Chronic Morphine Induces Adaptations in Opioid Receptor Signaling in a Thalamostriatal Circuit That Are Location Dependent, Sex Specific, and Regulated by µ-Opioid Receptor Phosphorylation.

Chronic opioid exposure induces tolerance to the pain-relieving effects of opioids but sensitization to some other effects. While the occurrence of these adaptations is well understood, the underlying cellular mechanisms are less clear. This study aimed to determine how chronic treatment with morphine, a prototypical opioid agonist, induced adaptations to subsequent morphine signaling in different subcellular contexts. Opioids acutely inhibit glutamatergic transmission from medial thalamic (MThal) inputs to the dorsomedial striatum (DMS) via activity at µ-opioid receptors (MORs). MORs are present in somatic and presynaptic compartments of MThal neurons terminating in the DMS. We investigated the effects of chronic morphine treatment on subsequent morphine signaling at MThal-DMS synapses and MThal cell bodies in male and female mice. Surprisingly, chronic morphine treatment increased subsequent morphine inhibition of MThal-DMS synaptic transmission (morphine facilitation) in male, but not female, mice. At MThal cell bodies, chronic morphine treatment decreased subsequent morphine activation of potassium conductance (morphine tolerance) in both male and female mice. In knock-in mice expressing phosphorylation-deficient MORs, chronic morphine treatment resulted in tolerance to, rather than facilitation of, subsequent morphine signaling at MThal-DMS terminals, suggesting phosphorylation deficiency unmasks adaptations that counter the facilitation observed at presynaptic terminals in wild-type mice. The results of this study suggest that the effects of chronic morphine exposure are not ubiquitous; rather adaptations in MOR function may be determined by multiple factors such as subcellular receptor distribution, influence of local circuitry, and sex.

Lineage-tracing reveals an expanded population of NPY neurons in the inferior colliculus.

Growing evidence suggests that neuropeptide signaling shapes auditory computations. We previously showed that neuropeptide Y (NPY) is expressed in the inferior colliculus (IC) by a population of GABAergic stellate neurons and that NPY regulates the strength of local excitatory circuits in the IC. NPY neurons were initially characterized using the NPY-hrGFP mouse, in which humanized renilla green fluorescent protein (hrGFP) expression indicates NPY expression at the time of assay, i.e., an expression-tracking approach. However, studies in other brain regions have shown that NPY expression can vary based on several factors, suggesting that the NPY-hrGFP mouse might miss NPY neurons not expressing NPY on the experiment date. Here, we hypothesized that neurons with the ability to express NPY represent a larger population of IC GABAergic neurons than previously reported. To test this hypothesis, we used a lineage-tracing approach to irreversibly tag neurons that expressed NPY at any point prior to the experiment date. We then compared the physiological and anatomical features of neurons labeled with this lineage-tracing approach to our prior data set, revealing a larger population of NPY neurons than previously found. In addition, we used optogenetics to test the local connectivity of NPY neurons and found that NPY neurons provide inhibitory synaptic input to other neurons in the ipsilateral IC. Together, our data expand the definition of NPY neurons in the IC, suggest that NPY expression might be dynamically regulated in the IC, and provide functional evidence that NPY neurons form local inhibitory circuits in the IC.NEW & NOTEWORTHY Across brain regions, neuropeptide Y (NPY) expression is dynamic and influenced by extrinsic and intrinsic factors. We previously showed that NPY is expressed by a class of inhibitory neurons in the auditory midbrain. Here, we find that this neuron class also includes neurons that previously expressed NPY, suggesting that NPY expression is dynamically regulated in the auditory midbrain. We also provide functional evidence that NPY neurons contribute to local inhibitory circuits in the auditory midbrain.

Lineage-tracing reveals an expanded population of NPY neurons in the inferior colliculus.

Growing evidence suggests that neuropeptide signaling shapes auditory computations. We previously showed that neuropeptide Y (NPY) is expressed in the inferior colliculus (IC) by a population of GABAergic stellate neurons and that NPY regulates the strength of local excitatory circuits in the IC. NPY neurons were initially characterized using the NPY-hrGFP reporter mouse, in which hrGFP expression indicates NPY expression at the time of assay, i.e., an expression-tracking approach. However, studies in other brain regions have shown that NPY expression can vary based on a range of factors, suggesting that the NPY-hrGFP mouse might miss NPY neurons not expressing NPY proximal to the experiment date. Here, we hypothesized that neurons with the ability to express NPY represent a larger population of IC GABAergic neurons than previously reported. To test this hypothesis, we used a lineage-tracing approach to irreversibly tag neurons that expressed NPY at any point prior to the experiment date. We then compared the physiological and anatomical features of neurons labeled with this lineage-tracing approach to our prior data set, revealing a larger population of NPY neurons than previously found. In addition, we used optogenetics to test the local connectivity of NPY neurons and found that NPY neurons routinely provide inhibitory synaptic input to other neurons in the ipsilateral IC. Together, our data expand the definition of NPY neurons in the IC, suggest that NPY expression might be dynamically regulated in the IC, and provide functional evidence that NPY neurons form local inhibitory circuits in the IC.

GluN2D-containing NMDA receptors enhance temporal integration in VIP neurons in the inferior colliculus.

Along the ascending auditory pathway, there is a broad shift from temporal coding, which is common in the lower auditory brainstem, to rate coding, which predominates in auditory cortex. This temporal-to-rate transition is particularly prominent in the inferior colliculus (IC), the midbrain hub of the auditory system, but the mechanisms that govern how individual IC neurons integrate information across time remain largely unknown. Here, we report the widespread expression of GluN2C and GluN2D mRNA in IC neurons. GluN2C/D-containing NMDA receptors are relatively insensitive to voltage-dependent Mg2+ block, and thus can activate at resting membrane potential. Using in situ hybridization and pharmacology, we show that VIP neurons in the IC express GluN2D-containing NMDA receptors that are activatable by ascending input from T-stellate cells in the anteroventral cochlear nucleus and commissural inputs from the contralateral IC. In addition, GluN2D-containing receptors have much slower kinetics than other NMDA receptors, and we found that GluN2D-containing receptors facilitate temporal summation in VIP neurons by prolonging the time window for synaptic integration. These results suggest that GluN2C/D-containing NMDA receptors support the shift from temporal to rate coding in the auditory system by facilitating the integration of ascending inputs.

Characterization of three cholinergic inputs to the cochlear nucleus.

Acetylcholine modulates responses throughout the auditory system, including at the earliest brain level, the cochlear nucleus (CN). Previous studies have shown multiple sources of cholinergic input to the CN but information about their relative contributions and the distribution of inputs from each source is lacking. Here, we used staining for cholinergic axons and boutons, retrograde tract tracing, and acetylcholine-selective anterograde tracing to characterize three sources of acetylcholine input to the CN in mice. Staining for cholinergic axons showed heavy cholinergic inputs to granule cell areas and the dorsal CN with lighter input to the ventral CN. Retrograde tract tracing revealed that cholinergic cells from the superior olivary complex, pontomesencephalic tegmentum, and lateral paragigantocellular nucleus send projections to the CN. When we selectively labeled cholinergic axons from each source to the CN, we found surprising similarities in their terminal distributions, with patterns that were overlapping rather than complementary. Each source heavily targeted granule cell areas and the dorsal CN (especially the deep dorsal CN) and sent light input into the ventral CN. Our results demonstrate convergence of cholinergic inputs from multiple sources in most regions of the CN and raise the possibility of convergence onto single CN cells. Linking sources of acetylcholine and their patterns of activity to modulation of specific cell types in the CN will be an important next step in understanding cholinergic modulation of early auditory processing.

Chronic morphine induces adaptations in opioid receptor signaling in a thalamo-cortico-striatal circuit that are projection-dependent, sex-specific and regulated by mu opioid receptor phosphorylation.

Chronic opioid exposure induces tolerance to the pain-relieving effects of opioids but sensitization to some other effects. While the occurrence of these adaptations is well-understood, the underlying cellular mechanisms are less clear. This study aimed to determine how chronic treatment with morphine, a prototypical opioid agonist, induced adaptations to subsequent morphine signaling in different subcellular contexts. Opioids acutely inhibit glutamatergic transmission from medial thalamic (MThal) inputs to the dorsomedial striatum (DMS) and anterior cingulate cortex (ACC) via activity at µ-opioid receptors (MORs). MORs are present in somatic and presynaptic compartments of MThal neurons terminating in both the DMS and ACC. We investigated the effects of chronic morphine treatment on subsequent morphine signaling at MThal-DMS synapses, MThal-ACC synapses, and MThal cell bodies in male and female mice. Surprisingly, chronic morphine treatment increased subsequent morphine inhibition of MThal-DMS synaptic transmission (morphine facilitation), but decreased subsequent morphine inhibition of transmission at MThal-ACC synapses (morphine tolerance) in a sex-specific manner; these adaptations were present in male but not female mice. Additionally, these adaptations were not observed in knockin mice expressing phosphorylation-deficient MORs, suggesting a role of MOR phosphorylation in mediating both facilitation and tolerance to morphine within this circuit. The results of this study suggest that the effects of chronic morphine exposure are not ubiquitous; rather adaptations in MOR function may be determined by multiple factors such as subcellular receptor distribution, influence of local circuitry and sex.

The effect of mechanosensitive channel MscL expression in cancer cells on 3D confined migration.

Metastatic cancer cells migrate through constricted spaces and experience significant compressive stress, but mechanisms enabling migration in confined geometries remain unclear. Cancer cell migration within confined 3-dimensional (3D) microfluidic channels has been shown to be distinct from 2D cell migration. However, whether 3D confined migration can be manipulated by mechanosensory components has not been examined in detail. In this work, we exogenously introduced a mechanosensitive channel of large conductance (MscL) into metastatic breast cancer cells MDA-MB-231. We discovered that inducing expression of a gain-of-function G22S mutant of MscL in MDA-MB-231 cells significantly reduced spontaneous lung metastasis without affecting the growth of orthotopic tumor implants. To further investigate the effects of G22S MscL on cell migration, we designed a microfluidic device with channels of various cross-sections ranging from a 2D planar environment to narrow 3D constrictions. Both MscL G22S and control breast cancer cells migrated progressively slower in more constricted environments. Migration of cells expressing MscL G22S did not differ from control cells, even though MscL was activated in cells in constricted channels of 3 µm width. Interestingly, we found MscL expressing cells to be more frequently "stuck" at the entrance of the 3 µm channels and failed to migrate into the microchannel. Our work demonstrates the possibility of engineering mechanotransduction for controlling confined cell migration.

Evaluating the Capacity of Human Gut Microorganisms to Colonize the Zebrafish Larvae (Danio rerio).

In this study we evaluated if zebrafish larvae can be colonized by human gut microorganisms. We tested two strategies: (1) through transplantation of a human fecal microbiota and (2) by successively transplanting aerotolerant anaerobic microorganisms, similar to the colonization in the human intestine during early life. We used conventionally raised zebrafish larvae harboring their own aerobic microbiota to improve the colonization of anaerobic microorganisms. The results showed with the fecal transplant, that some members of the human gut microbiota were transferred to larvae. Bacillus, Roseburia, Prevotella, Oscillospira, one unclassified genus of the family Ruminococcaceae and Enterobacteriaceae were detected in 3 days post fertilization (dpf) larvae; however only Bacillus persisted to 7 dpf. Successive inoculation of Lactobacillus, Bifidobacterium and Clostridioides did not improve their colonization, compared to individual inoculation of each bacterial species. Interestingly, the sporulating bacteria Bacillus clausii and Clostridioides difficile were the most persistent microorganisms. Their endospores persisted at least 5 days after inoculating 3 dpf larvae. However, when 5 dpf larvae were inoculated, the proportion of vegetative cells in larvae increased, revealing proliferation of the inoculated bacteria and better colonization of the host. In conclusion, these results suggest that it is feasible to colonize zebrafish larvae with some human bacteria, such as C. difficile and Bacillus and open an interesting area to study interactions between these microorganisms and the host.