Low-Frequency Stimulation of Trpv1-Lineage Peripheral Afferents Potentiates the Excitability of Spino-Periaqueductal Gray Projection Neurons.

High-threshold dorsal root ganglion (HT DRG) neurons fire at low frequencies during inflammatory injury, and low-frequency stimulation (LFS) of HT DRG neurons selectively potentiates excitatory synapses onto spinal neurons projecting to the periaqueductal gray (spino-PAG). Here, in male and female mice, we have identified an underlying peripheral sensory population driving this plasticity and its effects on the output of spino-PAG neurons. We provide the first evidence that Trpv1-lineage sensory neurons predominantly induce burst firing, a unique mode of neuronal activity, in lamina I spino-PAG projection neurons. We modeled inflammatory injury by optogenetically stimulating Trpv1+ primary afferents at 2 Hz for 2 min (LFS), as peripheral inflammation induces 1-2 Hz firing in high-threshold C fibers. LFS of Trpv1+ afferents enhanced the synaptically evoked and intrinsic excitability of spino-PAG projection neurons, eliciting a stable increase in the number of action potentials (APs) within a Trpv1+ fiber-induced burst, while decreasing the intrinsic AP threshold and increasing the membrane resistance. Further experiments revealed that this plasticity required Trpv1+ afferent input, postsynaptic G protein-coupled signaling, and NMDA receptor activation. Intriguingly, an inflammatory injury and heat exposure in vivo also increased APs per burst, in vitro These results suggest that inflammatory injury-mediated plasticity is driven though Trpv1+ DRG neurons and amplifies the spino-PAG pathway. Spinal inputs to the PAG could play an integral role in its modulation of heat sensation during peripheral inflammation, warranting further exploration of the organization and function of these neural pathways.

Myosin Vb mobilizes recycling endosomes and AMPA receptors for postsynaptic plasticity.

Learning-related plasticity at excitatory synapses in the mammalian brain requires the trafficking of AMPA receptors and the growth of dendritic spines. However, the mechanisms that couple plasticity stimuli to the trafficking of postsynaptic cargo are poorly understood. Here we demonstrate that myosin Vb (MyoVb), a Ca2+-sensitive motor, conducts spine trafficking during long-term potentiation (LTP) of synaptic strength. Upon activation of NMDA receptors and corresponding Ca2+ influx, MyoVb associates with recycling endosomes (REs), triggering rapid spine recruitment of endosomes and local exocytosis in spines. Disruption of MyoVb or its interaction with the RE adaptor Rab11-FIP2 abolishes LTP-induced exocytosis from REs and prevents both AMPA receptor insertion and spine growth. Furthermore, induction of tight binding of MyoVb to actin using an acute chemical genetic strategy eradicates LTP in hippocampal slices. Thus, Ca2+-activated MyoVb captures and mobilizes REs for AMPA receptor insertion and spine growth, providing a mechanistic link between the induction and expression of postsynaptic plasticity.

Loss of mitochondrial enzyme GPT2 causes early neurodegeneration in locus coeruleus.

Locus coeruleus (LC) is among the first brain areas to degenerate in Alzheimer's disease and Parkinson's disease; however, the underlying causes for the vulnerability of LC neurons are not well defined. Here we report a novel mechanism of degeneration of LC neurons caused by loss of the mitochondrial enzyme glutamate pyruvate transaminase 2 (GPT2). GPT2 Deficiency is a newly-recognized childhood neurometabolic disorder. The GPT2 enzyme regulates cell growth through replenishment of tricarboxylic acid (TCA) cycle intermediates and modulation of amino acid metabolism. In Gpt2-null mice, we observe an early loss of tyrosine hydroxylase (TH)-positive neurons in LC and reduced soma size at postnatal day 18. Gpt2-null LC shows selective positive Fluoro-Jade C staining. Neuron loss is accompanied by selective, prominent microgliosis and astrogliosis in LC. We observe reduced noradrenergic projections to and norepinephrine levels in hippocampus and spinal cord. Whole cell recordings in Gpt2-null LC slices show reduced soma size and abnormal action potentials with altered firing kinetics. Strikingly, we observe early decreases in phosphorylated S6 in Gpt2-null LC, preceding prominent p62 aggregation, increased LC3B-II to LC3B-I ratio, and neuronal loss. These data are consistent with a possible mechanism involving deficiency in protein synthesis and cell growth, associated subsequently with abnormal autophagy and neurodegeneration. As compared to the few genetic animal models with LC degeneration, loss of LC neurons in Gpt2-null mice is developmentally the earliest. Early neuron loss in LC in a model of human neurometabolic disease provides important clues regarding the metabolic vulnerability of LC and may lead to new therapeutic targets.

Long-Term Depression Induced by Optogenetically Driven Nociceptive Inputs to Trigeminal Nucleus Caudalis or Headache Triggers.

The peripheral trigeminovascular pathway mediates orofacial and craniofacial pain and projects centrally to the brainstem trigeminal nucleus caudalis (TNc). Sensitization of this pathway is involved in many pain conditions, but little is known about synaptic plasticity at its first central synapse. We have taken advantage of optogenetics to investigate plasticity selectively evoked at synapses of nociceptive primary afferents onto TNc neurons. Based on immunolabeling in the trigeminal ganglia, TRPV1-lineage neurons comprise primarily peptidergic and nonpeptidergic nociceptors. Optical stimulation of channelrhodopsin-expressing axons in the TRPV1/ChR2 mouse in TNc slices thus allowed us to activate a nociceptor-enriched subset of primary afferents. We recorded from lamina I/II neurons in acutely prepared transverse TNc slices, and alternately stimulated two independent afferent pathways, one with light-activated nociceptive afferents and the other with electrically-activated inputs. Low-frequency optical stimulation induced robust long-term depression (LTD) of optically-evoked EPSCs, but not of electrically-evoked EPSCs in the same neurons. Blocking NMDA receptors or nitric oxide synthase strongly attenuated LTD, whereas a cannabinoid receptor 1 antagonist had no effect. The neuropeptide PACAP-38 or the nitric oxide donors nitroglycerin or sodium nitroprusside are pharmacologic triggers of human headache. Bath application of any of these three compounds also persistently depressed optically-evoked EPSCs. Together, our data show that LTD of nociceptive afferent synapses on trigeminal nucleus neurons is elicited when the afferents are activated at frequencies consistent with the development of central sensitization of the trigeminovascular pathway.SIGNIFICANCE STATEMENT Animal models suggest that sensitization of trigeminovascular afferents plays a major role in craniofacial pain syndromes including primary headaches and trigeminal neuralgia, yet little is known about synaptic transmission and plasticity in the brainstem trigeminal nucleus caudalis (TNc). Here we used optogenetics to selectively drive a nociceptor-enriched population of trigeminal afferents while recording from superficial laminae neurons in the TNc. Low-frequency optical stimulation evoked robust long-term depression at TRPV1/ChR2 synapses. Moreover, application of three different headache trigger drugs also depressed TRPV1/ChR2 synapses. Synaptic depression at these primary afferent synapses may represent a newly identified mechanism contributing to central sensitization during headache.

Persistent but Labile Synaptic Plasticity at Excitatory Synapses.

Short-term synaptic plasticity contributes to many computations in the brain and allows synapses to keep a finite record of recent activity. Here we have investigated the mechanisms underlying an intriguing form of short-term plasticity termed labile LTP, at hippocampal and PFC synapses in male rats and male and female mice. In the hippocampus, labile LTP is triggered by high-frequency activation of presynaptic axons and is rapidly discharged with further activation of those axons. However, if the synapses are quiescent, they remain potentiated until further presynaptic activation. To distinguish labile LTP from NMDAR-dependent forms of potentiation, we blocked NMDARs in all experiments. Labile LTP was synapse-specific and was accompanied by a decreased paired pulse ratio, consistent with an increased release probability. Presynaptic Ca2+ and protein kinase activation during the tetanus appeared to be required for its initiation. Labile LTP was not reversed by a PKC inhibitor and did not require either RIM1α or synaptotagmin-7, proteins implicated in other forms of presynaptic short-term plasticity. Similar NMDAR-independent potentiation could be elicited at synapses in mPFC. Labile LTP allows for rapid information storage that is erased under controlled circumstances and could have a role in a variety of hippocampal and prefrontal cortical computations related to short-term memory.SIGNIFICANCE STATEMENT Changes in synaptic strength are thought to represent information storage relevant to particular nervous system tasks. A single synapse can exhibit multiple overlapping forms of plasticity that shape information transfer from presynaptic to postsynaptic neurons. Here we investigate the mechanisms underlying labile LTP, an NMDAR-independent form of plasticity induced at hippocampal synapses. The potentiation is maintained for long periods as long as the synapses are infrequently active, but with regular activation, the synapses are depotentiated. Similar NMDAR-independent potentiation can also be induced at L2/3-to-L5 synapses in mPFC. Labile LTP requires a rise in presynaptic Ca2+ and protein kinase activation but is unaffected in RIM1α or synaptotagmin-7 mutant mice. Labile LTP may contribute to short-term or working memory in hippocampus and mPFC.

Synaptic function and plasticity in identified inhibitory inputs onto VTA dopamine neurons.

Ventral tegmental area (VTA) dopaminergic neurons are key components of the reward pathway, and their activity is powerfully controlled by a diverse array of inhibitory GABAergic inputs. Two major sources of GABAergic nerve terminals within the VTA are local VTA interneurons and neurons in the rostromedial tegmental nucleus (RMTg). Here, using optogenetics, we compared synaptic properties of GABAergic synapses on VTA dopamine neurons using selective activation of afferents that originate from these two cell populations. We found little evidence of co-release of glutamate from either input, but RMTg-originating synaptic currents were reduced by strychnine, suggesting co-release of glycine and GABA. VTA-originating synapses displayed a lower initial release probability, and at higher frequency stimulation, short-term depression was more marked in VTA- but not RMTg-originating synapses. We previously reported that nitric oxide (NO)-induced potentiation of GABAergic synapses on VTA dopaminergic cells is lost after exposure to drugs of abuse or acute stress; in these experiments, multiple GABAergic afferents were simultaneously activated by electrical stimulation. Here we found that optogenetically-activated VTA-originating synapses on presumptive dopamine neurons also exhibited NO-induced potentiation, whereas RMTg-originating synapses did not. Despite providing a robust inhibitory input to the VTA, RMTg GABAergic synapses are most likely not those previously shown by our work to be persistently altered by addictive drugs and stress. Our work emphasises the idea that dopamine neuron excitability is controlled by diverse inhibitory inputs expected to exert varying degrees of inhibition and to participate differently in a range of behaviours.

Long-term potentiation of glycinergic synapses triggered by interleukin 1ß.

Long-term potentiation (LTP) is a persistent increase in synaptic strength required for many behavioral adaptations, including learning and memory, visual and somatosensory system functional development, and drug addiction. Recent work has suggested a role for LTP-like phenomena in the processing of nociceptive information in the dorsal horn and in the generation of central sensitization during chronic pain states. Whereas LTP of glutamatergic and GABAergic synapses has been characterized throughout the central nervous system, to our knowledge there have been no reports of LTP at mammalian glycinergic synapses. Glycine receptors (GlyRs) are structurally related to GABAA receptors and have a similar inhibitory role. Here we report that in the superficial dorsal horn of the spinal cord, glycinergic synapses on inhibitory GABAergic neurons exhibit LTP, occurring rapidly after exposure to the inflammatory cytokine interleukin-1 beta. This form of LTP (GlyR LTP) results from an increase in the number and/or change in biophysical properties of postsynaptic glycine receptors. Notably, formalin-induced peripheral inflammation in vivo potentiates glycinergic synapses on dorsal horn neurons, suggesting that GlyR LTP is triggered during inflammatory peripheral injury. Our results define a previously unidentified mechanism that could disinhibit neurons transmitting nociceptive information and may represent a useful therapeutic target for the treatment of pain.

Stress and VTA synapses: implications for addiction and depression.

While stressful experiences are a part of everyone's life, they can also exact a major toll on health. Stressful life experiences are associated with increased substance abuse, and there exists significant co-morbidity between mental illness and substance use disorders [N.D. Volkow & T.K. Li (2004) Nat. Rev. Neurosci., 5, 963-970; G. Koob & M.J. Kreek (2007) Am. J. Psych., 164, 1149-1159; R. Sinha (2008) Annals N.Y. Acad. Sci., 1141, 105-130]. The risk for development of mood or anxiety disorders after stress is positively associated with the risk for substance use disorders [R. Sinha (2008) Annals N.Y. Acad. Sci., 1141, 105-130], suggesting that there are common substrates for vulnerability to addictive and affective disorders. Understanding the molecular and physiological substrates of stress may lead to improved therapeutic interventions for the treatment of substance use disorders and mental illnesses.

Loss of interneuron LTD and attenuated pyramidal cell LTP in Trpv1 and Trpv3 KO mice.

TRPV (transient receptor potential, vanilloid) channels are a family of nonselective cation channels that are activated by a wide variety of chemical and physical stimuli. TRPV1 channels are highly expressed in sensory neurons in the peripheral nervous system. However, a number of studies have also reported TRPV channels in the brain, though their functions are less well understood. In the hippocampus, the TRPV1 channel is a novel mediator of long-term depression (LTD) at excitatory synapses on interneurons. Here we tested the role of other TRPV channels in hippocampal synaptic plasticity, using hippocampal slices from Trpv1, Trpv3 and Trpv4 knockout (KO) mice. LTD at excitatory synapses on s. radiatum hippocampal interneurons was attenuated in slices from Trpv3 KO mice (as well as in Trpv1 KO mice as previously reported), but not in slices from Trpv4 KO mice. A previous study found that in hippocampal area CA1, slices from Trpv1 KO mice have reduced tetanus-induced long-term potentiation (LTP) following high-frequency stimulation; here we confirmed this and found a similar reduction in Trpv3 KO mice. We hypothesized that the loss of LTD at the excitatory synapses on local inhibitory interneurons caused the attenuated LTP in the mutants. Consistent with this idea, blocking GABAergic inhibition rescued LTP in slices from Trpv1 KO and Trpv3 KO mice. Our findings suggest a novel role for TRPV3 channels in synaptic plasticity and provide a possible mechanism by which TRPV1 and TRPV3 channels modulate hippocampal output.

Opioids block long-term potentiation of inhibitory synapses.

Excitatory brain synapses are strengthened or weakened in response to specific patterns of synaptic activation, and these changes in synaptic strength are thought to underlie persistent pathologies such as drug addiction, as well as learning. In contrast, there are few examples of synaptic plasticity of inhibitory GABA (gamma-aminobutyric acid)-releasing synapses. Here we report long-term potentiation of GABA(A)-mediated synaptic transmission (LTP(GABA)) onto dopamine neurons of the rat brain ventral tegmental area, a region required for the development of drug addiction. This novel form of LTP is heterosynaptic, requiring postsynaptic NMDA (N-methyl-d-aspartate) receptor activation at glutamate synapses, but resulting from increased GABA release at neighbouring inhibitory nerve terminals. NMDA receptor activation produces nitric oxide, a retrograde signal released from the postsynaptic dopamine neuron. Nitric oxide initiates LTP(GABA) by activating guanylate cyclase in GABA-releasing nerve terminals. Exposure to morphine both in vitro and in vivo prevents LTP(GABA). Whereas brief treatment with morphine in vitro blocks LTP(GABA) by inhibiting presynaptic glutamate release, in vivo exposure to morphine persistently interrupts signalling from nitric oxide to guanylate cyclase. These neuroadaptations to opioid drugs might contribute to early stages of addiction, and may potentially be exploited therapeutically using drugs targeting GABA(A) receptors.

Somatodendritic Release of Cholecystokinin Potentiates GABAergic Synapses Onto Ventral Tegmental Area Dopamine Cells.

BACKGROUND: Neuropeptides are contained in nearly every neuron in the central nervous system and can be released not only from nerve terminals but also from somatodendritic sites. Cholecystokinin (CCK), among the most abundant neuropeptides in the brain, is expressed in the majority of midbrain dopamine neurons. Despite this high expression, CCK function within the ventral tegmental area (VTA) is not well understood. METHODS: We confirmed CCK expression in VTA dopamine neurons through immunohistochemistry and in situ hybridization and detected optogenetically induced CCK release using an enzyme-linked immunosorbent assay. To investigate whether CCK modulates VTA circuit activity, we used whole-cell patch clamp recordings in mouse brain slices. We infused CCK locally in vivo and tested food intake and locomotion in fasted mice. We also used in vivo fiber photometry to measure Ca2+ transients in dopamine neurons during feeding. RESULTS: Here we report that VTA dopamine neurons release CCK from somatodendritic regions, where it triggers long-term potentiation of GABAergic (gamma-aminobutyric acidergic) synapses. The somatodendritic release occurs during trains of optogenetic stimuli or prolonged but modest depolarization and is dependent on synaptotagmin-7 and T-type Ca2+ channels. Depolarization-induced long-term potentiation is blocked by a CCK2 receptor antagonist and mimicked by exogenous CCK. Local infusion of CCK in vivo inhibits food consumption and decreases distance traveled in an open field test. Furthermore, intra-VTA-infused CCK reduced dopamine cell Ca2+ signals during food consumption after an overnight fast and was correlated with reduced food intake. CONCLUSIONS: Our experiments introduce somatodendritic neuropeptide release as a previously unknown feedback regulator of VTA dopamine cell excitability and dopamine-related behaviors.

A novel non-CB1/TRPV1 endocannabinoid-mediated mechanism depresses excitatory synapses on hippocampal CA1 interneurons.

Endocannabinoids (eCBs) mediate various forms of synaptic plasticity at excitatory and inhibitory synapses in the brain. The eCB anandamide binds to several receptors including the transient receptor potential vanilloid 1 (TRPV1) and cannabinoid receptor 1 (CB1). We recently identified that TRPV1 is required for long-term depression at excitatory synapses on CA1 hippocampal stratum radiatum interneurons. Here we performed whole-cell patch clamp recordings from CA1 stratum radiatum interneurons in rat brain slices to investigate the effect of the eCB anandamide on excitatory synapses as well as the involvement of Group I metabotropic glutamate receptors (mGluRs), which have been reported to produce eCBs endogenously. Application of the nonhydrolysable anandamide analog R-methanandamide depressed excitatory transmission to CA1 stratum radiatum interneurons by ∼50%. The Group I mGluR agonist DHPG also depressed excitatory glutamatergic transmission onto interneurons to a similar degree, and this depression was blocked by the mGluR5 antagonist MPEP (10 µM) but not by the mGluR1 antagonist CPCCOEt (50 µM). Interestingly, however, neither DHPG-mediated nor R-methanandamide-mediated depression was blocked by the TRPV1 antagonist capsazepine (10 µM), the CB1 antagonist AM-251 (2 µM) or a combination of both, suggesting the presence of a novel eCB receptor or anandamide target at excitatory hippocampal synapses. DHPG also occluded R-methanandamide depression, suggesting the possibility that the two drugs elicit synaptic depression via a shared signaling mechanism. Collectively, this study illustrates a novel CB1/TRPV1-independent eCB pathway present in the hippocampus that mediates depression at excitatory synapses on CA1 stratum radiatum interneurons.

Synaptic plasticity and addiction.

Addiction is caused, in part, by powerful and long-lasting memories of the drug experience. Relapse caused by exposure to cues associated with the drug experience is a major clinical problem that contributes to the persistence of addiction. Here we present the accumulated evidence that drugs of abuse can hijack synaptic plasticity mechanisms in key brain circuits, most importantly in the mesolimbic dopamine system, which is central to reward processing in the brain. Reversing or preventing these drug-induced synaptic modifications may prove beneficial in the treatment of one of society's most intractable health problems.

Adolescent sleep shapes social novelty preference in mice.

Sleep disturbances frequently occur in neurodevelopmental disorders such as autism, but the developmental role of sleep is largely unexplored, and a causal relationship between developmental sleep defects and behavioral consequences in adulthood remains elusive. Here, we show that in mice, sleep disruption (SD) in adolescence, but not in adulthood, causes long-lasting impairment in social novelty preference. Furthermore, adolescent SD alters the activation and release patterns of dopaminergic neurons in the ventral tegmental area (VTA) in response to social novelty. This developmental sleep function is mediated by balanced VTA activity during adolescence; chemogenetic excitation mimics, whereas silencing rescues, the social deficits of adolescent SD. Finally, we show that in Shank3-mutant mice, improving sleep or rectifying VTA activity during adolescence ameliorates adult social deficits. Together, our results identify a critical role of sleep and dopaminergic activity in the development of social interaction behavior.

Selective control of synaptically-connected circuit elements by all-optical synapses.

Understanding percepts, engrams and actions requires methods for selectively modulating synaptic communication between specific subsets of interconnected cells. Here, we develop an approach to control synaptically connected elements using bioluminescent light: Luciferase-generated light, originating from a presynaptic axon terminal, modulates an opsin in its postsynaptic target. Vesicular-localized luciferase is released into the synaptic cleft in response to presynaptic activity, creating a real-time Optical Synapse. Light production is under experimenter-control by introduction of the small molecule luciferin. Signal transmission across this optical synapse is temporally defined by the presence of both the luciferin and presynaptic activity. We validate synaptic Interluminescence by multi-electrode recording in cultured neurons and in mice in vivo. Interluminescence represents a powerful approach to achieve synapse-specific and activity-dependent circuit control in vivo.

Hyperexcitable arousal circuits drive sleep instability during aging.

Sleep quality declines with age; however, the underlying mechanisms remain elusive. We found that hyperexcitable hypocretin/orexin (Hcrt/OX) neurons drive sleep fragmentation during aging. In aged mice, Hcrt neurons exhibited more frequent neuronal activity epochs driving wake bouts, and optogenetic activation of Hcrt neurons elicited more prolonged wakefulness. Aged Hcrt neurons showed hyperexcitability with lower KCNQ2 expression and impaired M-current, mediated by KCNQ2/3 channels. Single-nucleus RNA-sequencing revealed adaptive changes to Hcrt neuron loss in the aging brain. Disruption of Kcnq2/3 genes in Hcrt neurons of young mice destabilized sleep, mimicking aging-associated sleep fragmentation, whereas the KCNQ-selective activator flupirtine hyperpolarized Hcrt neurons and rejuvenated sleep architecture in aged mice. Our findings demonstrate a mechanism underlying sleep instability during aging and a strategy to improve sleep continuity.

Hot flash: TRPV channels in the brain.

TRPV1 (transient receptor potential, vanilloid) channels belong to a family of ligand-gated ion channels gated not only by the binding of certain lipophilic molecules but also by extracellular protons and physical stimuli such as heat or osmotic pressure changes. These nonselective cation channels are permeable to Na(+) and K(+) and are also very Ca(2+) permeable; in fact, TRPV1 is as Ca(2+) permeable as the NMDA receptor channel and can, thus, act as a trigger for Ca(2+)-mediated cell signaling. Although these channels are highly expressed in primary sensory afferents, accumulating evidence indicates that TRPV family channels are also present in the brain. Here, we review evidence that TRPV channels in the central nervous system might contribute to many basic neuronal functions including resting membrane potential, neurotransmitter release and synaptic plasticity.

Drugs of abuse and stress impair LTP at inhibitory synapses in the ventral tegmental area.

Synaptic plasticity in the ventral tegmental area (VTA) is modulated by drugs of abuse and stress and is hypothesized to contribute to specific aspects of addiction. Both excitatory and inhibitory synapses on dopamine neurons in the VTA are capable of undergoing long-term changes in synaptic strength. While the strengthening or weakening of excitatory synapses in the VTA has been widely examined, the role of inhibitory synaptic plasticity in brain reward circuitry is less established. Here, we investigated the effects of drugs of abuse, as well as acute stress, on long-term potentiation of GABAergic synapses onto VTA dopamine neurons (LTP(GABA)). Morphine (10 mg/kg i.p.) reduced the ability of inhibitory synapses in midbrain slices to express LTP(GABA) both at 2 and 24 h after drug exposure but not after 5 days. Cocaine (15 mg/kg i.p.) impaired LTP(GABA) 24 h after exposure, but not at 2 h. Nicotine (0.5 mg/kg i.p.) impaired LTP(GABA) 2 h after exposure, but not after 24 h. Furthermore, LTP(GABA) was completely blocked 24 h following brief exposure to a stressful stimulus, a forced swim task. Our data suggest that drugs of abuse and stress trigger a common modification to inhibitory plasticity, synergizing with their collective effect at excitatory synapses. Together, the net effect of addictive substances or stress is expected to increase excitability of VTA dopamine neurons, potentially contributing to the early stages of addiction.

Periaqueductal Gray and Rostromedial Tegmental Inhibitory Afferents to VTA Have Distinct Synaptic Plasticity and Opiate Sensitivity.

The ventral tegmental area (VTA) is a major target of addictive drugs and receives multiple GABAergic projections originating outside the VTA. We describe differences in synaptic plasticity and behavior when optogenetically driving two opiate-sensitive GABAergic inputs to the VTA, the rostromedial tegmental nucleus (RMTg), and the periaqueductal gray (PAG). Activation of GABAergic RMTg terminals in the VTA in vivo is aversive, and low-frequency stimulation induces long-term depression in vitro. Low-frequency stimulation of PAG afferents in vitro unexpectedly causes long-term potentiation. Opioid receptor activation profoundly depresses PAG and RMTg inhibitory synapses but prevents synaptic plasticity only at PAG synapses. Activation of the GABAergic PAG terminals in the VTA promotes immobility, and optogenetically-driven immobility is blocked by morphine. Our data reveal the PAG as a source of highly opioid-sensitive GABAergic afferents and support the idea that different GABAergic pathways to the VTA control distinct behaviors.

Endogenous Opsin 3 (OPN3) Protein Expression in the Adult Brain Using a Novel OPN3-mCherry Knock-In Mouse Model.

The opsins have been studied extensively for their functions in visual phototransduction; however, the mechanisms underlying extraocular opsin signaling remain poorly understood. The first mammalian extraocular opsin to be discovered, opsin 3 (OPN3), was found in the brain more than two decades ago, yet its function remains unknown. A significant hindrance to studying OPN3 has been a lack of specific antibodies against mammalian OPN3, resulting in an incomplete understanding of its expression in the brain. Although Opn3 promoter-driven reporter mice have been generated to examine general OPN3 localization, they lack the regulated expression of the endogenous protein and the ability to study its subcellular localization. To circumvent these issues, we have created a knock-in OPN3-mCherry mouse model in which the fusion protein OPN3-mCherry is expressed under the endogenous Opn3 promoter. Viable and fertile homozygotes for the OPN3-mCherry allele were used to create an extensive map of OPN3-mCherry expression across the adult mouse brain. OPN3-mCherry was readily visualized in distinct layers of the cerebral cortex (CTX), the hippocampal formation (HCF), distinct nuclei of the thalamus, as well as many other regions in both neuronal and non-neuronal cells. Our mouse model offers a new platform to investigate the function of OPN3 in the brain.