Neuroestrogens are synthesized within the brain and regulate social behavior, learning and memory, and cognition. In song sparrows, Melospiza melodia , 17β-estradiol (17β-E2) promotes aggressive behavior, including during the nonbreeding season when circulating steroid levels are low. Estrogens are challenging to measure because they are present at very low levels, and current techniques often lack the sensitivity required. Furthermore, current methods often focus on 17β-E2 and disregard other estrogens. Here, we developed and validated a method to measure four estrogens [estrone (E1), 17β-E2, 17α-estradiol (17α-E2), estriol (E3)] simultaneously in microdissected songbird brain, with high specificity, sensitivity, accuracy, and precision. We used liquid chromatography tandem mass spectrometry (LC-MS/MS), and to improve sensitivity, we derivatized estrogens using 1,2-dimethylimidazole-5-sulfonyl-chloride (DMIS). The straightforward protocol improved sensitivity by 10-fold for some analytes. There is substan...

The efferent pathway strengthens the auditory system for optimal performance by fine-tuning the response and protecting the inner ear from noise-induced damage. Although it has been well documented that efference helps defend against hair cell and synaptic extinction, the mechanisms of its otoprotective role have still not been established. Specifically, the effect of efference on an individual hair cell’s recovery from mechanical overstimulation has not been demonstrated. In the current work, we explored the impact of efferent stimulation on this recovery using in vitro preparations of hair cells situated in the sacculi of American bullfrogs ( Rana catesbeiana ). In the absence of efferent stimulus, exposure of a hair bundle to high-amplitude mechanical deflection detuned it from its oscillatory regime, with the extent of detuning dependent on the applied signal. Efferent actuation concomitant with the hair bundle’s relaxation from a high-amplitude deflection notably changed the recovery profile and often...

Opioid misuse among pregnant women is rapidly increasing in the United States. The number of maternal opioid-related diagnoses increased by 131% in the last 10 years, resulting in an increased number of infants exposed to opioids in utero and a subsequent increase in infants developing neonatal abstinence syndrome (NAS). The most prescribed treatment to combat maternal opioid use disorder is buprenorphine, a partial μ-opioid receptor agonist and κ-opioid receptor antagonist. Buprenorphine treatment effectively reduces NAS but has been associated with disrupted cortical development and neurodevelopmental consequences in childhood. Less is known about the long-term neurodevelopmental consequences following buprenorphine exposure in utero . Previous research has shown that gestational buprenorphine exposure can induce anxiety-like and depressive-like phenotypes in adult rats, suggesting that exposure to buprenorphine in utero may render individuals more susceptible to psychiatric illness in adulthood. A commo...

Drinking behavior has been used in fundamental research to study metabolism, motivation, decision-making and different aspects of health problems, such as anhedonia and alcohol use disorders. In the majority of studies, liquid intake is measured by weighing the bottles before and after the experiment. This method does not tell much about the drinking microstructure, e.g., licking bouts and periods of preference for each liquid, which could be valuable to understand drinking behavior. To improve data acquisition of drinking microstructure, companies have developed lickometer devices that acquire timestamps when animals approach or drink from a specific sipper. Nevertheless, commercially available devices have elevated costs. Here, we present a low-cost alternative for a lickometer system that allows wireless data acquisition of licks from eight cages with two sippers each. We ran a three-phase validation protocol to ensure (1) proper choice of the sensor to detect licks; (2) adaptation of the device to a wi...

CRISPR/Cas9-mediated knock-in methods enable the labeling of individual endogenous proteins to faithfully determine their spatiotemporal distribution in cells. However, reliable multiplexing of knock-in events in neurons remains challenging because of cross talk between editing events. To overcome this, we developed conditional activation of knock-in expression (CAKE), allowing efficient, flexible, and accurate multiplex genome editing. To diminish cross talk, CAKE is based on sequential, recombinase-driven guide RNA (gRNA) expression to control the timing of genomic integration of each donor sequence. We show that CAKE is broadly applicable in rat neurons to co-label various endogenous proteins, including cytoskeletal proteins, synaptic scaffolds, ion channels and neurotransmitter receptor subunits. To take full advantage of CAKE, we resolved the nanoscale co-distribution of endogenous synaptic proteins using super-resolution microscopy, demonstrating that their co-organization correlates with synapse siz...

Arginine vasopressin (AVP) serves as a neuromodulator in the brain. The hippocampus is one of the major targets for AVP as has been demonstrated that the hippocampus receives vasopressinergic innervation and expresses AVP receptors. The dentate gyrus (DG) granule cells (GCs) serve as a gate governing the inflow of information to the hippocampus. High densities of AVP receptors are expressed in the DG GCs. However, the roles and the underlying cellular and molecular mechanisms of AVP in the DG GCs have not been determined. We addressed this question by recording from the DG GCs in rat hippocampal slices. Our results showed that application of AVP concentration-dependently evoked an inward holding current recorded from the DG GCs. AVP depolarized the DG GCs and increased their action potential firing frequency. The excitatory effects of AVP were mediated by activation of V1a receptors and required the function of phospholipase Cβ (PLCβ). Whereas intracellular Ca2+ release and protein kinase C (PKC) activity ...

Foraging entails a complex balance between approach and avoidance alongside sensorimotor and homeostatic processes under the control of multiple cortical and subcortical areas. Recently, it has become clear that several thalamic nuclei located near the midline regulate motivated behaviors. However, one midline thalamic nucleus that project to key nodes in the foraging network, the central medial (CMT) nucleus, has received little attention so far. Therefore, the present study examined CMT contributions to foraging behavior using inactivation and unit recording techniques in male rats. Inactivation of CMT or the basolateral amygdala (BLA) with muscimol abolished the rats’ normally cautious behavior in the foraging task. Moreover, CMT neurons showed large but heterogeneous activity changes during the foraging task, with many neurons decreasing or increasing their discharge rates, with a modest bias for the latter. A generalized linear model revealed that the nature (inhibitory vs. excitatory) and relative ma...

Chemotherapy-induced peripheral neuropathy (CIPN) affects about 68% of patients undergoing chemotherapy, causing debilitating neuropathic pain and reducing quality of life. Cisplatin is a commonly used platinum-based chemotherapeutic drug known to cause CIPN, possibly by causing oxidative stress damage to primary sensory neurons. Metabotropic glutamate receptors (mGluRs) are widely hypothesized to be involved in pain processing and pain mitigation. Meclizine is an H1 histamine receptor antagonist known to have neuroprotective effects, including an anti-oxidative effect. Here, we used a mouse model of cisplatin-induced CIPN using male and female mice to test agonists of mGluR8 and group II mGluR as well as meclizine as interventions to reduce cisplatin-induced pain. We performed behavioral pain tests, and we imaged Ca2+ activity of the large population of DRG neurons in vivo . For the latter, we used a genetically-encoded Ca2+ indicator, Pirt-GCaMP3, which enabled us to monitor different drug interventions ...

Neuroestrogens are synthesized within the brain and regulate social behavior, learning and memory, and cognition. In song sparrows, Melospiza melodia , 17β-estradiol (17β-E2) promotes aggressive behavior, including during the non-breeding season when circulating steroid levels are low. Estrogens are challenging to measure because they are present at very low levels, and current techniques often lack the sensitivity required. Furthermore, current methods often focus on 17β-E2 and disregard other estrogens. Here, we developed and validated a method to measure four estrogens (estrone, 17β-E2, 17α-estradiol, estriol) simultaneously in microdissected songbird brain, with high specificity, sensitivity, accuracy, and precision. We used liquid chromatography tandem mass spectrometry (LC-MS/MS), and to improve sensitivity, we derivatized estrogens using 1,2-dimethylimidazole-5-sulfonyl-chloride (DMIS). The straightforward protocol improved sensitivity by 10-fold for some analytes. There is substantial regional vari...

Estrogen plays fundamental roles in nervous system development and function. Traditional studies examining the effect of estrogen in the brain have focused on the nuclear estrogen receptors (ERs), ERα and ERβ. Studies related to the extranuclear, membrane-bound G protein-coupled estrogen receptor (GPER/GPR30) have revealed a neuroprotective role for GPER in mature neurons. In this study, we investigated the differential effects of GPER activation in primary rat embryonic (E18) hippocampal and cortical neurons. Microscopy imaging, multielectrode array (MEA), and Ca2+ imaging experiments revealed that GPER activation with selective agonist, G-1, and non-selective agonist, 17β-estradiol (E2), increased neural growth, neural firing activity, and intracellular Ca2+ more profoundly in hippocampal neurons than in cortical neurons. The GPER-mediated Ca2+ rise in hippocampal neurons involve internal Ca2+ store release via activation of phospholipase C and extracellular entry via Ca2+ channels. Immunocytochemistry r...