Behavioral role of PACAP signaling reflects its selective distribution in glutamatergic and GABAergic neuronal subpopulations

  1. Limei Zhang
  2. Vito S. Hernández
  3. Charles R. Gerfen
  4. Sunny Z. Jiang
  5. Lilian Zavala
  6. Rafael A. Barrio
  7. Lee E. Eiden

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Abstract

The neuropeptide PACAP, acting as a co-transmitter, increases neuronal excitability, which may enhance anxiety and arousal associated with threat conveyed by multiple sensory modalities. The distribution of neurons expressing PACAP and its receptor, PAC1, throughout the mouse nervous system was determined, in register with expression of glutamatergic and GABAergic neuronal markers, to develop a coherent chemoanatomical picture of PACAP’s role in brain motor responses to sensory input. A circuit role for PACAP was tested by observing fos activation of brain neurons after olfactory threat cue in wild type and PACAP knockout mice. Neuronal activation, and behavioral response, were blunted in PACAP knock-out mice, accompanied by sharply down-regulated vesicular transporter expression in both GABAergic and glutamatergic neurons expressing PACAP and its receptor. This report signals a new perspective on the role of neuropeptide signaling in supporting excitatory and inhibitory neurotransmission in the nervous system within functionally coherent polysynaptic circuits.

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  1. eLife

    ###Reviewer #2:

    General assessment

    The manuscript of Zhang and colleagues studied the expression of PACAP and PAC1 mRNA in inhibitory and excitatory neurons in the entire mouse brain by using dual ISH method. Additionally, a behavioural test is carried out to provide a functional role for PACAP/PAC1 on olfaction and defensive behaviour followed by cFos examination of selected brain regions to indicate the role of PACAP and PAC1 in such behavioural outputs.

    Summary

    In my view, this study has two parts that could work separately.

    Part 1: the PACAP/PAC1 characterization is well designed and executed. The result description is lengthy and sometimes confusing. Figures and tables (including the supplementary information) are clear and informative. The authors decide to not show Vipr1/Vipr2 data, which should be reconsidered. Overall, this part of the manuscript represents a nice piece of work and surely will be very helpful to those who wish to work with PACAP/PAC1.

    Part 2: I think this part is the critical one in this manuscript. Starting from section 4, it uses part 1 of the manuscript to review the literature and build a neuronal circuit with PACAP/PAC1 that makes for behavioural processes. It is literally a review inside the results section. The schematic figures are interesting but also quite speculative regarding brain signalling since the authors did not perform any experiment to investigate the pathway of PACAP and the literature is scarce. Moreover, the role of Vip receptors were completely neglected here.

    Behavioural test: the authors decided for the predator odor paradigm based on the involvement of PACAP on the defensive circuit. However, a global PACAP KO is used instead of specifically targeting a brain region or a neuronal population. Not that this is not interesting, but the entire specificity applied in the first part of the study was not used to find a functional role for PACAP. Despite the cFos analysis demonstrating reduced activity in several brain regions in PACAP KO, the specific role of PACAP in such regions and the importance of each of the three PACAP receptors remained unknown. Also, the use of a global KO inhibits the understanding of the excitatory/inhibitory balance that perhaps the PACAP system may play a role. Moreover, due the specific requirement of the olfaction sense in this test (the considerable expression of PACAP on the olfactory bulb), it is not clear how much the olfaction function is affected in PACAP-deficient mice, and thus, consequently affect the defensive/fear circuit. Finally, is the change in locomotion found here due to a fear response or a hyperlocomotor activity?

  2. eLife

    ###Reviewer #1:

    Zhang/Hernandez et al provide a fascinating and comprehensive dataset of the distribution of PACAP (Adcyap1) and PAC1 (Adcyap1r1) mRNA expressing cells in most regions of the mouse brain. Using dual (two-colour) in situ hybridization (DISH) they go further than the Allen Institute ISH datasets by revealing the co-expression with common neurotransmitters (VGAT, VGLUT1, VGLUT2) as well as linking expression to a variety of physiologically and behaviourally relevant neural circuits. Among their observations, they observe a subpopulation of PACAP-expressing CA3 neurons, find that dentate mossy cells express PACAP with a particular septo-temporal distribution, as well as prominent expression in neurons of the bed nucleus of the anterior commissure. They report overlapping PACAP/PAC1 cell groups and also find that PACAP knockout mice exhibit impaired predator odour responsiveness and reduction in neurotransmitter expression in PACAP-related regions. This is a valuable and important study on PACAPergic brain regions in mice, especially relating to the hypothalamus, but would benefit from a reorganisation to improve the presentation of data, and further quantitative criteria to strengthen the observations.

    1. The paper would benefit from a reorganisation, especially when referring to figures and tables. There are a very large number of abbreviations. A list near the beginning of the manuscript would help the reader, and would also shorten the figure legends and improve readability/flow. For the non-expert, some areas should be labelled/highlighted separately or provide more information in the figures, e.g. line 184 'ACA and the entorhinal cortex' one has to search the figure legend, find the number then search the figure panels to find the location of these brain regions. Abbreviations and brain region names should be consistent, e.g. line 241, ACC is used in text, but ACA in figure and legend. Unless mistaken, Table S1 is not mentioned in the text. Figure 9 is first mentioned in the Discussion (line 780). Since these are valuable data, refer to this figure in the main Results section in terms of the knockout. Figure S1 is very informative, but requires a lot of searching to find the panel that is referred to in the text. In Figure S1-7/7-M, panels M1-4 are identical to Fig 1E-H and the scale bar in M3 is different to 1G.

    2. In several places there are anecdotal statements and it is not clear about the reproducibility of the results. The methods for quantification (including those mentioned in Table legends) should be included in Methods. For animals, please check and state the total number of mice and rats used in the study, and whether EGFP mice were also used (as referred to in line 191). In line 816, what is a group?

    For c-fos experiments, how were these cells counted, how many sections per mouse, what was the section thickness, how were the values calculated (mean, absolute numbers). Was fos counting done blind to genotype?

    Was there variation between animals in terms of expression levels/strength? Case/animal numbers in figures would help. It is not clear what is meant throughout by statements such as 'strongest'. Is this by density in cells or number/intensity of puncta? For example, section 3.1, retina. What is meant by 'higher percentage than previously reported' (line 148)? Is this referring to both previous reports in mice? Also see Engelund et al Cell Tissue Res 2010. How many samples and/or mice were examined and how were ganglion cells counted?

    Similarly, lines 174 and 182-183, cortical expression in different layers, how were the values of 80% obtained? Again line 196, 'highest expression level of PAC1 among all brain regions' is a strong claim, how was this quantified? Line 249-251, need references/evidence for observations of mouse claustrum percentages. Line 272, 'more than 90%'. Line 463, 'the highest expression of PACAP was observed in the MnPO'.

    Line 484 in terms of the olfactory pathways, is there evidence of co-transmission or is this a hypothesis?

    Some claims will need careful revision. E.g. in the Fig 5 legend, the last sentence contradicts line 286.

    In line 187, the finding that 100% of the 3 GABAergic subpopulations expressed PAC1 is a big claim, yet there is no quantification to back this up. How many brain regions were examined, how many mice, sections, counted cells etc.? If it just refers to the primary somatosensory cortex, was it all or some layers?

    Table 2 (also applies to parts of Table 1), do blank areas of the table mean not examined? Or should there be '-' in these areas? For example, the medial septal complex contains vglut2 expressing cells but the corresponding row/column is blank.

    Line 191-193, there is the claim that PACAP mRNA was not found in cell body layers, but in Table 1 it is reported that there is weak expression in VGLUT1+ cells. Since VGLUT1 cells are in the pyramidal cell layer, this seems contradictory. It would be helpful to have a higher power image of CA1 (as for rat in Fig S2). Could expression outside this layer be in subpopulations of GABAergic neurons? Were these examined (blank in Table 1)? DG is also missing from Table 1. PAC1 expression. Line 195, claims it is selective for VGAT cells. But there are clear examples of VGAT- cells in Fig S3B expressing PAC1. What are these?

    1. Suggestion about paracrine/autocrine signalling. Is there evidence in literature for such a role? This seems speculative without immunohistochemical evidence. Hannibal 2002, carried out at both the protein and mRNA levels, showed axon terminals in multiple regions. Can these be mapped to the regions that express PAC1 in mice? Is there any evidence or could the authors comment on the existence of presynaptic PACAP receptors? Expression of PAC1 mRNA does not imply that the cell would express the protein exclusively along its somatodendritic membrane. 'Classical' neurotransmission presumably could occur in PACAP/PAC1 rich regions via local axons in addition to long-range axons.

    2. The observation of PACAP in part of temporal CA3, which the authors refer to as CA3c, has in fact previously been defined as CA3vv, corresponding to the coch expressing domain (see Thompson et al Neuron 2008, Fanselow and Dong Neuron 2010). PACAP may indeed be an additional marker along with calretinin for this principal cell subpopulation, and they may want to revise their model or refer to these earlier papers.

    3. PACAP KO. Some clarification would be welcome in terms of animal cohorts. Please state the experimental unit (i.e. n=9 mice/group). In D, the freezing data show only 8 mice, was one pair excluded due to lack of freezing in an animal, as for jumping mice in C? In Ai, Aii, Bi, Bii, does this show the traces for the total time?

    In the separate experiment (lines 630-635), was n=3 a separate cohort of mice or from the N=18 total as stated in the methods? Is the n=3 per group or total mice? This may require an increased sample size for this claim, or show quantification/statistical tests. For this test, were experimenters also blind to the genotype? The last sentence is difficult to follow.

    For the behavioural tests, please include details about whether the wooden boxes, room and experimenter were familiar to the mice before the test (which could affect variability), whether mice were tested at the same time of day, and if KO and WT animals were housed together.

    In the Discussion, ~line 797, can the authors comment on or provide evidence of possible developmental changes / compensatory mechanisms occurring in the KO animals.

  3. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 2 of the manuscript.

    ###Summary:

    The manuscript of Zhang and colleagues studied the expression of PACAP and PAC1 mRNA in inhibitory and excitatory neurons in the entire mouse brain by using dual ISH method. Additionally, a behavioural test is carried out to provide a functional role for PACAP/PAC1 on olfaction and defensive behaviour followed by cFos examination of selected brain regions to indicate the role of PACAP and PAC1 in such behavioural outputs. The reviewers believe that this is a valuable and important study on PACAPergic brain regions in mice, especially relating to the hypothalamus, but would benefit from a major reorganisation to improve the presentation of data, and further quantitative criteria to strengthen the observations.

  4. Version published to 10.1101/2020.07.31.231795 on bioRxiv