 870 nm (Mai-Tai oscillator; Spectra-Physics, Santa Clara, CA). Images (256 × 256 pixels) were acquired at a frame rate of 2.3 Hz. Low quality images (when cells showed unclear boarders) were not used for analysis.</p><p>Imaging started at time 0, however high background signals (∼0.03 dF/F, lasting 2–3 s) were present at time 0 due to mechanical noise. Therefore, figures show the Ca<sup>2+</sup> transients starting from 10 s to demonstrate the neuronal activity that occurs in response to electrical stimulation (<xref ref-type="fig" rid="fig1 fig2 fig3">Figures 1–3</xref>, <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref> and <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>).</p></sec><sec id="s3-4"><title>Drug administration</title><p>SCH23390 (1 mg/kg), eticlopride (0.5 mg /kg), CPP (3 mg/kg), and CNQX (10 mg/kg) (Sigma–Aldrich) were administered by i.p. injection. The injected volume was adjusted to 1% of the animal's body weight.</p></sec><sec id="s3-5"><title>Microstimulation protocols</title><p>For VTA stimulation, a biphasic pulse of 1 ms duration was used in all experiments. To evoke the long-lasting Ca<sup>2+</sup> transients, 50–400 μA and 10–15 pulses were applied. The experimental protocol shown in <xref ref-type="fig" rid="fig1 fig2 fig3">Figures 1–3</xref> used 400 μA and 10 pulses. VTA stimulation with 50–400 μA and 10–15 pulses was used for the conditioning experiment (<xref ref-type="fig" rid="fig4">Figure 4</xref>). For the V2L, a biphasic pulse of 1 ms and 250 μs duration was used in the experiments shown in <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref> and <xref ref-type="fig" rid="fig4">Figure 4</xref>, respectively. Stimulation with 350 or 400 μA was used in <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>. To evoke dF/F values below 0.2, in response to V2L stimulation at ‘before’, current was adjusted (50–300 μA) in each animal (experiments shown in <xref ref-type="fig" rid="fig4">Figure 4</xref>).</p></sec><sec id="s3-6"><title>Data analysis</title><p>Data were analyzed with custom-written programs in MATLAB (Mathworks, Natick, MA) and ImageJ (NIH, Bethesda, MD). To remove motion artifacts from in vivo calcium imaging, the ImageJ plugin TurboReg for image alignment (<xref ref-type="bibr" rid="bib27">Thévenaz et al., 1998</xref>) was used. Individual cells were semi-automatically detected (<xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1</xref>). SR101-stained astrocytes were excluded from data analysis. For each cell, fluorescence change was defined as dF/F = (F1 − F0)/F0, where F1 is fluorescence at any time point, and F0 is the baseline fluorescence, defined as the median of fluorescence values measured within 40 s before and after the time point of F1 (for responses to VTA stimulation), or within 2.5 s before and after the time point of F1 (for responses to V2L stimulation). To calculate the decay time constant (tau) of Ca<sup>2+</sup> transients (<xref ref-type="fig" rid="fig3">Figure 3B,D</xref>), the following equation was used for fitting the time course of dF/F during the decay period: dF/F(t) = dF/F<sub>max</sub> × e<sup>−t/tau</sup>, where dF/F<sub>max</sub> is the peak dF/F value and t is elapsed time after dF/F peaks. To calculate the population average of dF/F shown in <xref ref-type="fig" rid="fig4">Figure 4</xref>, the averages across cells from the three V2L stimulations were averaged. Pattern similarity in <xref ref-type="fig" rid="fig4">Figure 4D</xref> was measured using cosine similarity (cosine of the angle between two vectors) in each pair of vectors, which are composed of the N-dimension of dF/F values of each neuronal response, where N is the total number of neurons. For the three repetitions of V2L stimulation at each time point, three cosine similarities were measured between the first and second, second and third, and first and third V2L stimulations, and then these three cosine similarities were averaged to show the pattern similarity of the time points. In <xref ref-type="fig" rid="fig4">Figure 4B</xref>, the result of post hoc tests revealed that dF/F values were significantly different in many of the comparisons between the time points in each conditioning paradigm (Ryan's test, p < 0.05). In <xref ref-type="fig" rid="fig4">Figure 4C</xref>, to simplify, the values of dF/F at 2 hr after conditioning were compared, and standardized by subtracting the ‘before’ values from ‘2 hr after’ among the different conditioning groups (dF/F<sub>(2 hr−before)</sub>).</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>I thank K. Wang for providing the research environment required to perform these experiments and for helpful discussions, G. Seabold, E. Tytell, and M. Yoshizawa for critically reading the manuscript, G. Ashida for helpful discussions, and K. Nakao and R. Renden for additional scientific comments on the manuscript.</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>The author declares that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>MI, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn></fn-group><fn-group content-type="ethics-information"><title>Ethics</title><fn fn-type="other"><p>Animal experimentation: All procedures were conducted according to the animal welfare guidelines of the NIH and approved by the NIH Animal Care and Use Committee (#GCP-01-07).</p></fn></fn-group></sec><sec sec-type="supplementary-material"><title>Additional files</title><supplementary-material id="SD1-data"><object-id 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pub-id-type="doi">10.7554/eLife.02726.012</article-id><title-group><article-title>Decision letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Häusser</surname><given-names>Michael</given-names></name><role>Reviewing editor</role><aff><institution>University College London</institution>, <country>United Kingdom</country></aff></contrib></contrib-group></front-stub><body><boxed-text><p>eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled “Phasic activation of VTA neurons increases response and pattern similarity in PFC neurons” for consideration at <italic>eLife.</italic> Your article has been favorably evaluated by Senior editor, a Reviewing editor, and 2 reviewers.</p><p>The following individuals responsible for the peer review of your submission have agreed to reveal their identity: Michael Hausser (Reviewing editor) and Jeremy Seamans (one of two peer reviewers).</p><p>The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.</p><p>In this manuscript, the author has investigated the contribution of dopamine to the modification of neuronal representations in the PFC (M2) by using in vivo two-photon Ca<sup>2+</sup> imaging techniques in awake mice. The author reported long-lasting (∼30sec) calcium transients in the PFC evoked by high-frequency electrical stimulation of the VTA, which depends on D1 receptors. In addition, the author showed that simultaneous activation of the VTA and V2L can cause long-term (∼2h) increases of calcium transients in the PFC to V2L inputs. This study is interesting and novel both in its approach and findings, and should make a significant contribution to the field. Several issues need to be addressed before publication.</p><p>Major comments:</p><p>1) <xref ref-type="fig" rid="fig4">Figure 4D</xref>: Analysis of pattern similarity differences. Before analyzing the pattern similarity of neuronal population activity, the author needs to check whether the increase of the population averages of dF/F values from 'Before' to '2h' with T1 conditioning (<xref ref-type="fig" rid="fig4">Figure 4A&B</xref>) is due to the increase of the numbers of the neurons activated by V2L stimulation, or due to the increase of the activity level of the neurons by V2L stimulation without changing the numbers of activated neurons. If the numbers of activated neurons by V2L stimulation was drastically changed, cosine similarity might not be a fair method for comparing the similarity of activity patterns across different conditions.</p><p>2) “Importantly, these long-lasting Ca<sup>2+</sup> transients were only detected in awake mice, but not in anesthetized ones (<xref ref-type="fig" rid="fig1">Figure 1E</xref>)...” This is a very interesting result, but also puzzling. If the mechanism of long-lasting calcium transients is due to D1 as the author showed, this effect should be observed also in the anesthetized state. It would be helpful if the author could speculate about the reason why the long-lasting calcium transients are state-dependent. Please also confirm whether the results in left ('Awake') and right ('under anesthesia') panels of <xref ref-type="fig" rid="fig1">Figure 1E</xref> were obtained from the same mice or the different ones.</p><p>3) There are serious reservations about the IP3 data. First, because the effect is so strong compared to the other manipulations and second because (by necessity) 2-APB was applied so differently from the other drugs. It was the only drug applied to the cortical surface (versus i.p.). Plus it was analyzed at the whole field level. Given these differences, either the data should be excluded from the manuscript, or more experiments should be done to confirm these conclusions.</p><p>4) In <xref ref-type="fig" rid="fig1">Figure 1E</xref> the author tries to make the point that the effect is seen in awake and not anesthetized mice. This needs to be quantified by group statistics.</p><p>5) <xref ref-type="fig" rid="fig3">Figure 3</xref> describes the effects of DA antagonists on the Ca transients. The author needs to do a more thorough job of characterizing what is changing (peak height, decay, duration etc.) and what is not. Same for glutamate and IP3 mediated effects.</p><p>6) In <xref ref-type="fig" rid="fig4">Figure 4D</xref>, observed increase in cosine similarity could simply be result from increased reliability of occurrence of calcium transients, not from increased population pattern similarity. It would be beneficial to analyse reliability of calcium transients in each experimental epochs.</p><p>7) The author states that 50-80 cells were analyzed per animal. Were these treated independently and the same way as cells from other animals? More importantly, what is the variance in the effect across the population?</p><p>8) “VTA neurons are mainly dopaminergic”: this description sounds incorrect. Dopaminergic neurons are up to 65% in VTA, and the rest are GABAergic and glutamatergic (Cohen JY et al Nature 2012; <xref ref-type="bibr" rid="bib6">Gorelova et al., Cereb Cortex 2012</xref>; <xref ref-type="bibr" rid="bib16">Nair-Roberts RG et al., Neuroscience 2008</xref>; etc.). The strong glutamatergic nature of the pathway is perfectly consistent with the effects of glutamate blockers on the transient responses (<xref ref-type="fig" rid="fig3s1">Figure 3–figure supplement 1C</xref>). The author should discuss the effects of non-dopaminergic afferents to M2 on the results reported in this paper.</p><p>9) Some portion of M2 pyramidal neurons have projections to the VTA (Watabe-Uchida M et al., Neuron. 2012). Has the author considered or ruled out the possibility of antidromic stimulation of M2 when using VTA electric stimulation?</p><p>10) In <xref ref-type="fig" rid="fig3">Figure 3A</xref> was water really used as a control?</p><p>[Editors' note: further revisions were requested prior to acceptance, as described below.]</p><p>Thank you for resubmitting your work entitled ”Phasic activation of ventral tegmental neurons increases response and pattern similarity in prefrontal neurons.” for further consideration at <italic>eLife.</italic> Your revised article has been favorably evaluated by a Senior editor, a Reviewing editor, and the original two reviewers. The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:</p><p>From the Reviewing editor:</p><p>The title should state “prefrontal cortex neurons" rather than just ”prefrontal neurons".</p><p>The Abstract needs some rewording: e.g. “By establishing a system of in vivo two-photon Ca<sup>2+</sup> imaging in awake mice” change to ”Using in vivo two-photon population Ca<sup>2+</sup> imaging in awake mice”; “Phasic stimulation of dopaminergic neurons in the ventral tegmental area (VTA) evoked long-lasting (∼30 sec) calcium transients in PFC” - in PFC neurons (specify layer or cell type if possible); "Pharmacological analysis revealed that this long-lasting calcium transients are regulated by dopamine D1 receptor-dependent pathway." Please clarify: the long-lasting enhancement of the transients, or the transients themselves?; “Furthermore, only visual sensory input applied 0.5 second before the VTA dopaminergic input could evoke higher Ca<sup>2+</sup> transients”; what does 'higher' mean?).</p><p><xref ref-type="fig" rid="fig1">Figure 1E</xref>: Ensure that the contrast to the line indicating the average is high enough. Clean up the axis labelling: why start from 10 s and not 0 s. The SI unit is “s” and not “sec”. A 0-line is nice, but doesn't replace an x-axis.</p><p><xref ref-type="fig" rid="fig1">Figure 1F</xref>: Drop the grid lines; the significance line should end centered on the data bars.</p><p><italic>Reviewer #1:</italic></p><p>The author has done a good job with the revisions. I would still suggest the following:</p><p>The text reads: ”In VTA, up to 65% of the neurons are dopaminergic and the others are GABAergic or glutamatergic (19)(20), and some DA neurons co-release glutamate (21)novel,, suggesting that glutamatergic/DA neurons might also contribute to the 5Hz responses.” Two points here. First, overall this is true for the VTA but the mesocortical projection which is what is being studied, is mainly glutamatergic not DAergic (See papers by Morales or Gorelova). Second, the results actually show that glutamate and not DA contributes the 5Hz response. Therefore, I cannot understand the equivocation when a firm conclusion can be drawn.</p><p>In the text and Figure 3–figure supplement 2: Once again I do not agree with including this in the paper. Not only for the reasons I mentioned in the last round but also because of the simple fact that just because IP3 and DA manipulations both decrease the Ca response, it in no way means they are related. To imply this given the existing data is not wise in my opinion.</p><p>In terms of H20 control I'm surprised that a hypo-osmotic solution didn't affect the cells and the Ca signal?</p><p>The document should be proofread to check that tenses are correct etc.</p><p><italic>Reviewer #2:</italic></p><p>1) Abstract. The sentence “Furthermore, ....” is a bit confusing, because “only visual sensory input applied 0.5 second before the VTA dopaminergic input ” is the protocol during the conditioning, and “evoke higher Ca<sup>2+</sup> transients and increase pattern similarity” is the effect 2 hours after the conditioning, but this timeline (or the experimental causality) is not clearly described.</p><p>2) <xref ref-type="fig" rid="fig1">Figure 1A</xref>. It would be helpful if the author can provide the picture or sketch of the injection site of the tracer as an insertion panel.</p><p>3) <xref ref-type="fig" rid="fig1">Figure 1E</xref>. “Population average of Ca<sup>2+</sup> transients (dF/F) in response to a 50-Hz-VTA”. Please provide the information of the duration or numbers of the pulses of the electrical stimulation, in the figure legend or main text. The author describes it in the Method section, but it is better to have the information earlier in the manuscript.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.02726.013</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>1) <xref ref-type="fig" rid="fig4">Figure 4D</xref>: Analysis of pattern similarity differences. Before analyzing the pattern similarity of neuronal population activity, the author needs to check whether the increase of the population averages of dF/F values from 'Before' to '2h' with T1 conditioning (<xref ref-type="fig" rid="fig4">Figure 4A&B</xref>) is due to the increase of the numbers of the neurons activated by V2L stimulation, or due to the increase of the activity level of the neurons by V2L stimulation without changing the numbers of activated neurons. If the numbers of activated neurons by V2L stimulation was drastically changed, cosine similarity might not be a fair method for comparing the similarity of activity patterns across different conditions</italic>.</p><p>Thank you for your suggestion. To address this comment, I have compared the percentage distributions of neurons pooled from all animals in the same group (V2L only, VTA only, T1 and T2; <xref ref-type="fig" rid="fig4s1">Figure4–figure supplement1A</xref>). The distribution shifted to a higher dF/F after 2h later in all groups (<xref ref-type="fig" rid="fig4s1">Figure4–figure supplement 1A</xref>; comparing ‘before’ and ‘2h’) and the largest shift was observed in T1 compared to other conditioning groups. To test whether the higher cosine similarity observed in T1 was only due to the increased number of high responsive cells, I focused on the data from T1 (1h after), VTA only (2h after) and T2 (2h after). Among these results, only T1 (1h after) had a significantly increased cosine similarity (<xref ref-type="fig" rid="fig4">Figure 4D</xref>), but its population probability distribution was not significantly different from that of VTA only (2h after) and T2 (2h after) (Two sample Kolmogorov-Smirnov test p=0.5128 and p=0.1418, respectively) (<xref ref-type="fig" rid="fig4s1">Figure 4–figure supplement 1A</xref>), suggesting that the increased cosine similarity in T1 is not only due to an increased number of high responsive cells. I also describe these points in more detail in the Results section.</p><p><italic>2) “Importantly, these long-lasting Ca</italic><sup><italic>2+</italic></sup> <italic>transients were only detected in awake mice, but not in anesthetized ones (<xref ref-type="fig" rid="fig1">Figure 1E</xref>)...” This is a very interesting result, but also puzzling. If the mechanism of long-lasting calcium transients is due to D1 as the author showed, this effect should be observed also in the anesthetized state. It would be helpful if the author could speculate about the reason why the long-lasting calcium transients are state-dependent. Please also confirm whether the results in left ('Awake') and right ('under anesthesia') panels of <xref ref-type="fig" rid="fig1">Figure 1E</xref> were obtained from the same mice or the different ones</italic>.</p><p>Isoflurane inhibits multiple voltage-dependent calcium channels (<xref ref-type="bibr" rid="bib25">Study 1994</xref>)(<xref ref-type="bibr" rid="bib9">Herring et al. 2009</xref>). Because I used a high dosage of isoflurane (4%), there is a possibility that the calcium current might be blocked. I described this point in the main text.</p><p>Thank you for the question in the last sentence. The same mice were used and I clarified this information in the figure legend for <xref ref-type="fig" rid="fig1">Figure 1E</xref>.</p><p><italic>3) There are serious reservations about the IP3 data. First, because the effect is so strong compared to the other manipulations and second because (by necessity) 2-APB was applied so differently from the other drugs. It was the only drug applied to the cortical surface (versus i.p.). Plus it was analyzed at the whole field level. Given these differences, either the data should be excluded from the manuscript, or more experiments should be done to confirm these conclusions.</italic></p><p>As the reviewers mentioned above, the drug administration procedure for 2-APB was different from the other drugs. Initially I tried i.p. injection with the concentration used for experiments, and ended up focusing on other organs rather than the brain, because I could not detect an inhibitory effect of 2-APB in Ca<sup>2+</sup> response in the brain. This suggests the drug does not cross the blood-brain barrier. As stated in the manuscript, the analysis was done according to whole field recordings, not at the single cell level, causing an inconsistency in experimental procedure. However, I would like to make the argument that it is important to consider a mechanism for the long-lasting calcium transients, and this experiment provides a potential source for the change seen with them. To address the reviewers’ concerns, I have moved the figure to the supplemental section and I described the procedure for this experiment more clearly in the main text of the Results and Material and methods sections.</p><p><italic>4) In <xref ref-type="fig" rid="fig1">Figure 1E</xref> the author tries to make the point that the effect is seen in awake and not anesthetized mice. This needs to be quantified by group statistics.</italic></p><p>I have added the statistical analysis recommended by the reviewers (<xref ref-type="fig" rid="fig1">Figure 1F</xref>).</p><p><italic>5) <xref ref-type="fig" rid="fig3">Figure 3</xref> describes the effects of DA antagonists on the Ca transients. The author needs to do a more thorough job of characterizing what is changing (peak height, decay, duration etc.) and what is not. Same for glutamate and IP3 mediated effects.</italic></p><p>Thank you for the above suggestions. I have added a more detailed description for the peak dF/F value and the decay (<xref ref-type="fig" rid="fig3">Figure 3B,D</xref>).</p><p>To further characterize the decay, the decay time constant (tau) was calculated by fitting the time course of dF/F during the decaying period using the following equation:</p><p>dF/F(t) = dF/F<sub>max</sub> * e <sup>-t/tau</sup>, where dF/F<sub>max</sub> is the peak dF/F value and t is the elapsed time after dF/F reached its peak. This is described in the Materials and methods section.</p><p><italic>6) In <xref ref-type="fig" rid="fig4">Figure 4D</xref>, observed increase in cosine similarity could simply be result from increased reliability of occurrence of calcium transients, not from increased population pattern similarity. It would be beneficial to analyse reliability of calcium transients in each experimental epochs.</italic></p><p>In response to the reviewers’ comment, I analyzed the reliability of the calcium transient occurrence across three repetitive V2L stimulations by calculating Cronbach’s alpha (<xref ref-type="fig" rid="fig4s1">Figure 4–figure supplement 1B</xref>). In all groups, Cronbach’s alpha of ‘Before’ and ‘2h’ are not significantly different, suggesting that the increase in cosine similarity may be caused by a change at the network level; not by an increased reliability of calcium transient occurrence. I added this analysis and results in the main text and a figure (<xref ref-type="fig" rid="fig4s1">Figure 4–figure supplement 1B</xref>).</p><p><italic>7) The author states that 50-80 cells were analyzed per animal. Were these treated independently and the same way as cells from other animals? More importantly, what is the variance in the effect across the population?</italic></p><p>Cells were treated independently and analyzed in the same way as cells from other animals. For an example, to test whether the difference in the number of cells analyzed per animal changes the result, I analyzed the correlation between the increase in population average of dF/F and the number of cells analyzed in each animal from the T1 conditioning group. The regression analysis showed no significant correlation between them (adjusted R<sup>2</sup> = -0.1144, standardized regression coefficient = -0.212, P = 0.614). So, I think as a whole, the number of cells analyzed per animal does not have major effect on the analysis across the animals.</p><p><italic>8) “VTA neurons are mainly dopaminergic”: this description sounds incorrect. Dopaminergic neurons are up to 65% in VTA, and the rest are GABAergic and glutamatergic (Cohen JY et al Nature 2012; <xref ref-type="bibr" rid="bib6">Gorelova et al., Cereb Cortex 2012</xref>; <xref ref-type="bibr" rid="bib16">Nair-Roberts RG et al., Neuroscience 2008</xref>; etc.). The strong glutamatergic nature of the pathway is perfectly consistent with the effects of glutamate blockers on the transient responses (<xref ref-type="fig" rid="fig3s1">Figure 3–figure supplement 1C</xref>). The author should discuss the effects of non-dopaminergic afferents to M2 on the results reported in this paper.</italic></p><p>Thank you for this comment. I have added points to address this observation in the main text.</p><p><italic>9) Some portion of M2 pyramidal neurons have projections to the VTA (Watabe-Uchida M et al., Neuron. 2012). Has the author considered or ruled out the possibility of antidromic stimulation of M2 when using VTA electric stimulation?</italic></p><p>I did not rule out antidromic effects on M2 neurons, however, long lasting Ca<sup>2+</sup> transients and short Ca<sup>2+</sup> transients were reduced by D1 and glutamate blocker, respectively, suggesting that these responses are mainly evoked by synaptic transmission.</p><p><italic>10) In <xref ref-type="fig" rid="fig3">Figure 3A</xref> was water really used as a control?</italic></p><p>I used H<sub>2</sub>O as a control because I diluted drugs for i.p. injection with H<sub>2</sub>O. The injected volume was adjusted to 1% of the animal’s body weight.</p><p><italic>[Editors' note: further revisions were requested prior to acceptance, as described below.]</italic></p><p><italic>The title should state “prefrontal cortex neurons” rather than just “prefrontal neurons”.</italic></p><p>Thanks for your comment. I corrected the title as suggested.</p><p><italic>The Abstract needs some rewording: e.g. “By establishing a system of in vivo two-photon Ca</italic><sup><italic>2+</italic></sup> <italic>imaging in awake mice” change to “Using in vivo two-photon population Ca</italic><sup><italic>2+</italic></sup> <italic>imaging in awake mice”; “Phasic stimulation of dopaminergic neurons in the ventral tegmental area (VTA) evoked long-lasting (∼30 sec) calcium transients in PFC” - in PFC neurons (specify layer or cell type if possible); “Pharmacological analysis revealed that this long-lasting calcium transients are regulated by dopamine D1 receptor-dependent pathway.” Please clarify: the long-lasting enhancement of the transients, or the transients themselves?; “Furthermore, only visual sensory input applied 0.5 second before the VTA dopaminergic input could evoke higher Ca</italic><sup><italic>2+</italic></sup> <italic>transients”; what does 'higher' mean?).</italic></p><p>I reworded the Abstract according to the reviewer’s comments and tried to maintain the word limit (150 words).</p><p><italic><xref ref-type="fig" rid="fig1">Figure 1E</xref>: Ensure that the contrast to the line indicating the average is high enough. Clean up the axis labelling: why start from 10 s and not 0 s. The SI unit is “s” and not “sec”. A 0-line is nice, but doesn't replace an x-axis.</italic></p><p>I corrected the line colors and width to ensure high contrast throughout the figures.</p><p>Concerning the reviewer’s comment about the x-axis, while the recordings were started at time 0, the first few seconds of recording showed high background signals (∼ 0.03 dF/F, lasting for 2-3 sec) which was due to mechanical noise. Therefore, I showed the calcium transients starting from 10s to only show the neuronal activity that occurred in response to the electrical stimulation. I have now included this information in the Material and methods.</p><p><italic><xref ref-type="fig" rid="fig1">Figure 1F</xref>: Drop the grid lines; the significance line should end centered on the data bars.</italic></p><p>Thanks, I made the recommended changes.</p><p>Reviewer #1:</p><p><italic>The author has done a good job with the revisions. I would still suggest the following:</italic></p><p><italic>The text reads: “In VTA, up to 65% of the neurons are dopaminergic and the others are GABAergic or glutamatergic (19)(20), and some DA neurons co-release glutamate (21)novel,, suggesting that glutamatergic/DA neurons might also contribute to the 5Hz responses.” Two points here. First, overall this is true for the VTA but the mesocortical projection which is what is being studied, is mainly glutamatergic not DAergic (See papers by Morales or Gorelova). Second, the results actually show that glutamate and not DA contributes the 5Hz response. Therefore, I cannot understand the equivocation when a firm conclusion can be drawn.</italic></p><p>Thank you for the comments. I rewrote the sentence to strengthen the conclusion that the 5Hz response is mainly due to glutamatergic neurons.</p><p><italic>In the text and Figure 3–figure supplement 2: Once again I do not agree with including this in the paper. Not only for the reasons I mentioned in the last round but also because of the simple fact that just because IP3 and DA manipulations both decrease the Ca response, it in no way means they are related. To imply this given the existing data is not wise in my opinion.</italic></p><p>Considering the reviewer’s concerns, I removed the section about the 2-APB experiments.</p><p><italic>In terms of H20 control I'm surprised that a hypo-osmotic solution didn't affect the cells and the Ca signal?</italic></p><p>Thanks for the observation. However, I feel that my experiments show that the H<sub>2</sub>O does not affect the Ca<sup>2+</sup> responses. Therefore, using H<sub>2</sub>O injections that are1% of the animal’s body weight has no noticeable effect.</p><p><italic>The document should be proofread to check that tenses are correct etc.</italic></p><p>I have had multiple readers check the document, and I hope to have made all of the appropriate corrections.</p><p>Reviewer #2:</p><p><italic>1) Abstract. The sentence “Furthermore, ....” is a bit confusing, because “only visual sensory input applied 0.5 second before the VTA dopaminergic input ” is the protocol during the conditioning, and “evoke higher Ca</italic><sup><italic>2+</italic></sup> <italic>transients and increase pattern similarity” is the effect 2 hours after the conditioning, but this timeline (or the experimental causality) is not clearly described.</italic></p><p>I rewrote sentence more clearly to address the reviewer’s concern.</p><p><italic>2) <xref ref-type="fig" rid="fig1">Figure 1A</xref>. It would be helpful if the author can provide the picture or sketch of the injection site of the tracer as an insertion panel.</italic></p><p>I added diagrams of the two injection sites in <xref ref-type="fig" rid="fig1">Figure1A,B</xref>.</p><p><italic>3) <xref ref-type="fig" rid="fig1">Figure 1E</xref>. “Population average of Ca</italic><sup><italic>2+</italic></sup> <italic>transients (dF/F) in response to a 50-Hz-VTA”. Please provide the information of the duration or numbers of the pulses of the electrical stimulation, in the figure legend or main text. The author describes it in the Method section, but it is better to have the information earlier in the manuscript.</italic></p><p>Thanks, this is a valid point. I added the stimulation protocol to the Figure legends to clarify the procedure used.</p></body></sub-article></article> |