Christophe D. Proulx

Engineering a memory with LTD and LTP

It has been proposed that memories are encoded by modification of synaptic strengths through cellular mechanisms such as long-term potentiation (LTP) and long-term depression (LTD). However, the causal link between these synaptic processes and memory has been difficult to demonstrate. Here we show that fear conditioning, a type of associative memory, can be inactivated and reactivated by LTD and LTP, respectively. We began by conditioning an animal to associate a foot shock with optogenetic stimulation of auditory inputs targeting the amygdala, a brain region known to be essential for fear conditioning. Subsequent optogenetic delivery of LTD conditioning to the auditory input inactivates memory of the shock. Then subsequent optogenetic delivery of LTP conditioning to the auditory input reactivates memory of the shock. Thus, we have engineered inactivation and reactivation of a memory using LTD and LTP, supporting a causal link between these synaptic processes and memory.

Synaptic potentiation onto habenula neurons in the learned helplessness model of depression

The cellular basis of depressive disorders is poorly understood. Recent studies in monkeys indicate that neurons in the lateral habenula (LHb), a nucleus that mediates communication between forebrain and midbrain structures, can increase their activity when an animal fails to receive an expected positive reward or receives a stimulus that predicts aversive conditions (that is, disappointment or anticipation of a negative outcome). LHb neurons project to, and modulate, dopamine-rich regions, such as the ventral tegmental area (VTA), that control reward-seeking behaviour and participate in depressive disorders. Here we show that in two learned helplessness models of depression, excitatory synapses onto LHb neurons projecting to the VTA are potentiated. Synaptic potentiation correlates with an animal’s helplessness behaviour and is due to an enhanced presynaptic release probability. Depleting transmitter release by repeated electrical stimulation of LHb afferents, using a protocol that can be effective for patients who are depressed, markedly suppresses synaptic drive onto VTA-projecting LHb neurons in brain slices and can significantly reduce learned helplessness behaviour in rats. Our results indicate that increased presynaptic action onto LHb neurons contributes to the rodent learned helplessness model of depression.

GABA/glutamate co-release controls habenula output and is modified by antidepressant treatment

A pathway that controls our mood A brain area called the lateral habenula is involved in negative motivation and may thus play a role in depression. Shabel et al. investigated synaptic transmission in a brain pathway to the lateral habenula that transmits disappointment signals. Surprisingly, they found the simultaneous release of two antagonistic substances, glutamate and GABA, from individual nerve cells. In an animal model of depression, GABA release was reduced in this pathway. A widely used antidepressant drug, however, increased GABA co-release. These results reveal an unusual synaptic mechanism that affects lateral habenula activity. This mechanism may be instrumental for regulating the emotional impact of disappointment. Science, this issue p. 1494 The relative level of excitation and inhibition controls activity of a brain region that is linked to depression. The lateral habenula (LHb), a key regulator of monoaminergic brain regions, is activated by negatively valenced events. Its hyperactivity is associated with depression. Although enhanced excitatory input to the LHb has been linked to depression, little is known about inhibitory transmission. We discovered that γ-aminobutyric acid (GABA) is co-released with its functional opponent, glutamate, from long-range basal ganglia inputs (which signal negative events) to limit LHb activity in rodents. At this synapse, the balance of GABA/glutamate signaling is shifted toward reduced GABA in a model of depression and increased GABA by antidepressant treatment. GABA and glutamate co-release therefore controls LHb activity, and regulation of this form of transmission may be important for determining the effect of negative life events on mood and behavior.

Biological properties and functional determinants of the urotensin II receptor

The urotensin II receptor (UT) is a member of the G protein-coupled receptor (GPCR) family and binds the cyclic undecapeptide urotensin II (U-II) as well as the octapeptide urotensin II-related peptide (URP). The active UT mediates pleiotropic effects through various signal transduction pathways, including coupling to G proteins and activating the mitogen-activated protein kinase pathway. Several highly conserved residues and motifs of class A GPCRs that are important for activity are found in UT. This review highlights some of the putative roles of these motifs in the binding, activation and desensitization of UT.

Mutational Analysis of the Conserved Asp2.50 and ERY Motif Reveals Signaling Bias of the Urotensin II Receptor

Class A (rhodopsin-like) G protein-coupled receptors possess conserved residues and motifs that are important for their specific activity. In the present study, we examined the role of residue Asp972.50 as well as residues Glu1473.49, Arg1483.50, and Tyr1493.51 of the ERY motif on the functionality of the urotensin II receptor (UT). Mutations D972.50A, R1483.50A, and R1483.50H abolished the ability of UT to activate phospholipase C, whereas mutations E1473.49A and Y1493.51A reduced the ability to activate PLC by 50%. None of the mutants exhibited constitutive activity. However, R1483.50A and R1483.50H promoted ERK1/2 activation, which was abolished by 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478), an inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase activity. Both these mutants were capable of directly activating EGFR, which confirmed that they activated the mitogen-activated protein kinase (MAPK) pathway by a Gαq/11-independent transactivation of EGFR. The D972.50A, R1483.50A, and R1483.50H mutants did not readily internalize and did not promote translocation or colocalize with β-arrestin2-GFP. Finally, the agonist-induced internalization of the E1473.49A mutant receptor was significantly increased compared with wild-type receptor. This study highlights the major contribution of the conserved Asp2.50 residue to the functionality of the UT receptor. The Arg residue in the ERY motif of UT is an important structural element in signaling crossroads that determine whether Gαq/11-dependent and -independent events can occur.

Involvement of a cytoplasmic-tail serine cluster in urotensin II receptor internalization

Most G-protein-coupled receptors that undergo agonist-dependent internalization require the presence of specific cytoplasmic-tail residues to initiate interactions with proteins of the endocytic machinery. Here we show that the UT receptor (urotensin II receptor) undergoes internalization, and that specific serine residues of the receptors cytoplasmic tail participate in this process. We first observed a time-dependent increase in internalization of the UT receptor expressed in COS-7 cells following binding of the agonist urotensin II. This sequestration was significantly reduced in the presence of sucrose, demonstrating that the agonist-activated UT receptor is internalized in part by clathrin-coated pits. Moreover, the sequestered receptor was co-localized in endocytic vesicles with beta-arrestin1 and beta-arrestin2. To assess whether specific regions of the receptors cytoplasmic tail were involved in internalization, five UT receptor mutants were constructed. In four constructs the receptors cytoplasmic tail was truncated at various positions (UTDelta367, UTDelta363, UTDelta350 and UTDelta336), and in the other four adjacent serine residues at positions 364-367 were replaced by Ala (Mut4S). Each mutant, except UTDelta367, demonstrated a significantly reduced internalization rate, thereby revealing the importance of specific serine residues within the cytoplasmic tail of the UT receptor for its ability to be internalized efficiently.

Photolabelling the urotensin II receptor reveals distinct agonist- and partial-agonist-binding sites.

The mechanism by which GPCRs (G-protein-coupled receptors) undergo activation is believed to involve conformational changes following agonist binding. We have used photoaffinity labelling to identify domains within GPCRs that make contact with various photoreactive ligands in order to better understand the activation mechanism. Here, a series of four agonist {[Bpa1]U-II (Bpa is p-benzoyl-L-phenylalanine), [Bpa2]U-II, [Bpa3]U-II and [Bpa4]U-II} and three partial agonist {[Bpa1Pen5D-Trp7Orn8]U-II (Pen is penicillamine), [Bpa2Pen5D-Trp7Orn8]U-II and [Pen5Bpa6D-Trp7Orn8]U-II} photoreactive urotensin II (U-II) analogues were used to identify ligand-binding sites on the UT receptor (U-II receptor). All peptides bound the UT receptor expressed in COS-7 cells with high affinity (Kd of 0.3-17.7 nM). Proteolytic mapping and mutational analysis led to the identification of Met288 of the third extracellular loop of the UT receptor as a binding site for all four agonist peptides. Both partial agonists containing the photoreactive group in positions 1 and 2 also cross-linked to Met288. We found that photolabelling with the partial agonist containing the photoreactive group in position 6 led to the detection of transmembrane domain 5 as a binding site for that ligand. Interestingly, this differs from Met184/Met185 of the fourth transmembrane domain that had been identified previously as a contact site for the full agonist [Bpa6]U-II. These results enable us to better map the binding pocket of the UT receptor. Moreover, the data also suggest that, although structurally related agonists or partial agonists may dock in the same general binding pocket, conformational changes induced by various states of activation may result in slight differences in spatial proximity within the cyclic portion of U-II analogues.

Genetic Disruption of Circadian Rhythms in the Suprachiasmatic Nucleus Causes Helplessness, Behavioral Despair, and Anxiety-like Behavior in Mice

BACKGROUND Major depressive disorder is associated with disturbed circadian rhythms. To investigate the causal relationship between mood disorders and circadian clock disruption, previous studies in animal models have employed light/dark manipulations, global mutations of clock genes, or brain area lesions. However, light can impact mood by noncircadian mechanisms; clock genes have pleiotropic, clock-independent functions; and brain lesions not only disrupt cellular circadian rhythms but also destroy cells and eliminate important neuronal connections, including light reception pathways. Thus, a definitive causal role for functioning circadian clocks in mood regulation has not been established. METHODS We stereotactically injected viral vectors encoding short hairpin RNA to knock down expression of the essential clock gene Bmal1 into the brains master circadian pacemaker, the suprachiasmatic nucleus (SCN). RESULTS In these SCN-specific Bmal1-knockdown (SCN-Bmal1-KD) mice, circadian rhythms were greatly attenuated in the SCN, while the mice were maintained in a standard light/dark cycle, SCN neurons remained intact, and neuronal connections were undisturbed, including photic inputs. In the learned helplessness paradigm, the SCN-Bmal1-KD mice were slower to escape, even before exposure to inescapable stress. They also spent more time immobile in the tail suspension test and less time in the lighted section of a light/dark box. The SCN-Bmal1-KD mice also showed greater weight gain, an abnormal circadian pattern of corticosterone, and an attenuated increase of corticosterone in response to stress. CONCLUSIONS Disrupting SCN circadian rhythms is sufficient to cause helplessness, behavioral despair, and anxiety-like behavior in mice, establishing SCN-Bmal1-KD mice as a new animal model of depression.

A neural pathway controlling motivation to exert effort

Significance The lateral habenula, a brain region that has been implicated in depression, receives inputs from brain nuclei associated with basic emotions and drives. In this report, using fiber photometry and optogenetics on behaving rats, we show that one major lateral habenula output pathway controls the motivation to exert effort in both aversive and appetitive contexts. Overactivity of this pathway could contribute to the reduced motivation seen in human depression. The neural mechanisms conferring reduced motivation, as observed in depressed individuals, is poorly understood. Here, we examine in rodents if reduced motivation to exert effort is controlled by transmission from the lateral habenula (LHb), a nucleus overactive in depressed-like states, to the rostromedial tegmental nucleus (RMTg), a nucleus that inhibits dopaminergic neurons. In an aversive test wherein immobility indicates loss of effort, LHb→RMTg transmission increased during transitions into immobility, driving LHb→RMTg increased immobility, and inhibiting LHb→RMTg produced the opposite effects. In an appetitive test, driving LHb→RMTg reduced the effort exerted to receive a reward, without affecting the reward’s hedonic property. Notably, LHb→RMTg stimulation only affected specific aspects of these motor tasks, did not affect all motor tasks, and promoted avoidance, indicating that LHb→RMTg activity does not generally reduce movement but appears to carry a negative valence that reduces effort. These results indicate that LHb→RMTg activity controls the motivation to exert effort and may contribute to the reduced motivation in depression.