Disruption of the CRF₁ receptor eliminates morphine-induced sociability deficits and firing of oxytocinergic neurons in male mice

Reviewer #1 (Public review):

Summary:

The use of antalarmin, a selective CRF1 receptor antagonist, prevents the deficits in sociability in (acutely) morphine-treated males, but not in females. In addition, cell-attached experiments show a rescue to control levels of the morphine-induced increased firing in PVN neurons from morphine-treated males. Similar results are obtained in CRF receptor 1-/- male mice, confirming the involvement of CRF receptor 1-mediated signaling in both sociability deficits and neuronal firing changes in morphine-treated male mice.

Strengths:

The experiments and analyses appear to be performed to a high standard, and the manuscript is well written and the data clearly presented. The main finding, that CRF-receptor plays a role in sociability deficits occurring after acute morphine administration, is an important contribution to the field.

Weaknesses:

The link between the effect of pharmacological and genetic modulation of CRF 1 receptor on sociability and on PVN neuronal firing, is less well supported by the data presented. No evidence of causality is provided.

Major points:

(1) The results of behavioral tests and the neural substrate are purely correlative. To find causality would be important to selectively delete or re-express CRF1 receptor sequence in the VPN. Re-expressing the CRF1 receptor in the VPN of male mice and testing them for social behavior and for neuronal firing would be the easier step in this direction.

(2) It would be interesting to discuss the relationship between morphine dose and CRF1 receptor expression.

(3) It would be important to show the expression levels of CRF1 receptors in PVN neurons in controls and morphine-treated mice, both males and females.

(4) It would be important to discuss the mechanisms by which CRF1 receptor controls the firing frequency of APV+/OXY+ neurons in the VPN of male mice.

Minor points:

(1) The phase of the estrous cycles in which females are analyzed for both behavior and electrophysiology should be stated.

(2) It would be important to show the statistical analysis between sexes.

Reviewer #2 (Public review):

This manuscript reports a series of studies that sought to identify a biological basis for morphine-induced social deficits. This goal has important translational implications and is, at present, incompletely understood in the field. The extant literature points to changes in periventricular CRF and oxytocin neurons as critical substrates for morphine to alter social behavior. The experiments utilize mice, administered morphine prior to a sociability assay. Both male and female mice show reduced sociability in this procedure. Pretreatment with the CRF1 receptor antagonist, antalarmin, clearly abolished the morphine effect in males, and the data are compelling. Consistently, CRF1-/- male mice appeared to be spared of the effect of morphine (while wild-type and het mice had reduced sociability). The same experiment was reported as non-feasible in females due to the effect of dose on exploratory behavior per se. Seeking a neural correlate of the behavioral pharmacology, acute cell-attached recordings of PVN neurons were made in acute slices from mice pretreated with morphine or anatalarmin. Morphine increased firing frequencies, and both antalarmin and CRF1-/- mice were spared of this effect. Increasing confidence that this is a CRF1 mediated effect, there is a gene deletion dose effect where het's had an intermediate response to morphine. In general, these experiments are well-designed and sufficiently powered to support the authors' inferences. A final experiment repeated the cell-attached recordings with later immunohistochemical verification of the recorded cells as oxytocin or vasopressin positive. Here the data are more nuanced. The majority of sampled cells were positive for both oxytocin and vasopressin, in cells obtained from males, morphine pretreatment increased firing in this population and was CRF1 dependent, however in females the effect of morphine was more modest without sensitivity to CRF1. Given that only ~8 cells were only immunoreactive for oxytocin, it may be premature to attribute the changes in behavior and physiology strictly to oxytocinergic neurons. In sum, the data provide convincing behavioral pharmacological evidence and a regional (and possibly cellular) correlation of these effects suggesting that morphine leads to sociality deficits via CRF interacting with oxytocin in the hypothalamus. While this hypothesis remains plausible, the current data do not go so far as directly testing this mechanism in a site or cell-specific way. With regard to the presentation of these data and their interpretation, the manuscript does not sufficiently draw a clear link between mu-opioid receptors, their action on CRF neurons of the PVN, and the synaptic connectivity to oxytocin neurons. Importantly, sex, cell, and site-specific variations in the CRF are well established (see Valentino & Bangasser) yet these are not reviewed nor are hypotheses regarding sex differences articulated at the outset. The manuscript would have more impact on the field if the implications of the sex-specific effects evident here were incorporated into a larger literature.

With regards to the model proposed in the discussion, it seems that there is an assumption that ip morphine or antalarmin have specific effects on the PVN and that these mediate behavior - but this is impossible to assume and there are many meaningful alternatives (for example, both MOR and CRF modulation of the raphe or accumbens are worth exploration). While it is up to the authors to conduct additional studies, a demonstration that the physiology findings are in fact specific to the PVN would greatly increase confidence that the pharmacology is localized here. Similarly, direct infusion of antalarmin to the PVN, or cell-specific manipulation of OT neurons (OT-cre mice with inhibitory dreadds) combined with morphine pre-exposure would really tie the correlative data together for a strong mechanistic interpretation.

Because the work is framed as informing a clinical problem, the discussion might have increased impact if the authors describe how the acute effects of CRF1 antagonists and morphine might change as a result of repeated use or withdrawal.

Reviewer #3 (Public review):

Summary:

In the current manuscript, Piccin et al. identify a role for CRF type 1 receptors in morphine-induced social deficits using a 3-chamber social interaction task in mice. They demonstrate that pre-treatment with a CRFR1 antagonist blocks morphine-induced social deficits in male, but not female, mice, and this is associated with the CRF R1 antagonist blocking morphine-induced increases in PVN neuronal excitability in male but not female mice. They followed up by using a transgenic mouse CRFR1 knockout mouse line. CRFR1 genetic deletion also blocked morphine-induced social deficits, similar to the pharmacological approach, in male mice. This was also associated with morphine-induced increases in PVN neuronal excitability being blocked in CRFR1 knockout mice. Interestingly they found that the pharmacological antagonism of the CRFR1 specifically blocked morphine-induced increases in oxytocin/AVP neurons in the PVN in male mice.

Strengths:

The authors used both male and female mice where possible and the studies were fairly well controlled. The authors provided sufficient methodological detail and detailed statistical information. They also examined measures of locomotion in all of the behavioral tasks to separate changes in sociability from overall changes in locomotion. The experiments were well thought out and well controlled. The use of both the pharmacological and genetic approaches provides converging lines of evidence for the role of CRFR1 in morphine-induced social deficits. Additionally, they have identified the PVN as a potential site of action for these CRFR1 effects.

Weaknesses:

While the authors included both sexes they analyzed them independently. This was done for simplicity's sake as they have multiple measures but there are several measures where the number of factors is reduced and the inclusion of sex as a factor would be possible. Additionally, single doses of both the CRFR1 antagonist and morphine are used within an experiment without justification for the doses. In fact, a lower dose of morphine was needed for the genetic CRFR1 mouse line. This would suggest that the dose of morphine being used is likely causing some aversion that may be more present in the females, as they have lower overall time in the ROI areas of both the object and the mouse following morphine exposure. As for the discussion, the authors do not sufficiently address why CRFR1 has an effect in males but not females and what might be driving that difference, or why male and female mice have different distribution of PVN cell types during the recordings. Additionally, the authors attribute their effect to CRF and CRFR1 within the PVN but do not consider the role of extrahypothalamic CRF and CRFR1. While the PVN does contain the largest density of CRF neurons there are other CRF neurons, notably in the central amygdala and BNST, that have been shown to play important roles in the impact of stress on drug-related behavior. This also holds true for the expression of CRFR1 in other regions of the brain, including the VTA, which is important for drug-related behavior and social behavior. The treatments used in the current manuscript were systemic or brain-wide deletion of CRFR1. Therefore, the authors should consider that the effects could be outside the PVN.