Dear Editor,
Please, find below the itemized list of the corrections brought to our ms (N°NPP-07-0364RR) on the basis of the last reviewers comments.
Reviewer #1
1. There is only one major concern about the question.... "that recent studies... of changes in 5-HT RNAm and protein expression induced by memory formation and drugs..., eventually lead to arborization? The authors in the rebuttal letter stated that: "It is likely they might contribute. The point is that we do not know how these 5-HT protein and RNAm receptors are expressed in the mouse brain so that we cannot speculate on their role in promoting dendritic spine growth in mice treated with the 5-HT4Rs agonist". However, in the new introduction authors mentioned that "there is not yet evidence that such compounds enhance learning-induced structural plasticity" (last lines of the first paragraph). This last statement seems speculative and/or not justified by the available evidence.
This statement has been deleted along with the following reference:
Izquierdo I, Medina JH (1995) Correlation between the pharmacology of long-term potentiation and the pharmacology of memory. Neurobiol Learn Mem 63:19-32
Associate editor
1. I was surprised that the authors did the control experiment reviewer #3 requested (i.e. to check spine densities in cortical regions unassociated with olfactory memory) but make no mention of these results in the Discussion.
A sentence in the discussion section (1st paragraph) states now that, in mice trained or pseudo trained in the OTM,“the compound was not found modify spine density on pyramidal neurons laying in the primary visual cortex area, a region unrelated to the memory pathways”
2. I am still concerned about the small number of experimental subjects that make up each group. A concern is that the effect sizes reported are not huge and this statistic is only reported in the legends to Figures 1 and 3 yet they refer to significant effects in figures 2 and 4 as well but give no values in the figure legends.
In the revised version of the manuscript the sample size issue was addressed replicating experiments. The main effect (SL65.0155 enhancing learning-induced spine growth)was observed (i) across different batchesthus increasing its internal validity and (ii) on several related measurements (behaviour and morphology of apical, oblique and basal dendrites). Keeping in mind that the C57Bl/6 strain is an inbred strain (showingminimal inter-individual variability in behaviour and brain characteristics) it is rather customary using 6-8 per group. Moreover, unbiased sampling procedures applied throughout the experiment (blind counting of spines, random selection of the brains to be processed for spine analysis) made us comfortable with the validity of the statistical results.
The statistical values are now reported also in figures 2 and 4. In addition, new symbols indicating the correct p-level for post-hoc tests have been added to figure 2.
Minor changes
1. Text (Typographical errors corrected)
Page 8, paragraph title: correct concentration for SL65.0155 is 0.01mg/Kg
Page 8, line 8 and last line: correct name is RS39604
Page 8, line 9: correct name is SL65.0155
2. Figures
Symbols indicating p level have been changed on figure 2 according to post–hoc results described in the results section.
3. Acknowledgment section has been added
We thank the reviewers for their suggestions along with their positive comments and hope that our manuscript can be now considered for publication in Neuropsychopharmacology.
Martine Ammassari-Teule
Corresponding author