Ketamine rescues anhedonia by cell-type and input specific adaptations in the Nucleus Accumbens.

Ketamine's role in providing a rapid and sustained antidepressant response, particularly for patients unresponsive to conventional treatments, is increasingly recognized. A core symptom of depression, anhedonia, or the loss of enjoyment or interest in previously pleasurable activities, is known to be significantly alleviated by ketamine. While several hypotheses have been proposed regarding the mechanisms by which ketamine alleviates anhedonia, the specific circuits and synaptic changes responsible for its sustained therapeutic effects are not yet understood. Here, we show that the nucleus accumbens (NAc), a major hub of the reward circuitry, is essential for ketamine's effect in rescuing anhedonia in mice subjected to chronic stress, a critical risk factor in the genesis of depression in humans. Specifically, a single exposure to ketamine rescues stress-induced decreased strength of excitatory synapses on NAc D1 dopamine receptor-expressing medium spiny neurons (D1-MSNs). By using a novel cell-specific pharmacology method, we demonstrate that this cell-type specific neuroadaptation is necessary for the sustained therapeutic effects of ketamine. To test for causal sufficiency, we artificially mimicked ketamine-induced increase in excitatory strength on D1-MSNs and found that this recapitulates the behavioral amelioration induced by ketamine. Finally, to determine the presynaptic origin of the relevant glutamatergic inputs for ketamine-elicited synaptic and behavioral effects, we used a combination of opto- and chemogenetics. We found that ketamine rescues stress-induced reduction in excitatory strength at medial prefrontal cortex and ventral hippocampus inputs to NAc D1-MSNs. Chemogenetically preventing ketamine-evoked plasticity at those unique inputs to the NAc reveals a ketamine-operated input-specific control of hedonic behavior. These results establish that ketamine rescues stress-induced anhedonia via cell-type-specific adaptations as well as information integration in the NAc via discrete excitatory synapses.

Structural Plasticity of Dopaminergic Neurons Requires the Activation of the D3R-nAChR Heteromer and the PI3K-ERK1/2/Akt-Induced Expression of c-Fos and p70S6K Signaling Pathway.

We have previously shown that the heteromer composed by the dopamine D3 receptor (D3R) and the nicotinic acetylcholine receptor (nAChR) (D3R-nAChR heteromer) is expressed in dopaminergic neurons, activated by nicotine and represents the molecular unit that, in these neurons, contributes to the modulation of critical events such as structural plasticity and neuroprotection. We now extended this study by investigating the D3R-nAChR heteromer properties using various cell models such as transfected HEK293 cells, primary cultures of mouse dopaminergic neurons and human dopaminergic neurons derived from induced pluripotent stem cells.We found that the D3R-nAChR heteromer is the molecular effector that transduces the remodeling properties not only associated with nicotine but also with D3R agonist stimulation: neither nAChR nor D3R, in fact, when express as monomers, are able to elicit these effects. Moreover, strong and sustained activation of the PI3K-ERK1/2/Akt pathways is coupled with D3R-nAChR heteromer stimulation, leading to the expression of the immediate-early gene c-Fos and to sustained phosphorylation of cytosolic p70 ribosomal S6 kinase (p70S6K), critical for dendritic remodeling. By contrast, while D3R stimulation results in rapid and transient activation of both Erk1/2 and Akt, that is PI3K-dependent, stimulation of nAChR is associated with persistent activation of Erk1/2 and Akt, in a PI3K-independent way. Thus, the D3R-nAChR heteromer and its ability to trigger the PI3K-ERK1/2/Akt signaling pathways may represent a novel target for preserving dopaminergic neurons healthy and for conferring neuronal protection against injuries.

Striatal Astrocytes Shape Behavioral Flexibility via Regulation of the Glutamate Transporter EAAT2.

BACKGROUND: Striatal circuits must be modulated for behavioral flexibility, the ability to adapt to environmental changes. Striatal astrocytes contribute to circuit neuromodulation by controlling the activity of ambient neurotransmitters. In particular, extracellular glutamate levels are tightly controlled by the astrocytic glutamate transporter EAAT2, influencing synaptic functioning and neural network activity. However, it remains unclear if EAAT2 responds to environmental cues to specifically shape action control. METHODS: To investigate the relationship between behavioral flexibility and experience-dependent regulation of EAAT2 expression in the dorsal striatum, mice were trained on an instrumental task. We manipulated EAAT2 expression using chemogenetic activation of astrocytic Gq signaling or in vivo morpholinos and determined the ability to adapt to novel environmental contingencies. RESULTS: The loss of behavioral flexibility with task overtraining is associated with the upregulation of EAAT2, which results in enhanced glutamate clearance and altered modulation of glutamatergic neurotransmission in the lateral part of the dorsal striatum. Interfering with EAAT2 upregulation in this striatal area preserves behavioral flexibility. CONCLUSIONS: Astrocytes are emerging as critical regulators of striatal functions. This work demonstrates that plasticity of EAAT2 expression in the lateral part of the dorsal striatum shapes behavior, thus providing novel mechanistic insights into how flexibility in action control is regulated.

Cooperative synaptic and intrinsic plasticity in a disynaptic limbic circuit drive stress-induced anhedonia and passive coping in mice.

Stress promotes negative affective states, which include anhedonia and passive coping. While these features are in part mediated by neuroadaptations in brain reward circuitry, a comprehensive framework of how stress-induced negative affect may be encoded within key nodes of this circuit is lacking. Here, we show in a mouse model for stress-induced anhedonia and passive coping that these phenomena are associated with increased synaptic strength of ventral hippocampus (VH) excitatory synapses onto D1 medium spiny neurons (D1-MSNs) in the nucleus accumbens medial shell (NAcmSh), and with lateral hypothalamus (LH)-projecting D1-MSN hyperexcitability mediated by decreased inwardly rectifying potassium channel (IRK) function. Stress-induced negative affective states are prevented by depotentiation of VH to NAcmSh synapses, restoring Kir2.1 function in D1R-MSNs, or disrupting co-participation of these synaptic and intrinsic adaptations in D1-MSNs. In conclusion, our data provide strong evidence for a disynaptic pathway controlling maladaptive emotional behavior.

µ-Opioid receptor-induced synaptic plasticity in dopamine neurons mediates the rewarding properties of anabolic androgenic steroids.

Anabolic androgenic steroids (AAS) have medical utility but are often abused, and the effects of AAS on reward circuits in the brain have been suggested to lead to addiction. We investigated the previously reported correlations between AAS and the endogenous µ-opioid system in the rewarding properties of AAS in mice. We found that a single injection of a supraphysiological dose of natural or synthetic AAS strengthened excitatory synaptic transmission in putative ventral tegmental area (VTA) dopaminergic neurons. This effect was associated with the activation of µ-opioid receptors (MORs) and an increase in ß-endorphins released into the VTA and the plasma. Irreversible blockade of MORs in the VTA counteracted two drug-seeking behaviors, locomotor activity and place preference. These data suggest that AAS indirectly stimulate a dopaminergic reward center of the brain through activation of endogenous opioid signaling and that this mechanism mediates the addictive effects of AAS.

The novel hybrid agonist HyNDA-1 targets the D3R-nAChR heteromeric complex in dopaminergic neurons.

In this paper, we designed, synthesized and tested a small set of three new derivatives potentially targeting the D3R-nAChR heteromer, a receptor complex recently identified and characterized as the molecular entity that, in dopaminergic neurons, mediates the neurotrophic effects of nicotine. By means of a partially rigidified spacer of variable length, we incorporated in the new compounds (1a-c) the pharmacophoric substructure of a known ß2-subunit-containing nAChR agonist (A-84543) and that of the D2/D3R agonist drug ropinirole. All the compounds retained the ability to bind with high affinity both ß2-subunit-containing nAChR and D3R. Compound 1a, renamed HyNDA-1, which is characterized by the shortest linker moiety, was the most interesting ligand. We found, in fact, that HyNDA-1 significantly modulated structural plasticity on both mice and human dopaminergic neurons, an effect strongly prevented by co-incubating this ligand with either nAChR or D3R antagonists. Moreover, the neurotrophic effects of HyNDA-1 were specifically lost by disrupting the complex with specific interfering peptides. Interestingly, by using the Bioluminescence Resonance Energy Transfer 2 (BRET2) assay in HEK-293 transfected cells, we also found that HyNDA-1 has the ability to increase the affinity of interaction between nAChR and D3R. Overall, our results indicate that the neurotrophic effects of HyNDA-1 are mediated by activation of the D3R-nAChR heteromeric complex specifically expressed on dopaminergic neurons.

Dopamine D3 and acetylcholine nicotinic receptor heteromerization in midbrain dopamine neurons: Relevance for neuroplasticity.

Activation of nicotinic acetylcholine receptors (nAChR) promotes the morphological remodeling of cultured dopamine (DA) neurons, an effect requiring functional DA D3 receptors (D3R). The aim of this study was to investigate the mechanisms mediating D3R-nAChR cross-talk in the modulation of DA neuron structural plasticity. By using bioluminescence resonance energy transfer2 (BRET2) and proximity ligation assay (PLA), evidence for the existence of D3R-nAChR heteromers has been obtained. In particular, BRET2 showed that the D3R directly and specifically interacts with the ß2 subunit of the nAChR. The D3R-nAChR complex was also identified in cultured DA neurons and in mouse Substantia Nigra/Ventral Tegmental Area by PLA. Cell permeable interfering peptides, containing highly charged amino acid sequences from the third intracellular loop of D3R (TAT-D3R) or the second intracellular loop of the ß2 subunit (TAT-ß2), were developed. Both peptides, but not their scrambled counterparts, significantly reduced the BRET2 signal generated by D3R-GFP2 and ß2-Rluc. Similarly, the PLA signal was undetectable in DA neurons exposed to the interfering peptides. Moreover, interfering peptides abolished the neurotrophic effects of nicotine on DA neurons. Taken together these data first demonstrate that a D3R-nAChR heteromer is present in DA neurons and represents the functional unit mediating the neurotrophic effects of nicotine.

Neuroprotective effects of the Sigma-1 receptor (S1R) agonist PRE-084, in a mouse model of motor neuron disease not linked to SOD1 mutation.

The identification of novel molecular targets crucially involved in motor neuron degeneration/survival is a necessary step for the development of hopefully more effective therapeutic strategies for amyotrophic lateral sclerosis (ALS) patients. In this view, S1R, an endoplasmic reticulum (ER)-resident receptor with chaperone-like activity, has recently attracted great interest. S1R is involved in several processes leading to acute and chronic neurodegeneration, including ALS pathology. Treatment with the S1R agonist PRE-084 improves locomotor function and motor neuron survival in presymptomatic and early symptomatic mutant SOD1-G93A ALS mice. Here, we tested the efficacy of PRE-084 in a model of spontaneous motor neuron degeneration, the wobbler mouse (wr) as a proof of concept that S1R may be regarded as a key therapeutic target also for ALS cases not linked to SOD1 mutation. Increased staining for S1R was detectable in morphologically spared cervical spinal cord motor neurons of wr mice both at early (6th week) and late (12th week) phases of clinical progression. S1R signal was also detectable in hypertrophic astrocytes and reactive microglia of wr mice. Chronic treatment with PRE-084 (three times a week, for 8weeks), starting at symptom onset, significantly increased the levels of BDNF in the gray matter, improved motor neuron survival and ameliorated paw abnormality and grip strength performance. In addition, the treatment significantly reduced the number of reactive astrocytes whereas, that of CD11b+ microglial cells was increased. A deeper evaluation of microglial markers revealed significant increased number of cells positive for the pan-macrophage marker CD68 and of CD206+ cells, involved in tissue restoration, in the white matter of PRE-084-treated mice. The mRNA levels of TNF-α and IL-1ß were not affected by PRE-084 treatment. Thus, our results support pharmacological manipulation of S1R as a promising strategy to cure ALS and point to increased availability of growth factors and modulation of astrocytosis and of macrophage/microglia as part of the mechanisms involved in S1R-mediated neuroprotection.