Optogenetic excitation in the ventral tegmental area of glutamatergic or cholinergic inputs from the laterodorsal tegmental area drives reward.

Converging evidence shows that ventral tegmental area (VTA) dopamine neurons receive laterodorsal tegmental nucleus (LDTg) cholinergic and glutamatergic inputs. To test the behavioral consequences of selectively driving the two sources of excitatory LDTg input to the VTA, channelrhodopsin-2 (ChR2) was expressed in LDTg cholinergic neurons of ChAT::Cre mice (ChAT-ChR2 mice) or in LDTg glutamatergic neurons of VGluT2::Cre mice (VGluT2-ChR2 mice). Mice were tested in a 3-chamber place preference apparatus where entry into a light-paired chamber resulted in VTA light stimulation of LDTg-cholinergic or LDTg-glutamatergic axons for the duration of a chamber stay. ChAT-ChR2 mice spent more time in the light-paired chamber and subsequently showed conditioned place preference for the light-paired chamber in the absence of light. VGluT2-ChR2 mice, entered the light-paired chamber significantly more times than a light-unpaired chamber, but remained in the light-paired chamber for short time periods and did not show a conditioned place preference. When each entry into the light-paired chamber resulted in a single train of VTA light stimulation, VGluT2-ChR2 mice entered the light-paired chamber significantly more times than the light-unpaired chamber, but spent approximately equal amounts of time in the two chambers. VTA excitation of LDTg-glutamatergic inputs may be more important for reinforcement of initial chamber entry while VTA excitation of LDTg-cholinergic inputs may be more important for the rewarding effects of chamber stays. We suggest that LDTg-cholinergic and LDTg-glutamatergic inputs to the VTA each contribute to the net rewarding effects of exciting LDTg axons in the VTA.

Glutamate inputs from the laterodorsal tegmental nucleus to the ventral tegmental area are essential for the induction of cocaine sensitization in male mice.

RATIONALE: Drug-induced potentiation of ventral tegmental area (VTA) glutamate signaling contributes critically to the induction of sensitization - an enhancement in responding to a drug following exposure which is thought to reflect neural changes underlying drug addiction. The laterodorsal tegmental nucleus (LDTg) provides one of several sources of glutamate input to the VTA. OBJECTIVE: We used optogenetic techniques to test either the role of LDTg glutamate cells or their VTA afferents in the development of cocaine sensitization in male VGluT2::Cre mice. These were inhibited using halorhodopsin during each of five daily cocaine exposure injections. The expression of locomotor sensitization was assessed following a cocaine challenge injection 1-week later. RESULTS: The locomotor sensitization seen in control mice was absent in male mice subjected to inhibition of LDTg-VTA glutamatergic circuitry during cocaine exposure. As sensitization of nucleus accumbens (NAcc) dopamine (DA) overflow is also induced by this drug exposure regimen, we used microdialysis to measure NAcc DA overflow on the test for sensitization. Consistent with the locomotor sensitization results, inhibition of LDTg glutamate afferents to the VTA during cocaine exposure prevented the sensitization of NAcc DA overflow observed in control mice. CONCLUSIONS: These data identify the LDTg as the source of VTA glutamate critical for the development of cocaine sensitization in male mice. Accordingly, the LDTg may give rise to the synapses in the VTA at which glutamatergic plasticity, known to contribute to the enhancement of addictive behaviors, occurs.

Sex-specific nicotine sensitization and imprinting of self-administration in rats inform GWAS findings on human addiction phenotypes.

Repeated nicotine exposure leads to sensitization (SST) and enhances self-administration (SA) in rodents. However, the molecular basis of nicotine SST and SA and their biological relevance to the mounting genome-wide association study (GWAS) loci of human addictive behaviors are poorly understood. Considering a gateway drug role of nicotine, we modeled nicotine SST and SA in F1 progeny of inbred rats (F344/BN) and conducted integrative genomics analyses. We unexpectedly observed male-specific nicotine SST and a parental effect of SA only present in paternal F344 crosses. Transcriptional profiling in the ventral tegmental area (VTA) and nucleus accumbens (NAc) core and shell further revealed sex- and brain region-specific transcriptomic signatures of SST and SA. We found that genes associated with SST and SA were enriched for those related to synaptic processes, myelin sheath, and tobacco use disorder or chemdependency. Interestingly, SST-associated genes were often downregulated in male VTA but upregulated in female VTA, and strongly enriched for smoking GWAS risk variants, possibly explaining the male-specific SST. For SA, we found widespread region-specific allelic imbalance of expression (AIE), of which genes showing AIE bias toward paternal F344 alleles in NAc core were strongly enriched for SA-associated genes and for GWAS risk variants of smoking initiation, likely contributing to the parental effect of SA. Our study suggests a mechanistic link between transcriptional changes underlying the NIC SST and SA and human nicotine addiction, providing a resource for understanding the neurobiology basis of the GWAS findings on human smoking and other addictive phenotypes.

Rewarding effects of M4 but not M3 muscarinic cholinergic receptor antagonism in the rostromedial tegmental nucleus.

The rostromedial tegmental nucleus (RMTg) receives inputs from the laterodorsal tegmental and pedunculopontine tegmental nuclei, the two principle brainstem cholinergic nuclei. We tested the effects of RMTg M3 and M4 muscarinic cholinergic receptor antagonism in a conditioned place preference (CPP) paradigm in mice. RMTg infusions of the M3 muscarinic cholinergic receptor antagonist 1,1-Dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP) do not result in the acquisition of CPP but increase locomotor activation. By contrast, RMTg infusions of the M4 muscarinic cholinergic receptor antagonist Tropicamide result in the acquisition of CPP but do not increase locomotor activation. The rewarding effects of RMTg Tropicamide infusions are dopamine-dependent as systemic pre-treatment with the broad-spectrum dopamine receptor antagonist flupenthixol prevents the acquisition of CPP induced by RMTg Tropicamide infusions. Under conditions of systemic dopamine receptor blockade, RMTg Tropicamide infusions significantly increase locomotor activation. These data provide further support for an important role of endogenous cholinergic input to the RMTg in reward function and suggest that the contributions of RMTg cholinergic input to rewarding and locomotor-activating effects involve differential contributions of RMTg M4 and M3 muscarinic receptors, respectively.

M5 muscarinic receptor knockout mice show reduced morphine-induced locomotion but increased locomotion after cholinergic antagonism in the ventral tegmental area.

M(5) muscarinic receptors are the only muscarinic receptor subtype expressed by mesencephalic dopamine neurons and provide an important excitatory input to mesolimbic and nigrostriatal dopamine systems. Here, we studied locomotion induced by systemic morphine (3, 10, and 30 mg/kg i.p.) in M(5) knockout mice of the C57BL/6 (B6) and CD1 x 129SvJ background strains. M(5) knockout mice of both strains showed reduced locomotion in response to 30 mg/kg morphine. B6 M(5) knockout mice were less sensitive to naltrexone in either the antagonism of morphine-induced locomotion or in the reduction of locomotion by naltrexone alone. This suggests that M(5) knockout mice are less sensitive to the effects of either exogenous or endogenous opiates on locomotion and that spontaneous locomotion in B6 mice is sustained by endogenous opiates. In B6 wild-type mice, ventral tegmental area (VTA) pretreatment with the muscarinic receptor antagonist atropine (3 microg bilateral), but not the nicotinic receptor antagonist mecamylamine (5 microg bilateral), reduced locomotion in response to 30 mg/kg morphine to a similar extent as systemic M(5) knockout, suggesting that reduced morphine-induced locomotion in M(5) knockout mice is due to the loss of M(5) receptors on VTA dopamine neurons. In contrast, in M(5) knockout mice, but not in wild-type mice, either intra-VTA atropine or mecamylamine alone increased locomotion by almost 3 times relative to saline and potentiated morphine-induced locomotion. Therefore, in M(5) knockout mice, blockade of either VTA muscarinic or nicotinic receptors increased locomotion, suggesting that in the absence of VTA M(5) receptors, VTA cholinergic inputs inhibit locomotion.

Muscarinic cholinergic receptor antagonists in the VTA and RMTg have opposite effects on morphine-induced locomotion in mice.

The ventral tegmental area (VTA) and the rostromedial tegmental nucleus (RMTg) each contribute to opiate reward and each receive inputs from the laterodorsal tegmental and pedunculopontine tegmental nuclei, the two principle brainstem cholinergic cell groups. We compared the contributions of VTA or RMTg muscarinic cholinergic receptors to locomotion induced by morphine infusions into the same sites. VTA co-infusion of atropine completely blocked VTA morphine-induced locomotion providing additional support for the important role of VTA muscarinic cholinergic receptors in the stimulant effects of opiates. By contrast, RMTg co-infusion of atropine increased RMTg morphine-induced locomotion. Furthermore, RMTg co-infusion of the M3-selective antagonist 4-DAMP, but not the M4-selective antagonist Tropicamide, strongly increased RMTg morphine-induced locomotion. RMTg infusions of 4-DAMP, but not of Tropicamide, by themselves strongly increased drug-free locomotion. Muscarinic cholinergic receptors in the RMTg thus also contribute to the stimulant effects of morphine, but in a way opposite to those in VTA. We suggest that the net effect of endogenous cholinergic input to the RMTg on drug-free and on RMTg morphine-induced locomotion is inhibitory.

Opioid-induced rewards, locomotion, and dopamine activation: A proposed model for control by mesopontine and rostromedial tegmental neurons.

Opioids, such as morphine or heroin, increase forebrain dopamine (DA) release and locomotion, and support the acquisition of conditioned place preference (CPP) or self-administration. The most sensitive sites for these opioid effects in rodents are in the ventral tegmental area (VTA) and rostromedial tegmental nucleus (RMTg). Opioid inhibition of GABA neurons in these sites is hypothesized to lead to arousing and rewarding effects through disinhibition of VTA DA neurons. We review findings that the laterodorsal tegmental (LDTg) and pedunculopontine tegmental (PPTg) nuclei, which each contain cholinergic, GABAergic, and glutamatergic cells, are important for these effects. LDTg and/or PPTg cholinergic inputs to VTA mediate opioid-induced locomotion and DA activation via VTA M5 muscarinic receptors. LDTg and/or PPTg cholinergic inputs to RMTg also modulate opioid-induced locomotion. Lesions or inhibition of LDTg or PPTg neurons reduce morphine-induced increases in forebrain DA release, acquisition of morphine CPP or self-administration. We propose a circuit model that links VTA and RMTg GABA with LDTg and PPTg neurons critical for DA-dependent opioid effects in drug-naïve rodents.

Operant responding for optogenetic excitation of LDTg inputs to the VTA requires D1 and D2 dopamine receptor activation in the NAcc.

Behavioral studies in rats and mice indicate that laterodorsal tegmental nucleus (LDTg) inputs to the ventral tegmental area (VTA) importantly contribute to reward function. Further evidence from anesthetized rat and mouse preparations suggests that these LTDg inputs may exert this effect by regulating mesolimbic dopamine (DA) signaling. Direct evidence supporting this possibility remains lacking however. To address this lack, rat LDTg neurons were transfected with adeno-associated viral vectors encoding channelrhodopsin2 and eYFP (ChR2) or eYFP alone (eYFP) and rats were subsequently trained to lever press for intracranial self-stimulation (ICSS) of the inputs of these neurons to the VTA. First, we found that DA overflow in the forebrain nucleus accumbens (NAcc) increased maximally during ICSS to approximately 240% of baseline levels in ChR2, but not in eYFP, rats. Based on these findings, we next tested the contribution of NAcc D1 and D2 DA receptors to the reinforcing effects of optogenetic excitation of LDTg inputs to the VTA. Microinjecting SCH23390 or raclopride, D1 and D2 DA receptor antagonists respectively, into the NAcc significantly reduced operant responding for this stimulation. Together these results demonstrate for the first time that optogenetic ICSS of LDTg inputs to the VTA increases DA overflow in the NAcc and requires activation of D1 and D2 DA receptors in this site.

Optogenetic excitation of LDTg axons in the VTA reinforces operant responding in rats.

The laterodorsal tegmental nucleus (LDTg) importantly contributes to regulating firing activity of midbrain dopamine neurons and forebrain dopamine levels. Whether excitation of LDTg afferents to the ventral tegmental area (VTA) can reinforce operant behavior in rats is not known. Rats received adeno-associated viral vectors encoding channelrhodopsin2 (ChR2) and EYFP or EYFP only into the LDTg and were implanted with bilateral optic probes aimed at the VTA. LDTg ChR2-infected rats, but not LDTg EYFP-infected rats acquired lever pressing to obtain photostimulation into the VTA. During reversal testing, where contingencies between response levers were reversed, LDTg ChR2-infected rats learned to press the alternate, now reinforced, lever within one session. Following pretreatment with the broad-spectrum dopamine receptor blocker flupenthixol LDTg ChR2-infected rats initiated lever-pressing with normal latencies and lever-pressed normally for the first ten minutes of the session. Lever-pressing rates were strongly reduced thereafter. These results provide further support for an important role of LDTg inputs to the VTA in appetitively motivated behaviors.

Increased latencies to initiate cocaine self-administration following laterodorsal tegmental nucleus lesions.

Cholinergic input to the ventral tegmental area (VTA), origin of the mesocorticolimbic dopamine system that is critical for cocaine reward, is important for both cocaine seeking and cocaine taking. The laterodorsal tegmental nucleus (LDTg) provides one of the two major sources of excitatory cholinergic input to the VTA, but little is known of the role of the LDTg in cocaine reward. LDTg cholinergic cells express urotensin-II receptors and here we used local microinjections of a conjugate of the endogenous ligand for these receptors with diphtheria toxin (Dtx::UII) to lesion the cholinergic cells of the LDTg in rats previously trained to self-administer cocaine (1mg/kg/infusion, i.v.). Lesioned rats showed long latencies to initiate cocaine self-administration after treatment with the toxin, which resulted in a reduction in cocaine intake per session. Priming injections reduced latencies to initiate responding for cocaine in lesioned rats, and once they began to respond the rats regulated their moment-to-moment cocaine intake within normal limits. Thus we conclude that while LDTg cholinergic cell loss does not significantly alter the rewarding effects of cocaine, LDTg lesions can reduce the rat's responsiveness to cocaine-predictive stimuli.

Stages of memory in the nematode Caenorhabditis elegans.

Studies on the cellular basis of learning and memory have revealed several distinct phases of memory that can be distinguished by their time course. In addition to the traditional short-term and long-term memory distinction, several other phases of memory have been identified: forms of intermediate-term memory, at least two seperable forms of long-term memory, and possibly several forms of short-term memory. This article presents the contributions made by research on phases of memory for habituation in the nematode Caenorhabditis elegans. Through behavioral, neural circuit and genetic analyses of habituation, research using this simple organism has provided insights into different memory phases. Studying experience-dependent plasticity of this behavior has not only provided corroborating evidence for the existence of short-, intermediate-, and long-term forms of memory, as have been demonstrated in both Aplysia and Drosophila, but also has revealed the possible existence of multiple forms of short-term memory.

Lesions of cholinergic pedunculopontine tegmental nucleus neurons fail to affect cocaine or heroin self-administration or conditioned place preference in rats.

Cholinergic input to the ventral tegmental area (VTA) is known to contribute to reward. Although it is known that the pedunculopontine tegmental nucleus (PPTg) provides an important source of excitatory input to the dopamine system, the specific role of PPTg cholinergic input to the VTA in cocaine reward has not been previously determined. We used a diphtheria toxin conjugated to urotensin-II (Dtx::UII), the endogenous ligand for urotensin-II receptors expressed by PPTg cholinergic but not glutamatergic or GABAergic cells, to lesion cholinergic PPTg neurons. Dtx::UII toxin infusion resulted in the loss of 95.78 (±0.65)% of PPTg cholinergic cells but did not significantly alter either cocaine or heroin self-administration or the development of cocaine or heroin conditioned place preferences. Thus, cholinergic cells originating in PPTg do not appear to be critical for the rewarding effects of cocaine or of heroin.

Acute food deprivation reverses morphine-induced locomotion deficits in M5 muscarinic receptor knockout mice.

Lesions of the pedunculopontine tegmental nucleus (PPT), one of two sources of cholinergic input to the ventral tegmental area (VTA), block conditioned place preference (CPP) for morphine in drug-naïve rats. M5 muscarinic cholinergic receptors, expressed by midbrain dopamine neurons, are critical for the ability of morphine to increase nucleus accumbens dopamine levels and locomotion, and for morphine CPP. This suggests that M5-mediated PPT cholinergic inputs to VTA dopamine neurons critically contribute to morphine-induced dopamine activation, reward and locomotion. In the current study we tested whether food deprivation, which reduces PPT contribution to morphine CPP in rats, could also reduce M5 contributions to morphine-induced locomotion in mice. Acute 18-h food deprivation reversed the phenotypic differences usually seen between non-deprived wild-type and M5 knockout mice. That is, food deprivation increased morphine-induced locomotion in M5 knockout mice but reduced morphine-induced locomotion in wild-type mice. Food deprivation increased saline-induced locomotion equally in wild-type and M5 knockout mice. Based on these findings, we suggest that food deprivation reduces the contribution of M5-mediated PPT cholinergic inputs to the VTA in morphine-induced locomotion and increases the contribution of a PPT-independent pathway. The contributions of cholinergic, dopaminergic and GABAergic neurons to the effects of acute food deprivation are discussed.

Emotion enhanced retention of cognitive skill learning.

Ample evidence suggests that emotional arousal enhances declarative/episodic memory. By contrast, there is little evidence that emotional enhancement of memory (EEM) extends to procedural skill based memory. We examined remote EEM (1.5-month delay) for cognitive skill learning using the weather prediction (WP) probabilistic classification task. Participants viewed interleaved emotionally arousing or neutral pictures during WP acquisition. Arousal retarded initial WP acquisition. While participants in the neutral condition showed substantial forgetting of WP learning across the 1.5-month delay interval, the arousal condition showed no evidence of forgetting across the same time period. Thus, arousal during encoding determined the mnemonic fate of cognitive skill learning. Emotional enhancement of WP retention was independent of verbally stated knowledge of WP learning and EEM for the picture contexts in which learning took place. These results reveal a novel demonstration of EEM for cognitive skill learning, and suggest that emotional arousal may in parallel enhance the neural systems that support procedural learning and its declarative context.

M5 muscarinic receptors mediate striatal dopamine activation by ventral tegmental morphine and pedunculopontine stimulation in mice.

Opiates, like other addictive drugs, elevate forebrain dopamine levels and are thought to do so mainly by inhibiting GABA neurons near the ventral tegmental area (VTA), in turn leading to a disinhibition of dopamine neurons. However, cholinergic inputs from the laterodorsal (LDT) and pedunculopontine (PPT) tegmental nucleus to the VTA and substantia nigra (SN) importantly contribute, as either LDT or PPT lesions strongly attenuate morphine-induced forebrain dopamine elevations. Pharmacological blockade of muscarinic acetylcholine receptors in the VTA or SN has similar effects. M5 muscarinic receptors are the only muscarinic receptor subtype associated with VTA and SN dopamine neurons. Here we tested the contribution of M5 muscarinic receptors to morphine-induced dopamine elevations by measuring nucleus accumbens dopamine efflux in response to intra-VTA morphine infusion using in vivo chronoamperometry. Intra-VTA morphine increased nucleus accumbens dopamine efflux in urethane-anesthetized wildtype mice starting at 10 min after infusion. These increases were absent in M5 knockout mice and were similarly blocked by pre-treatment with VTA scopolamine in wildtype mice. Furthermore, in wildtype mice electrical stimulation of the PPT evoked an initial, short-lasting increase in striatal dopamine efflux, followed 5 min later by a second prolonged increase in dopamine efflux. In M5 knockout mice, or following systemic pre-treatment with scopolamine in wildtype mice, the prolonged increase in striatal dopamine efflux was absent. The time course of increased accumbal dopamine efflux in wildtype mice following VTA morphine was consistent with both the prolonged M5-mediated excitation of striatal dopamine efflux following PPT electrical stimulation and accumbal dopamine efflux following LDT electrical stimulation. Therefore, M5 receptors appear critical for prolonged PPT excitation of dopamine efflux and for dopamine efflux induced by intra-VTA morphine.

Effects of emotional arousal on multiple memory systems: evidence from declarative and procedural learning.

Extensive evidence documents emotional modulation of hippocampus-dependent declarative memory in humans. However, little is known about the emotional modulation of striatum-dependent procedural memory. To address how emotional arousal influences declarative and procedural memory, the current study utilized (1) a picture recognition and (2) a weather prediction (WP) task (a probabilistic classification learning task), which have been shown to rely on hippocampal- and striatum-based memory systems, respectively. Observers viewed arousing or neutral pictures after (Experiment 1) or during (Experiment 2) WP training trials. A 1-wk delayed picture recognition memory test revealed enhanced declarative memory for arousing compared with neutral pictures. Arousal during encoding impaired initial WP acquisition but did not influence retention when tested after a 1-wk delay. Data from a subsequent 3-mo delayed test, however, suggested that arousal during acquisition may enhance remote WP retention. These results suggest a potential dissociation between how readily emotional arousal influences hippocampus-dependent and striatum-dependent memory systems in humans.