Probes for CRE

Key targets of both the therapeutic and abused properties of opioids are μ-opioid receptors (MORs). Despite years of research investigating the biochemistry and signal transduction pathways associated with MOR activation, we do not fully understand the cellular mechanisms underlying opioid addiction. Given that addictive opioids such as morphine, oxycodone, heroin, and fentanyl all activate MORs, and current therapies such as naloxone and buprenorphine block this activation, the availability of tools to mechanistically investigate opioid-mediated cellular and behavioral phenotypes are necessary. Therefore, we derived, validated, and applied a novel MOR-specific Cre mouse line, inserting a T2A cleavable peptide sequence and the Cre coding sequence into the MOR 3'UTR. Importantly, this line shows specificity and fidelity of MOR expression throughout the brain and with respect to function, there were no differences in behavioral responses to morphine when compared to wild type mice, nor are there any alterations in Oprm1 gene expression or receptor density. To assess Cre recombinase activity, MOR-Cre mice were crossed with the floxed GFP-reporters, RosaLSLSun1-sfGFP or RosaLSL-GFP-L10a. The latter allowed for cell type specific RNA sequencing via TRAP (Translating Ribosome Affinity Purification) of striatal MOR+ neurons following opioid withdrawal. The breadth of utility of this new tool will greatly facilitate the study of opioid biology under varying conditions.

Serotonin and oxytocin influence aggressive and anxiety-like behaviors, though it is unclear how the two may interact. That the oxytocin receptor is expressed in the serotonergic raphe nuclei suggests a mechanism by which the two neurotransmitters may cooperatively influence behavior. We hypothesized that oxytocin acts on raphe neurons to influence serotonergically-mediated anxiety-like, aggressive and parental care behaviors. We eliminated expression of the oxytocin receptor in raphe neurons by crossing mice expressing Cre recombinase under control of the serotonin transporter promoter (Slc6a4) with our conditional oxytocin receptor knockout line. The knockout mice generated by this cross are normal across a range of behavioral measures: there are no effects for either sex on locomotion in an open-field, olfactory habituation/dishabituation or, surprisingly, anxiety-like behaviors in the elevated O and plus mazes. There was a profound deficit in male aggression: only one of 11 raphe oxytocin receptor knockouts showed any aggressive behavior, compared to eight of 11 wildtypes. In contrast, female knockouts displayed no deficits in maternal behavior or aggression. Our results show that oxytocin, via its effects on raphe neurons, is a key regulator of resident-intruder aggression in males but not maternal aggression. Furthermore, this reduction in male aggression is quite different from the effects reported previously after forebrain or total elimination of oxytocin receptors. Finally, we conclude that when constitutively eliminated, oxytocin receptors expressed by serotonin cells do not contribute to baseline anxiety-like behaviors or maternal care.

Sleep disturbances are strongly associated with cardiovascular diseases. Baroreflex, a basic cardiovascular regulation mechanism, is modulated by sleep-wake states. Here, we show that neurons at key stages of baroreflex pathways also promote sleep. Using activity-dependent genetic labeling, we tagged neurons in the nucleus of the solitary tract (NST) activated by blood pressure elevation and confirmed their barosensitivity with optrode recording and calcium imaging. Chemogenetic or optogenetic activation of these neurons promoted non-REM sleep in addition to decreasing blood pressure and heart rate. GABAergic neurons in the caudal ventrolateral medulla (CVLM)-a downstream target of the NST for vasomotor baroreflex-also promote non-REM sleep, partly by inhibiting the sympathoexcitatory and wake-promoting adrenergic neurons in the rostral ventrolateral medulla (RVLM). Cholinergic neurons in the nucleus ambiguous-a target of the NST for cardiac baroreflex-promoted non-REM sleep as well. Thus, key components of the cardiovascular baroreflex circuit are also integral to sleep-wake brain-state regulation.

The Temporomandibular joint (TMJ) is one of the most complex joints in the human body. TMJ is composed of the temporal bone, a disc and a movable mandibular condyle with abundant tendon attachments. Tendon has been thought to play the sole function of transmitting muscle forces to stabilize joints, yet it is largely unclear why tendon undergoes ectopic ossification in trauma or diseases, and whether it has any direct contribution to skeletal formation. This study aimed to investigate the full biological significance of tendon in TMJ growth. We first discovered that the TMJ condyle is composed of a well-established cartilage head and an overlooked “bony head” that grows after birth and continuously expands throughout the lifespan with little signs of remodeling. Mouse X-ray images (Fig.1a) showed little change in the cartilage head’s volume but a continuous expansion in the bony head’s mass with a low mineral content from 1 to 5 months (Fig.1b). Toluidine blue staining showed TMJ condyle had a large area of tendon attachment extending down to ramus (Fig.1c, white dotted line in lower magnification), defined by regions of tendon, interface, and TFB (Fig.1c1). The TFB morphology was distinct from endosteum-formed bone (EFB, Fig.1c1), cartilage-formed bone (CFB, Fig.1c2, rich in cartilage residual), or periosteum-formed bone (PFB, Fig.1c3) in cell shape and distribution, and ECM. TEM images further revealed that the osteocytes in the TFB were large in size, irregular in shape, had small nuclei but numerous ERs and Golgi complexes, and were embedded in ECM rich in fibropositors. In contrast, the osteocytes in EFB, CFB or PFB were spindle-shaped with larger nuclei but fewer ERs and Golgi complexes (Fig.1d). To reveal the cell source of the bony head, cell lineage tracing were used. Tracing data showed that most CFB cells originate from Col10a1+ hypertrophic chondrocytes, whereas the interface and TFB were derived from Scx+ cells (Fig.1e). RNAscope displayed high levels of Thbs4 (Thrombospondin-4, a tendon marker) and SOST (a potent inhibitor of Wnt signaling secreted by osteocytes) mRNA in TFB at bony head (Fig.1f). The Scx-CreERT2 tracing combined with IHC staining showed TFB maintained a mixed ECM of bone (Col1), cartilage (Aggrecan) and tendon (Periostin, Fig.1g). To further determine the role of tendon lineage in condyle expansion, we generated Scx-CreERT2; R26RDTA (carrying a loxP-flanked stop cassette associated with an attenuated diphtheria toxin fragment A, DTA, for the ablation of cells when Cre is active). Deletion of Scx+ cells greatly reduced the size of bony head (Fig.1h) and the thickness of interface with few Scx+/Col1+ bone cells in P28 DTA mice (Fig.1i); In conclusion, our study tendon cells, beyond their conventional role in joint movement, are key players for the postnatal growth and expansion of TMJ condyle (Fig.1j).