High fat diet allows food-predictive stimuli to energize action performance in the absence of hunger, without distorting insulin signaling on accumbal cholinergic interneurons.

Obesity can disrupt how food-predictive stimuli control action performance and selection. These two forms of control recruit cholinergic interneurons (CIN) located in the nucleus accumbens core (NAcC) and shell (NAcS), respectively. Given that obesity is associated with insulin resistance in this region, we examined whether interfering with CIN insulin signaling disrupts how food-predictive stimuli control actions. To interfere with insulin signaling we used a high-fat diet (HFD) or genetic excision of the insulin receptor (InsR) from cholinergic cells. HFD left intact the capacity of food-predictive stimuli to energize performance of an action earning food when mice were tested hungry. However, it allowed this energizing effect to persist when the mice were tested sated. This persistence was linked to NAcC CIN activity but was not associated with distorted CIN insulin signaling. Accordingly, InsR excision had no effect on how food-predicting stimuli control action performance. Next, we found that neither HFD nor InsR excision altered the capacity of food-predictive stimuli to guide action selection. Yet, this capacity was associated with changes in NAcS CIN activity. These results indicate that insulin signaling on accumbal CINs does not modulate how food-predictive stimuli control action performance and selection. However, they show that HFD allows food-predictive stimuli to energize performance of an action earning food in the absence of hunger.

Basal forebrain cholinergic signaling in the basolateral amygdala promotes strength and durability of fear memories.

The basolateral amygdala (BLA) complex receives dense cholinergic projections from the nucleus basalis of Meynert (NBM) and the horizontal limb of the diagonal band of Broca (HDB). The present experiments examined whether these projections regulate the formation, extinction, and renewal of fear memories. This was achieved by employing a Pavlovian fear conditioning protocol and optogenetics in transgenic rats. Silencing NBM projections during fear conditioning weakened the fear memory produced by that conditioning and abolished its renewal after extinction. By contrast, silencing HDB projections during fear conditioning had no effect. Silencing NBM or HDB projections during extinction enhanced the loss of fear produced by extinction, but only HDB silencing prevented renewal. Next, we found that systemic blockade of nicotinic acetylcholine receptors during fear conditioning mimicked the effects produced by silencing NBM projections during fear conditioning. However, this blockade had no effect when given during extinction. These findings indicate that basal forebrain cholinergic signaling in the BLA plays a critical role in fear regulation by promoting strength and durability of fear memories. We concluded that cholinergic compounds may improve treatments for post-traumatic stress disorder by durably stripping fear memories from their fear-eliciting capacity.

Basolateral Amygdala Drives a GPCR-Mediated Striatal Memory Necessary for Predictive Learning to Influence Choice.

Predictive learning exerts a powerful influence over choice between instrumental actions. Nevertheless, how this learning is encoded in a sufficiently stable manner to influence choices that can occur much later in time is unclear. Here, we report that the basolateral amygdala (BLA) encodes predictive learning and establishes the memory necessary for future choices by driving the accumulation of delta-opioid receptors (DOPRs) on the somatic membrane of cholinergic interneurons in the nucleus accumbens shell (NAc-S). We found that the BLA controls DOPR accumulation via its influence on substance P release in the NAc-S, and that although DOPR accumulation is not necessary for predictive learning per se, it is necessary for the influence of this learning on later choice between actions. This study uncovers, therefore, a novel GPCR-based form of memory that is established by predictive learning and is necessary for such learning to guide the selection and execution of specific actions.

Effects of repeated cocaine exposure on habit learning and reversal by N-acetylcysteine.

Exposure to drugs of abuse can result in a loss of control over both drug- and nondrug-related actions by accelerating the transition from goal-directed to habitual control, an effect argued to reflect changes in glutamate homeostasis. Here we examined whether exposure to cocaine accelerates habit learning and used in vitro electrophysiology to investigate its effects on measures of synaptic plasticity in the dorsomedial (DMS) and dorsolateral (DLS) striatum, areas critical for actions and habits, respectively. We then administered N-acetylcysteine (NAC) in an attempt to normalize glutamate homeostasis and hence reverse the cellular and behavioral effects of cocaine exposure. Rats received daily injections of cocaine (30 mg/kg) for 6 days and were then trained to lever press for a food reward. We used outcome devaluation and whole-cell patch-clamp electrophysiology to assess the behavioral and cellular effects of cocaine exposure. We then examined the ability of NAC to reverse the effects of cocaine exposure on these measures. Cocaine treatment produced a deficit in goal-directed action, as assessed by outcome devaluation, and increased the frequency of spontaneous and miniature excitatory postsynaptic currents (EPSCs) in the DMS but not in the DLS. Importantly, NAC treatment both normalized EPSC frequency and promoted goal-directed control in cocaine-treated rats. The promotion of goal-directed control has the potential to improve treatment outcomes in human cocaine addicts.

δ-opioid and dopaminergic processes in accumbens shell modulate the cholinergic control of predictive learning and choice.

Decision-making depends on the ability to extract predictive information from the environment to guide future actions. Outcome-specific Pavlovian-instrumental transfer (PIT) provides an animal model of this process in which a stimulus predicting a particular outcome biases choice toward actions earning that outcome. Recent evidence suggests that cellular adaptations of δ-opioid receptors (DORs) on cholinergic interneurons (CINs) in the nucleus accumbens shell (NAc-S) are necessary for PIT. Here we found that modulation of DORs in CINs critically influences D1-receptor (D1R)-expressing projection neurons in the NAc-S to promote PIT. First, we assessed PIT-induced changes in signaling processes in dopamine D1- and D2-receptor-expressing neurons using drd2-eGFP mice, and found that PIT-related signaling was restricted to non-D2R-eGFP-expressing neurons, suggesting major involvement of D1R-neurons. Next we confirmed the role of D1Rs pharmacologically: the D1R antagonist SCH-23390, but not the D2R antagonist raclopride, infused into the NAc-S abolished PIT in rats, an effect that depended on DOR activity. Moreover, asymmetrical infusion of SCH-23390 and the DOR antagonist naltrindole into the NAc-S also abolished PIT. DOR agonists were found to sensitize the firing responses of CINs in brain slices prepared immediately after the PIT test. We confirmed the opioid-acetylcholinergic influence over D1R-neurons by selectively blocking muscarinic M4 receptors in the NAc-S, which tightly regulate the activity of D1Rs, a treatment that rescued the deficit in PIT induced by naltrindole. We describe a model of NAc-S function in which DORs modulate CINs to influence both D1R-neurons and stimulus-guided choice between goal-directed actions.

Learning-related translocation of δ-opioid receptors on ventral striatal cholinergic interneurons mediates choice between goal-directed actions.

The ability of animals to extract predictive information from the environment to inform their future actions is a critical component of decision-making. This phenomenon is studied in the laboratory using the pavlovian-instrumental transfer protocol in which a stimulus predicting a specific pavlovian outcome biases choice toward those actions earning the predicted outcome. It is well established that this transfer effect is mediated by corticolimbic afferents on the nucleus accumbens shell (NAc-S), and recent evidence suggests that δ-opioid receptors (DORs) play an essential role in this effect. In DOR-eGFP knock-in mice, we show a persistent, learning-related plasticity in the translocation of DORs to the somatic plasma membrane of cholinergic interneurons (CINs) in the NAc-S during the encoding of the specific stimulus-outcome associations essential for pavlovian-instrumental transfer. We found that increased membrane DOR expression reflected both stimulus-based predictions of reward and the degree to which these stimuli biased choice during the pavlovian-instrumental transfer test. Furthermore, this plasticity altered the firing pattern of CINs increasing the variance of action potential activity, an effect that was exaggerated by DOR stimulation. The relationship between the induction of membrane DOR expression in CINs and both pavlovian conditioning and pavlovian-instrumental transfer provides a highly specific function for DOR-related modulation in the NAc-S, and it is consistent with an emerging role for striatal CIN activity in the processing of predictive information. Therefore, our results reveal evidence of a long-term, experience-dependent plasticity in opioid receptor expression on striatal modulatory interneurons critical for the cognitive control of action.

Prolonged stimulation of µ-opioid receptors produces ß-arrestin-2-mediated heterologous desensitization of α(2)-adrenoceptor function in locus ceruleus neurons.

Prolonged agonist stimulation of the µ-opioid receptor (MOR) initiates receptor regulatory events that rapidly attenuate receptor-mediated signaling (homologous desensitization). Emerging evidence suggests that persistent MOR stimulation can also reduce responsiveness of effectors to other G-protein-coupled receptors, termed heterologous desensitization. However, the mechanisms by which heterologous desensitization is triggered by MOR stimulation are unclear. This study used whole-cell patch-clamp recordings of ligand activated G-protein-activated inwardly rectifying potassium channel currents in mouse brain slices containing locus ceruleus (LC) neurons to determine the effects of prolonged stimulation of MOR on α(2)-adrenoceptor (α(2)-AR) function. The results show distinct and sequential development of homologous and heterologous desensitization during persistent stimulation of MOR in LC neurons with Met(5)-enkephalin (ME). ME stimulation of MOR promoted rapid homologous desensitization that reached a steady state after 5 min and partially recovered over 30 min. Longer stimulation of MOR (10 min) induced heterologous desensitization of α(2)-AR function that exhibited slower recovery than homologous desensitization. Heterologous (but not homologous) desensitization required ß-arrestin-2 (ßarr-2) because it was nearly abolished in ßarr-2-knockout (ko) mice. Heterologous (but not homologous) desensitization was also prevented by inhibition of ERK1/2 and c-Src signaling in wild-type (wt) mouse LC neurons. Heterologous desensitization may be physiologically relevant during exposure to high doses of opioids because α(2)-AR-mediated slow inhibitory postsynaptic currents were depressed in wt but not ßarr-2 ko LC neurons after prolonged exposure to opioids. Together, these findings demonstrate a novel mechanism by which ßarr-2 can regulate postsynaptic responsiveness to neurotransmitter release.

Striatal cholinergic interneurons display activity-related phosphorylation of ribosomal protein S6.

Cholinergic interneurons (CINs) provide the main source of acetylcholine to all striatal regions, and strongly modulate dopaminergic actions through complex regulation of pre- and post-synaptic acetylcholine receptors. Although striatal CINs have a well-defined electrophysiological profile, their biochemical properties are poorly understood, likely due to their low proportion within the striatum (2-3%). We report a strong and sustained phosphorylation of ribosomal protein S6 on its serine 240 and 244 residues (p-Ser²4°â»²44-S6rp), a protein integrant of the ribosomal machinery related to the mammalian target of the rapamycin complex 1 (mTORC1) pathway, which we found to be principally expressed in striatal CINs in basal conditions. We explored the functional relevance of this cellular event by pharmacologically inducing various sustained physiological activity states in CINs and assessing the effect on the levels of S6rp phosphorylation. Cell-attached electrophysiological recordings from CINs in a striatal slice preparation showed an inhibitory effect of tetrodotoxin (TTX) on action potential firing paralleled by a decrease in the p-Ser²4°â»²44-S6rp signal as detected by immunofluorescence after prolonged incubation. On the other hand, elevation in extracellular potassium concentration and the addition of apamin generated an increased firing rate and a burst-firing activity in CINs, respectively, and both stimulatory conditions significantly increased Ser²4°â»²44-S6rp phosphorylation above basal levels when incubated for one hour. Apamin generated a particularly large increase in phosphorylation that was sensitive to rapamycin. Taken together, our results demonstrate for the first time a link between the state of neuronal activity and a biochemical signaling event in striatal CINs, and suggest that immunofluorescence can be used to estimate the cellular activity of CINs under different pharmacological and/or behavioral conditions.

Drug-induced GABA transporter currents enhance GABA release to induce opioid withdrawal behaviors.

Neurotransmitter transporters can affect neuronal excitability indirectly via modulation of neurotransmitter concentrations or directly via transporter currents. A physiological or pathophysiological role for transporter currents has not been described. We found that GABA transporter 1 (GAT-1) cation currents directly increased GABAergic neuronal excitability and synaptic GABA release in the periaqueductal gray (PAG) during opioid withdrawal in rodents. In contrast, GAT-1 did not indirectly alter GABA receptor responses via modulation of extracellular GABA concentrations. Notably, we found that GAT-1-induced increases in GABAergic activity contributed to many PAG-mediated signs of opioid withdrawal. Together, these data support the hypothesis that GAT-1 activity directly produces opioid withdrawal signs through direct hyperexcitation of GABAergic PAG neurons and nerve terminals, which presumably enhances GABAergic inhibition of PAG output neurons. These data provide, to the best of our knowledge, the first evidence that dysregulation of a neurotransmitter transporter current is important for the maladaptive plasticity that underlies opiate withdrawal.

Dendritic function of tau mediates amyloid-beta toxicity in Alzheimer's disease mouse models.

Alzheimer's disease (AD) is characterized by amyloid-beta (Abeta) and tau deposition in brain. It has emerged that Abeta toxicity is tau dependent, although mechanistically this link remains unclear. Here, we show that tau, known as axonal protein, has a dendritic function in postsynaptic targeting of the Src kinase Fyn, a substrate of which is the NMDA receptor (NR). Missorting of tau in transgenic mice expressing truncated tau (Deltatau) and absence of tau in tau(-/-) mice both disrupt postsynaptic targeting of Fyn. This uncouples NR-mediated excitotoxicity and hence mitigates Abeta toxicity. Deltatau expression and tau deficiency prevent memory deficits and improve survival in Abeta-forming APP23 mice, a model of AD. These deficits are also fully rescued with a peptide that uncouples the Fyn-mediated interaction of NR and PSD-95 in vivo. Our findings suggest that this dendritic role of tau confers Abeta toxicity at the postsynapse with direct implications for pathogenesis and treatment of AD.

Functional coupling of mu-receptor-Galphai-tethered proteins in AtT20 cells.

Opioid efficacy on mu-receptor may be influenced by various Gi/o-G-protein subunits interacting with intracellular face of receptor. Pertussis toxin-insensitive Galphai1 and Galphai2 subunits tethered with mu-receptor were stably transfected into AtT20 cells to (i) determine coupling of different alpha-subunits on opioid efficacy, and (ii) determine coupling to downstream effectors, for example, calcium and potassium channels. After pertussis toxin, stimulation of [35S]GTP-gamma-S incorporation persisted. Both constructs were able to couple to native calcium and potassium channels, with endomorphins 1 and 2 equally effective. However, pertussis toxin abolished opioid actions on calcium and potassium channels suggesting strong coupling to endogenous G-proteins, and that differences in coupling efficacy to Galphai1 and Galphai2 previously observed are restricted to initial step of signaling cascade.

Characterization of neurons in the rat central nucleus of the amygdala: cellular physiology, morphology, and opioid sensitivity.

The central nucleus of the amygdala (CeA) orchestrates autonomic and other behavioral and physiological responses to conditioned stimuli that are aversive or elicit fear. As a related CeA function is the expression of hypoalgesia induced by conditioned stimuli or systemic morphine administration, we examined postsynaptic opioid modulation of neurons in each major CeA subdivision. Following electrophysiological recording, biocytin-filled neurons were precisely located in CeA regions identified by chemoarchitecture (enkephalin-immunoreactivity) and cytoarchitecture (DAPI nuclear staining) in fixed adult rat brain slices. This revealed a striking distribution of physiological types, as 92% of neurons in capsular CeA were classified as late-firing, whereas no neurons in the medial CeA were of this class. In contrast, 60% or more of neurons in the lateral and medial CeA were low-threshold bursting neurons. Mu-opioid receptor (MOPR) agonists induced postsynaptic inhibitory potassium currents in 61% of CeA cells, and this ratio was maintained in each subdivision and for each physiological class of neuron. However, MOPR agonists more frequently inhibited bipolar/fusiform cells than triangular or multipolar neurons. A subpopulation of MOPR-expressing neurons were also inhibited by delta opioid receptor agonists, whereas a separate population were inhibited kappa opioid receptors (KOPR). The MOPR agonist DAMGO inhibited 9/9 CeM neurons with projections to the parabrachial nucleus identified by retrograde tracer injection. These data support models of striatopallidal organization that have identified striatal-like and pallidal-like CeA regions. Opioids can directly inhibit output from each subdivision by activating postsynaptic MOPRs or KOPRs on distinct subpopulations of opioid-sensitive neurons.

Enhanced c-Fos in periaqueductal grey GABAergic neurons during opioid withdrawal.

The ventrolateral subdivision of periaqueductal grey is crucial for expression of opioid withdrawal signs. Previous investigations suggest that interneurons rather than the medullary projecting output neurons in this midbrain region are hyperexcited during opioid withdrawal. In the present study, transgenic mice with glutamic acid decarboxylase-containing neurons coupled to enhanced green fluorescent protein expression were used as a marker for GABAergic neurons and for studying opioid withdrawal. We found that these transgenic mice exhibited a full range of opioid withdrawal signs on abrupt cessation of chronic opioid action. Consistent with previous studies, c-Fos expression was also robustly enhanced (two-fold) in the ventrolateral periaqueductal grey. Furthermore, about one third (30%) of glutamic acid decarboxylase-containing neurons coupled to enhanced green fluorescent protein in the ventrolateral periaqueductal grey were stained c-Fos positive (i.e. a four-fold increase from control mice). These results indicate hyperexcitation of GABAergic neurons in the ventrolateral periaqueductal grey during opioid withdrawal.

Opioid tolerance in periaqueductal gray neurons isolated from mice chronically treated with morphine.

The midbrain periaqueductal gray (PAG) is a major site of opioid analgesic action, and a significant site of cellular adaptations to chronic morphine treatment (CMT). We examined mu-opioid receptor (MOP) regulation of voltage-gated calcium channel currents (I(Ca)) and G-protein-activated K channel currents (GIRK) in PAG neurons from CMT mice. Mice were injected s.c. with 300 mg kg(-1) of morphine base in a slow release emulsion three times over 5 days, or with emulsion alone (vehicles). This protocol produced significant tolerance to the antinociceptive effects of morphine in a test of thermal nociception. Voltage clamp recordings were made of I(Ca) in acutely isolated PAG neurons and GIRK in PAG slices. The MOP agonist DAMGO (Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol enkephalin) inhibited I(Ca) in neurons from CMT mice (230 nM) with a similar potency to vehicle (150 nM), but with a reduced maximal effectiveness (37% inhibition in vehicle neurons, 27% in CMT neurons). Inhibition of I(Ca) by the GABA(B) agonist baclofen was not altered by CMT. Met-enkephalin-activated GIRK currents recorded in PAG slices were significantly smaller in neurons from CMT mice than vehicles, while GIRK currents activated by baclofen were unaltered. These data demonstrate that CMT-induced antinociceptive tolerance is accompanied by homologous reduction in the effectiveness of MOP agonists to inhibit I(Ca) and activate GIRK. Thus, a reduction in MOP number and/or functional coupling to G proteins accompanies the characteristic cellular adaptations to CMT previously described in PAG neurons.

Induction of delta-opioid receptor function in the midbrain after chronic morphine treatment.

Delta-opioid receptor (DOPr) activation fails to produce cellular physiological responses in many brain regions, including the periaqueductal gray (PAG), despite neural expression of high densities of the receptor. Previous histochemical studies have demonstrated that a variety of stimuli, including chronic morphine treatment, induce the translocation of DOPr from intracellular pools to the surface membrane of CNS neurons. PAG neurons in slices taken from untreated mice exhibited mu-opioid receptor (MOPr) but not DOPr-mediated presynaptic inhibition of GABAergic synaptic currents. In contrast, after 5-6 d of chronic morphine treatment, DOPr stimulation inhibited synaptic GABA release onto most neurons. Shorter exposure to morphine in vitro (upto 4 h) or in vivo (18 h) did not induce functional DOPr responses. DOPr-mediated presynaptic inhibition could not be induced in slices from untreated animals by increasing synaptic activity in vitro using high extracellular potassium concentrations or activation of protein kinase A. Induction of functional DOPr signaling by chronic morphine required MOPr expression, because no DOPr receptor responses were observed in MOPr knock-out mice. DOPr agonists also had no effect on miniature IPSCs in beta-arrestin-2 knock-out mice after chronic morphine. These results suggest that induction of DOPr-mediated actions in PAG by chronic morphine requires prolonged MOPr stimulation and expression of beta-arrestin-2.