The Paraventricular Thalamic Nucleus and Its Projections in Regulating Reward and Context Associations.

The paraventricular thalamic nucleus (PVT) is a brain region that mediates aversive and reward-related behaviors as shown in animals exposed to fear conditioning, natural rewards, or drugs of abuse. However, it is unknown whether manipulations of the PVT, in the absence of external factors or stimuli (e.g., fear, natural rewards, or drugs of abuse), are sufficient to drive reward-related behaviors. Additionally, it is unknown whether drugs of abuse administered directly into the PVT are sufficient to drive reward-related behaviors. Here, using behavioral as well as pathway and cell-type specific approaches, we manipulate PVT activity as well as the PVT-to-nucleus accumbens shell (NAcSh) neurocircuit to explore reward phenotypes. First, we show that bath perfusion of morphine (10 µM) caused hyperpolarization of the resting membrane potential, increased rheobase, and decreased intrinsic membrane excitability in PVT neurons that project to the NAcSh. Additionally, we found that direct injections of morphine (50 ng) in the PVT of mice were sufficient to generate conditioned place preference (CPP) for the morphine-paired chamber. Mimicking the inhibitory effect of morphine, we employed a chemogenetic approach to inhibit PVT neurons that projected to the NAcSh and found that pairing the inhibition of these PVT neurons with a specific context evoked the acquisition of CPP. Lastly, using brain slice electrophysiology, we found that bath-perfused morphine (10 µM) significantly reduced PVT excitatory synaptic transmission on both dopamine D1 and D2 receptor-expressing medium spiny neurons in the NAcSh, but that inhibiting PVT afferents in the NAcSh was not sufficient to evoke CPP.

Anterior cingulate cortex and its projections to the ventral tegmental area regulate opioid withdrawal, the formation of opioid context associations and context-induced drug seeking.

Clinical evidence suggests that there are correlations between activity within the anterior cingulate cortex (ACC) following re-exposure to drug-associated contexts and drug craving. However, there are limited data contributing to our understanding of ACC function at the cellular level during re-exposure to drug-context associations as well as whether the ACC is directly related to context-induced drug seeking. Here, we addressed this issue by employing our novel behavioral procedure capable of measuring the formation of drug-context associations as well as context-induced drug-seeking behavior in male mice (8-12 weeks of age) that orally self-administered oxycodone. We found that mice escalated oxycodone intake during the long-access training sessions and that conditioning with oxycodone was sufficient to evoke conditioned place preference (CPP) and drug-seeking behaviors. Additionally, we found that thick-tufted, but not thin-tufted pyramidal neurons (PyNs) in the ACC as well as ventral tegmental area (VTA)-projecting ACC neurons had increased intrinsic membrane excitability in mice that self-administered oxycodone compared to controls. Moreover, we found that global inhibition of the ACC or inhibition of VTA-projecting ACC neurons was sufficient to significantly reduce oxycodone-induced CPP, drug seeking, and spontaneous opioid withdrawal. These results demonstrate a direct role of ACC activity in mediating context-induced opioid seeking among other behaviors, including withdrawal, that are associated with the DSM-V criteria of opioid use disorder.

Anterior cingulate cortex is necessary for spontaneous opioid withdrawal and withdrawal-induced hyperalgesia in male mice.

The anterior cingulate cortex (ACC) is implicated in many pathologies, including depression, anxiety, substance-use disorders, and pain. There is also evidence from brain imaging that the ACC is hyperactive during periods of opioid withdrawal. However, there are limited data contributing to our understanding of ACC function at the cellular level during opioid withdrawal. Here, we address this issue by performing ex vivo electrophysiological analysis of thick-tufted, putative dopamine D2 receptor expressing, layer V pyramidal neurons in the ACC (ACC L5 PyNs) in a mouse model of spontaneous opioid withdrawal. We found that escalating doses of morphine (20, 40, 60, 80, and 100 mg/kg, i.p. on days 1-5, respectively) injected twice daily into male C57BL/6 mice evoked withdrawal behaviors and an associated withdrawal-induced mechanical hypersensitivity. Brain slices prepared 24 h following the last morphine injection showed increases in ACC L5 thick-tufted PyN-intrinsic membrane excitability, increases in membrane resistance, reductions in the rheobase, and reductions in HCN channel-mediated currents (IH). We did not observe changes in intrinsic or synaptic properties on thin-tufted, dopamine D1-receptor-expressing ACC L5 PyNs recorded from male Drd1a-tdTomato transgenic mice. In addition, we found that chemogenetic inhibition of the ACC blocked opioid-induced withdrawal and withdrawal-induced mechanical hypersensitivity. These results demonstrate that spontaneous opioid withdrawal alters neuronal properties within the ACC and that ACC activity is necessary to control behaviors associated with opioid withdrawal and withdrawal-induced mechanical hypersensitivity. The ability of the ACC to regulate both withdrawal behaviors and withdrawal-induced mechanical hypersensitivity suggests overlapping mechanisms between two seemingly distinguishable behaviors. This commonality potentially suggests that the ACC is a locus for multiple withdrawal symptoms.

Ketamine Blocks Morphine-Induced Conditioned Place Preference and Anxiety-Like Behaviors in Mice.

Patients suffering from opioid use disorder often relapse during periods of abstinence, which is posited to be caused by negative affective states that drive motivated behaviors. Here, we explored whether conditioning mice with morphine in a conditioned place preference (CPP) training paradigm evoked anxiety-like behavior during morphine abstinence. To do this, mice were conditioned with morphine (10 mg/kg, i.p.) for 5 days. Twenty-four hours following conditioning, anxiety levels were tested by measuring time in the open arms of the elevated plus-maze. The next day, mice were placed in the three-compartment chamber to measure morphine-induced CPP. Our results show that following morphine conditioning, mice spent significantly less time in the open arm of the elevated plus-maze and expressed robust morphine CPP on CPP test day. Furthermore, we found that an acute treatment with (R,S)-ketamine (10 mg/kg, i.p.), a medication demonstrating promise for preventing anxiety-related phenotypes, 30 min before testing on post-conditioning day 1, increased time spent in the open arm of the elevated plus-maze in saline- and morphine-conditioned mice. Additionally, we found that the second injection of ketamine 30 min before CPP tests on post-conditioning day 2 prevented morphine-induced CPP, which lasted for up to 28 days post-conditioning. Furthermore, we found that conditioning mice with 10% (w/v) sucrose using an oral self-administration procedure did not evoke anxiety-like behavior, but elicited robust CPP, which was attenuated by ketamine treatment 30 min before CPP tests. Overall, our results suggest that the ketamine-induced block of morphine CPP may not be attributed solely to alleviating negative affective states, but potentially through impaired memory of morphine-context associations.

Transferrin and H-ferritin involvement in brain iron acquisition during postnatal development: impact of sex and genotype.

Iron delivery to the developing brain is essential for energy and metabolic support needed for processes such as myelination and neuronal development. Iron deficiency, especially in the developing brain, can result in a number of long-term neurological deficits that persist into adulthood. There is considerable debate that excess access to iron during development may result in iron overload in the brain and subsequently predispose individuals to age-related neurodegenerative diseases. There is a significant gap in knowledge regarding how the brain acquires iron during development and how biological variables such as development, genetics, and sex impact brain iron status. In this study, we used a mouse model expressing a mutant form of the iron homeostatic regulator protein HFE, (Hfe H63D), the most common gene variant in Caucasians, to determine impact of the mutation on brain iron uptake. Iron uptake was assessed using 59 Fe bound to either transferrin or H-ferritin as the iron carrier proteins. We demonstrate that at postnatal day 22, mutant mice brains take up greater amounts of iron compared with wildtype. Moreover, we introduce H-ferritin as a key protein in brain iron transport during development and identify a sex and genotype effect demonstrating female mutant mice take up more iron by transferrin, whereas male mutant mice take up more iron from H-ferritin at PND22. Furthermore, we begin to elucidate the mechanism for uptake using immunohistochemistry to profile the regional distribution and temporal expression of transferrin receptor and T-cell immunoglobulin and mucin domain 2, the latter is the receptor for H-ferritin. These data demonstrate that sex and genotype have significant effects on iron uptake and that regional receptor expression may play a large role in the uptake patterns during development. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/ Cover Image for this issue: doi: 10.1111/jnc.14731.

Timing of Morphine Administration Differentially Alters Paraventricular Thalamic Neuron Activity.

The paraventricular thalamic nucleus (PVT) is a brain region involved in regulating arousal, goal-oriented behaviors, and drug seeking, all key factors playing a role in substance use disorder. Given this, we investigated the temporal effects of administering morphine, an opioid with strongly addictive properties, on PVT neuronal function in mice using acute brain slices. Here, we show that morphine administration and electrophysiological recordings that occur during periods of animal inactivity (light cycle) elicit increases in PVT neuronal function during a 24-h abstinence time point. Furthermore, we show that morphine-induced increases in PVT neuronal activity at 24-h abstinence are occluded when morphine administration and recordings are performed during an animals' active state (dark cycle). Based on our electrophysiological results combined with previous findings demonstrating that PVT neuronal activity regulates drug-seeking behaviors, we investigated whether timing morphine administration with periods of vigilance (dark cycle) would decrease drug-seeking behaviors in an animal model of substance use disorder. We found that context-induced morphine-seeking behaviors were intact regardless of the time morphine was administered (e.g., light cycle or dark cycle). Our electrophysiological results suggest that timing morphine with various states of arousal may impact the firing of PVT neurons during abstinence. Although, this may not impact context-induced drug-seeking behaviors.

Adenosine receptor expression in the adult zebrafish retina.

Adenosine is an endogenous nucleoside in the central nervous system that acts on adenosine receptors. These are G protein-coupled receptors that have four known subtypes: A1, A2A, A2B, and A3 receptors. In the present study, we aimed to map the location of the adenosine receptor subtypes in adult wild-type zebrafish retina using in situ hybridization and immunohistochemistry. A1R, A2AR, and A2BR mRNA were detected in the ganglion cell layer (GCL), the inner nuclear layer (INL), the outer nuclear layer (ONL), and the outer segment (OS). A3R mRNA was detected in the GCL, ONL, and OS. A1R-immunoreactivity was expressed as puncta in the INL and in the outer plexiform layer (OPL). A1Rs were located within the cone pedicle and contiguous to horizontal cell tips in the OPL. A2AR-immunoreactivity was expressed as puncta in the GCL, inner plexiform layer (IPL), INL, and outer retina. A2AR puncta in the outer retina were situated around the ellipsoids and nuclei of cones, and weakly around the rod nuclei. A1Rs and A2ARs were clustered around ON cone bipolar cell terminals and present in the OFF lamina of the INL but were not expressed on mixed rod/cone response bipolar cell terminals. A2BR-immunoreactivity was mainly localized to the Müller cells, while A3Rs were found to be expressed in retinal ganglion cells of the GCL, INL, ONL, and OS. In summary, all four adenosine receptor subtypes were localized in the zebrafish retina and are in agreement with expression patterns shown in retinas from other species.

Morphine Differentially Alters the Synaptic and Intrinsic Properties of D1R- and D2R-Expressing Medium Spiny Neurons in the Nucleus Accumbens.

Exposure to opioids reshapes future reward and motivated behaviors partially by altering the functional output of medium spiny neurons (MSNs) in the nucleus accumbens shell. Here, we investigated how morphine, a highly addictive opioid, alters synaptic transmission and intrinsic excitability on dopamine D1-receptor (D1R) expressing and dopamine D2-receptor (D2R) expressing MSNs, the two main output neurons in the nucleus accumbens shell. Using whole-cell electrophysiology recordings, we show, that 24 h abstinence following repeated non-contingent administration of morphine (10 mg/kg, i.p.) in mice reduces the miniature excitatory postsynaptic current (mEPSC) frequency and miniature inhibitory postsynaptic current (mIPSC) frequency on D2R-MSNs, with concomitant increases in D2R-MSN intrinsic membrane excitability. We did not observe any changes in synaptic or intrinsic changes on D1R-MSNs. Last, in an attempt to determine the integrated effect of the synaptic and intrinsic alterations on the overall functional output of D2R-MSNs, we measured the input-output efficacy by measuring synaptically-driven action potential firing. We found that both D1R-MSN and D2R-MSN output was unchanged following morphine treatment.

Neuronal mechanisms mediating pathological reward-related behaviors: A focus on silent synapses in the nucleus accumbens.

The compulsive drive to seek drugs despite negative consequences relies heavily on drug-induced alterations that occur within the reward neurocircuit. These alterations include changes in neuromodulator and neurotransmitter systems that ultimately lock behaviors into an inflexible and permanent state. To provide clinicians with improved treatment options, researchers are trying to identify, as potential targets of therapeutic intervention, the neural mechanisms mediating an "addictive-like state". Here, we discuss how drug-induced generation of silent synapses in the nucleus accumbens may be a potential therapeutic target capable of reversing drug-related behaviors.