Metabolic cues impact non-oscillatory intergeniculate leaflet and ventral lateral geniculate nucleus: standard versus high-fat diet comparative study.

The intergeniculate leaflet and ventral lateral geniculate nucleus (IGL/VLG) are subcortical structures involved in entrainment of the brain's circadian system to photic and non-photic (e.g. metabolic and arousal) cues. Both receive information about environmental light from photoreceptors, exhibit infra-slow oscillations (ISO) in vivo, and connect to the master circadian clock. Although current evidence demonstrates that the IGL/VLG communicate metabolic information and are crucial for entrainment of circadian rhythms to time-restricted feeding, their sensitivity to food intake-related peptides has not been investigated yet. We examined the effect of metabolically relevant peptides on the spontaneous activity of IGL/VLG neurons. Using ex vivo and in vivo electrophysiological recordings as well as in situ hybridisation, we tested potential sensitivity of the IGL/VLG to anorexigenic and orexigenic peptides, such as cholecystokinin, glucagon-like peptide 1, oxyntomodulin, peptide YY, orexin A and ghrelin. We explored neuronal responses to these drugs during day and night, and in standard vs. high-fat diet conditions. We found that IGL/VLG neurons responded to all the substances tested, except peptide YY. Moreover, more neurons responded to anorexigenic drugs at night, while a high-fat diet affected the IGL/VLG sensitivity to orexigenic peptides. Interestingly, ISO neurons responded to light and orexin A, but did not respond to the other food intake-related peptides. In contrast, non-ISO cells were activated by metabolic peptides, with only some being responsive to light. Our results show for the first time that peptides involved in the body's energy homeostasis stimulate the thalamus and suggest functional separation of the IGL/VLG cells. KEY POINTS: The intergeniculate leaflet and ventral lateral geniculate nucleus (IGL/VLG) of the rodent thalamus process various signals and participate in circadian entrainment. In both structures, cells exhibiting infra-slow oscillatory activity as well as non-rhythmically firing neurons being observed. Here, we reveal that only one of these two groups of cells responds to anorexigenic (cholecystokinin, glucagon-like peptide 1 and oxyntomodulin) and orexigenic (ghrelin and orexin A) peptides. Neuronal responses vary depending on the time of day (day vs. night) and on the diet (standard vs. high-fat diet). Additionally, we visualised receptors to the tested peptides in the IGL/VLG using in situ hybridisation. Our results suggest that two electrophysiologically different subpopulations of IGL/VLG neurons are involved in two separate functions: one related to the body's energy homeostasis and one associated with the subcortical visual system.

Recruitment of inhibitory neuronal pathways regulating dopaminergic activity for the control of cocaine seeking.

Drug seeking is associated with the ventral tegmental area (VTA) dopaminergic (DA) activity. Previously, we have shown that brief optogenetic inhibition of VTA DA neurons with 1 s pulses delivered every 9 s attenuates cocaine seeking under extinction conditions in rats without producing overt signs of dysphoria or locomotor sedation. Whether recruitment of neuronal pathways inhibiting VTA neuronal activity would suppress drug seeking remains unknown. Here, we asked if optogenetic stimulation of the lateral habenula (LHb) efferents in the rostromedial tegmental nucleus (RMTg) as well as RMTg efferents in VTA would reduce drug seeking. To investigate this, we measured how recruitment of elements of this inhibitory pathway affects cocaine seeking in male rats under extinction conditions. The effectiveness of brief optogenetic manipulations was confirmed electrophysiologically at the level of electrical activity of VTA DA neurons. Real-time conditioned place aversion (RT-CPA) and open field tests were performed to control for potential dysphoric/sedating effects of brief optogenetic stimulation of LHb-RMTg-VTA circuitry. Optogenetic stimulation of either RMTg or LHb inhibited VTA DAergic neuron firing, whereas similar stimulation of RMTg efferents in VTA or LHb efferents in RMTg reduced cocaine seeking under extinction conditions. Moreover, stimulation of LHb-RMTg efferents produced an effect that was maintained 24 h later, during cocaine seeking test without stimulation. This effect was specific, as brief optogenetic stimulation did not affect locomotor activity and was not aversive. Our results indicate that defined inhibitory pathways can be recruited to inhibit cocaine seeking, providing potential new targets for non-pharmacological treatment of drug craving.

High-Fat-Diet-Evoked Disruption of the Rat Dorsomedial Hypothalamic Clock Can Be Prevented by Restricted Nighttime Feeding.

Obesity is a growing health problem for modern society; therefore, it has become extremely important to study not only its negative implications but also its developmental mechanism. Its links to disrupted circadian rhythmicity are indisputable but are still not well studied on the cellular level. Circadian food intake and metabolism are controlled by a set of brain structures referred to as the food-entrainable oscillator, among which the dorsomedial hypothalamus (DMH) seems to be especially heavily affected by diet-induced obesity. In this study, we evaluated the effects of a short-term high-fat diet (HFD) on the physiology of the male rat DMH, with special attention to its day/night changes. Using immunofluorescence and electrophysiology we found that both cFos immunoreactivity and electrical activity rhythms become disrupted after as few as 4 weeks of HFD consumption, so before the onset of excessive weight gain. This indicates that the DMH impairment is a possible factor in obesity development. The DMH cellular activity under an HFD became increased during the non-active daytime, which coincides with a disrupted rhythm in food intake. In order to explore the relationship between them, a separate group of rats underwent time-restricted feeding with access to food only during the nighttime. Such an approach completely abolished the disruptive effects of the HFD on the DMH clock, confirming its dependence on the feeding schedule of the animal. The presented data highlight the importance of a temporally regulated feeding pattern on the physiology of the hypothalamic center for food intake and metabolism regulation, and propose time-restricted feeding as a possible prevention of the circadian dysregulation observed under an HFD.

Alpha1-adrenergic receptor blockade in the ventral tegmental area attenuates acquisition of cocaine-induced pavlovian associative learning.

Activity of the alpha1-adrenergic receptor (α1-AR) in the ventral tegmental area (VTA) modulates dopaminergic activity, implying its modulatory role in the behavioral functions of the dopamine (DA) system. Indeed, intra-VTA α1-AR blockade attenuates conditioned stimulus dependent behaviors such as drug seeking responses signifying a role of the noradrenergic signaling in the VTA in conditioned behaviors. Importantly, the role of the VTA α1-AR activity in Pavlovian associative learning with positive outcomes remains unknown. Here, we aimed to examine how intra-VTA α1-AR blockade affects acquisition of cocaine-induced Pavlovian associative learning in the conditioned place preference (CPP) paradigm. The impact of α1-AR blockade on cocaine-reinforced operant responding and cocaine-evoked ultrasonic vocalizations (USVs) was also studied. In addition, both α1-AR immunoreactivity in the VTA and its role in phasic DA release in the nucleus accumbens (NAc) were assessed. We demonstrated cellular localization of α1-AR expression in the VTA, providing a neuroanatomical substrate for the α1-AR mechanism. We showed that prazosin (α1-AR selective antagonist; 1 µg/0.5 µl) microinfusion attenuated electrically evoked DA transients in the NAc and dose-dependently (0.1-1 µg/0.5 µl) prevented the acquisition of cocaine CPP but did not affect cocaine-reinforced operant responding nor cocaine-induced positive affective state (measured as USVs). We propose that the VTA α1-AR signaling is necessary for the acquisition of Pavlovian associative learning but does not encode hedonic value. Thus, α1-AR signaling in the VTA might underlie salience encoding of environmental stimuli and reflect an ability of alerting/orienting functions, originating from bottom-up information processing to guide behaviors.

Racing and Pacing in the Reward System: A Multi-Clock Circadian Control Over Dopaminergic Signalling.

Level of motivation, responsiveness to rewards and punishment, invigoration of exploratory behaviours, and motor performance are subject to daily fluctuations that emerge from circadian rhythms in neuronal activity of the midbrain's dopaminergic system. While endogenous circadian rhythms are weak in the ventral tegmental area and substantia nigra pars compacta, daily changes in expression of core clock genes, ion channels, neurotransmitter receptors, dopamine-synthesising enzymes, and dopamine transporters, accompanied by changes in electrical activity, are readily observed in these nuclei. These processes cause dopamine levels released in structures innervated by midbrain dopaminergic neurons (e.g., the striatum) to oscillate in a circadian fashion. Additionally, growing evidence show that the master circadian clock located in the suprachiasmatic nucleus of the hypothalamus (SCN) rhythmically influences the activity of the dopaminergic system through various intermediate targets. Thus, circadian changes in the activity of the dopaminergic system and concomitant dopamine release observed on a daily scale are likely to be generated both intrinsically and entrained by the master clock. Previous studies have shown that the information about the value and salience of stimuli perceived by the animal is encoded in the neuronal activity of brain structures innervating midbrain dopaminergic centres. Some of these structures themselves are relatively autonomous oscillators, while others exhibit a weak endogenous circadian rhythm synchronised by the SCN. Here, we place the dopaminergic system as a hub in the extensive network of extra-SCN circadian oscillators and discuss the possible consequences of its daily entrainment for animal physiology and behaviour.

Bidirectional Communication between the Pontine Nucleus Incertus and the Medial Septum Is Carried Out by Electrophysiologically-Distinct Neuronal Populations.

Theta oscillations are key brain rhythm involved in memory formation, sensorimotor integration, and control of locomotion and behavioral states. Generation and spatiotemporal synchronization of theta oscillations rely on interactions between brain nuclei forming a large neural network, which includes pontine nucleus incertus (NI). Here we identified distinct populations of NI neurons, based on the relationship of their firing to hippocampal waves, with a special focus on theta oscillations, and the direction and type of interaction with the medial septum (MS) in male, urethane-anesthetized rats. By recording NI neuronal firing and hippocampal LFP, we described NI neurons that fire action potentials in a theta phase-independent or theta phase-locked and delta wave-independent or delta wave-locked manner. Among hippocampal activity-independent NI neurons, irregular, slow-firing, and regular, fast-firing cells were observed, while hippocampal oscillation-/wave-locked NI neurons were of a bursting or nonbursting type. By projection-specific optotagging, we revealed that only fast-firing theta phase-independent NI neurons innervate the MS, rarely receiving feedback information. In contrast, the majority of theta-bursting NI neurons were inhibited by MS stimulation, and this effect was mediated by direct GABAergic input. Described NI neuronal populations differ in reciprocal connections with the septohippocampal system, plausibly forming separate neuronal loops. Our results suggest that theta phase-independent NI neurons participate in theta rhythm generation through direct innervation of the MS, while theta-bursting NI neurons further transmit the rhythmic signal received from the MS to stabilize and/or strengthen rhythmic activity in other structures.SIGNIFICANCE STATEMENT The generation and spatiotemporal synchronization of theta oscillations rely on interactions between nuclei forming a large neural network, part of which is the pontine nucleus incertus (NI). Here we describe that within NI there are populations of neurons that can be distinguished based on the relationship of their firing to hippocampal theta oscillations and delta waves. We show that these neuronal populations largely do not have reciprocal connections with the septohippocampal system, but form separate neuronal loops. Our results suggest that medial septum (MS)-projecting, fast-firing, theta phase-independent NI neurons may participate in theta rhythm generation through direct innervation of the MS, while theta-bursting NI neurons may further transmit the rhythmic signal received from the MS to other structures.

Rhythmic neuronal activities of the rat nucleus of the solitary tract are impaired by high-fat diet - implications for daily control of satiety.

Temporal partitioning of daily food intake is crucial for survival and involves the integration of internal circadian states and external influences such as the light-dark cycle and dietary composition. These intrinsic and extrinsic factors are interdependent with misalignment of circadian rhythms promoting body weight gain, while consumption of a calorie-dense diet elevates the risk of obesity and blunts circadian rhythms. Recently, we defined the circadian properties of the dorsal vagal complex of the brainstem, a structure implicated in the control of food intake and autonomic tone, but whether and how 24 h rhythms in this area are influenced by diet remains unresolved. Here we focused on a key structure of this complex, the nucleus of the solitary tract (NTS). We used a combination of immunohistochemical and electrophysiological approaches together with daily monitoring of body weight and food intake to interrogate how the neuronal rhythms of the NTS are affected by a high-fat diet. We report that short-term consumption of a high-fat diet increases food intake during the day and blunts NTS daily rhythms in neuronal discharge. Additionally, we found that a high-fat diet dampens NTS responsiveness to metabolic neuropeptides, and decreases orexin immunoreactive fibres in this structure. These alterations occur without prominent body weight gain, suggesting that a high-fat diet acts initially to reduce activity in the NTS to disinhibit mechanisms that suppress daytime feeding. KEY POINTS: The dorsal vagal complex of the rodent hindbrain possesses intrinsic circadian timekeeping mechanisms In particular, the nucleus of the solitary tract (NTS) is a robust circadian oscillator, independent of the master suprachiasmatic clock Here, we reveal that rat NTS neurons display timed daily rhythms in their neuronal activity and responsiveness to ingestive cues These daily rhythms are blunted or eliminated by a short-term high-fat diet, together with increased consumption of calories during the behaviourally quiescent day Our results help us better understand the circadian control of satiety by the brainstem and its malfunctioning under a high-fat diet.

Daily changes in neuronal activities of the dorsal motor nucleus of the vagus under standard and high-fat diet.

KEY POINTS: Recently, we found that the dorsal vagal complex displays autonomous circadian timekeeping properties  The dorsal motor nucleus of the vagus (DMV) is an executory part of this complex - a source of parasympathetic innervation of the gastrointestinal tract  Here, we reveal daily changes in the neuronal activities of the rat DMV, including firing rate, intrinsic excitability and synaptic input - all of these peaking in the late day  Additionally, we establish that short term high-fat diet disrupts these daily rhythms, boosting the variability in the firing rate, but blunting the DMV responsiveness to ingestive cues  These results help us better understand daily control over parasympathetic outflow and provide evidence on its dependence on the high-fat diet ABSTRACT: The suprachiasmatic nuclei (SCN) of the hypothalamus function as the brain's primary circadian clock, but circadian clock genes are also rhythmically expressed in several extra-SCN brain sites where they can exert local temporal control over physiology and behaviour. Recently, we found that the hindbrain dorsal vagal complex possesses strong daily timekeeping capabilities, with the area postrema and nucleus of the solitary tract exhibiting the most robust clock properties. The possibility that the executory part of this complex - the dorsal motor nucleus of the vagus (DMV) - also exhibits daily changes has not been extensively studied. The DMV is the source of vagal efferent motoneurons that regulate gastric motility and emptying and consequently influence meal size and energy homeostasis. We used a combination of multi-channel electrophysiology and patch clamp recordings to gain insight into effects of time of day and diet on these DMV cells. We found that DMV neurons increase their spontaneous activity, excitability and responsiveness to metabolic neuromodulators at late day and this was paralleled with an enhanced synaptic input to these neurons. A high-fat diet typically damps circadian rhythms, but we found that consumption of a high-fat diet paradoxically amplified daily variation of DMV neuronal activity, while blunting the neurons responsiveness to metabolic neuromodulators. In summary, we show for the first time that DMV neural activity changes with time of day, with this temporal variation modulated by diet. These findings have clear implications for our understanding of the daily control of vagal efferents and parasympathetic outflow.

Intrinsic circadian timekeeping properties of the thalamic lateral geniculate nucleus.

Circadian rhythmicity in mammals is sustained by the central brain clock-the suprachiasmatic nucleus of the hypothalamus (SCN), entrained to the ambient light-dark conditions through a dense retinal input. However, recent discoveries of autonomous clock gene expression cast doubt on the supremacy of the SCN and suggest circadian timekeeping mechanisms devolve to local brain clocks. Here, we use a combination of molecular, electrophysiological, and optogenetic tools to evaluate intrinsic clock properties of the main retinorecipient thalamic center-the lateral geniculate nucleus (LGN) in male rats and mice. We identify the dorsolateral geniculate nucleus as a slave oscillator, which exhibits core clock gene expression exclusively in vivo. Additionally, we provide compelling evidence for intrinsic clock gene expression accompanied by circadian variation in neuronal activity in the intergeniculate leaflet and ventrolateral geniculate nucleus (VLG). Finally, our optogenetic experiments propose the VLG as a light-entrainable oscillator, whose phase may be advanced by retinal input at the beginning of the projected night. Altogether, this study for the first time demonstrates autonomous timekeeping mechanisms shaping circadian physiology of the LGN.

Daily coordination of orexinergic gating in the rat superior colliculus-Implications for intrinsic clock activities in the visual system.

The orexinergic system delivers excitation for multiple brain centers to facilitate behavioral arousal, with its malfunction resulting in narcolepsy, somnolence, and notably, visual hallucinations. Since the circadian clock underlies the daily arousal, a timed coordination is expected between the orexin system and its target subcortical visual system, including the superior colliculus (SC). Here, we use a combination of electrophysiological, immunohistochemical, and molecular approaches across 24 h, together with the neuronal tract-tracing methods to investigate the daily coordination between the orexin system and the rodent SC. Higher orexinergic input was found to occur nocturnally in the superficial layers of the SC, in time for nocturnal silencing of spontaneous firing in this visual brain area. We identify autonomous daily and circadian expression of clock genes in the SC, which may underlie these day-night changes. Additionally, we establish the lateral hypothalamic origin of the orexin innervation to the SC and that the SC neurons robustly respond to orexin A via OX2 receptor in both excitatory and GABAA receptor-dependent inhibitory manners. Together, our evidence elucidates the combination of intrinsic and extrinsic clock mechanisms that shape the daily function of the visual layers of the SC.

Orexin A excites the rat olivary pretectal nucleus via OX2 receptor in a daily manner.

Pronounced environmental changes between the day and night led to evolution of specialised mechanisms organising their daily physiology, named circadian clocks. Currently, it has become clear that the master clock in the suprachiasmatic nuclei of the hypothalamus is not an exclusive brain site to generate daily rhythms. Indeed, several brain areas, including the subcortical visual system have been recently shown to change their neuronal activity across the daily cycle. Here we focus our investigation on the olivary pretectal nucleus (OPN) - a retinorecipient structure primarily involved in the pupillary light reflex. Using the multi-electrode array technology ex vivo we provide evidence for OPN neurons to elevate their firing during the behaviourally quiescent light phase. Additionally, we report the robust responsivity to orexin A via the identified OX2 receptor in this pretectal centre, with higher responsiveness noted during the night. Interestingly, we likewise report a daily variation in the response to PAC1 receptor activation, with implications for the convergence of orexinergic and visual input on the same OPN neurons. Altogether, our report is first to suggest a daily modulation of the OPN activity via intrinsic and extrinsic mechanisms, organising its temporal physiology.

Phasic Neuronal Firing in the Rodent Nucleus of the Solitary Tract ex vivo.

Phasic pattern of neuronal activity has been previously described in detail for magnocellular vasopressin neurons in the hypothalamic paraventricular and supraoptic nuclei. This characteristic bistable pattern consists of alternating periods of electrical silence and elevated neuronal firing, implicated in neuropeptide release. Here, with the use of multi-electrode array recordings ex vivo, we aimed to study the firing pattern of neurons in the nucleus of the solitary tract (NTS) - the brainstem hub for homeostatic, cardio-vascular, and metabolic processes. Our recordings from the mouse and rat hindbrain slices reveal the phasic activity pattern to be displayed by a subset of neurons in the dorsomedial NTS subjacent to the area postrema (AP), with the inter-spike interval distribution closely resembling that reported for phasic magnocellular vasopressin cells. Additionally, we provide interspecies comparison, showing higher phasic frequency and firing rate of phasic NTS cells in mice compared to rats. Further, we describe daily changes in their firing rate and pattern, peaking at the middle of the night. Last, we reveal these phasic cells to be sensitive to α 2 adrenergic receptors activation and to respond to electrical stimulation of the AP. This study provides a comprehensive description of the phasic neuronal activity in the rodent NTS and identifies it as a potential downstream target of the AP noradrenergic system.

Superior Colliculus Controls the Activity of the Rostromedial Tegmental Nuclei in an Asymmetrical Manner.

Dopaminergic (DA) neurons of the midbrain are involved in controlling orienting and approach of animals toward relevant external stimuli. The firing of DA neurons is regulated by many brain structures; however, the sensory input is provided predominantly by the ipsilateral superior colliculus (SC). It is suggested that SC also innervates the contralateral rostromedial tegmental nucleus (RMTg)-the main inhibitory input to DA neurons. Therefore, this study aimed to describe the physiology and anatomy of the SC-RMTg pathway. To investigate the anatomic connections within the circuit of interest, anterograde, retrograde, and transsynaptic tract-tracing studies were performed on male Sprague Dawley rats. We have observed that RMTg is monosynaptically innervated predominantly by the lateral parts of the intermediate layer of the contralateral SC. To study the physiology of this neuronal pathway, we conducted in vivo electrophysiological experiments combined with optogenetics; the activity of RMTg neurons was recorded using silicon probes, while either contralateral or ipsilateral SC was optogenetically stimulated. Obtained results revealed that activation of the contralateral SC excites the majority of RMTg neurons, while stimulation of the ipsilateral SC evokes similar proportions of excitatory or inhibitory responses. Consequently, single-unit recordings showed that the activation of RMTg neurons innervated by the contralateral SC, or stimulation of contralateral SC-originating axon terminals within the RMTg, inhibits midbrain DA neurons. Together, the anatomy and physiology of the discovered brain circuit suggest its involvement in the orienting and motivation-driven locomotion of animals based on the direction of external sensory stimuli.SIGNIFICANCE STATEMENT Dopaminergic neurons are the target of predominantly ipsilateral, excitatory innervation originating from the superior colliculus. However, we demonstrate in our study that SC inhibits the activity of dopaminergic neurons on the contralateral side of the brain via the rostromedial tegmental nucleus. In this way, sensory information received by the animal from one hemifield could induce opposite effects on both sides of the dopaminergic system. It was shown that the side to which an animal directs its behavior is a manifestation of asymmetry in dopamine release between left and right striatum. Animals tend to move oppositely to the hemisphere with higher striatal dopamine concentration. This explains how the above-described circuit might guide the behavior of animals according to the direction of incoming sensory stimuli.

Circadian actions of orexins on the retinorecipient lateral geniculate complex in rat.

KEY POINTS: Rhythmic processes in living organisms are controlled by biological clocks. The orexinergic system of the lateral hypothalamus carries circadian information to provide arousal for the brain during the active phase. Here, we show that orexins exert an excitatory action in three parts of the lateral geniculate nucleus (LGN), in particular upon directly retinorecipient neurons in the non-image forming visual structures. We provide evidence for the high nocturnal levels of orexins with stable circadian expression of predominant orexin receptor 2 in the LGN. Our data additionally establish the convergence of orexinergic and pituitary adenylate cyclase (PAC)-activating peptide/PAC1 receptor systems (used by melanopsin-expressing retinal ganglion cells), which directly regulates responses to the retinal input. These results help us better understand circadian orexinergic control over the non-image forming subcortical visual system, forming the animal's preparedness for the behaviourally active night. ABSTRACT: The orexinergic system of the lateral hypothalamus is tightly interlinked with the master circadian clock and displays daily variation in activity to provide arousal-related excitation for the plethora of brain structures in a circadian manner. Here, using a combination of electrophysiological, optogenetic, histological, molecular and neuronal tracing methods, we explore a particular link between orexinergic and visual systems in rat. The results of the present study demonstrate that orexinergic fibre density at the area of subcortical visual system exerts a clear day to night variability, reaching a maximum at behaviourally active night. We also show pronounced electrophysiological activations of neurons in the lateral geniculate nucleus by orexin A through 24 h, via identified distinct orexin receptors, with the ventrolateral geniculate displaying a daily cycle of responsiveness. In addition, for the first time, we provide a direct evidence for orexins to act on retinorecipient neurons with a high convergence of orexinergic and putatively retinal pituitary adenylate cyclase (PAC)-activating peptide/PAC1 receptor systems. Altogether, the present study ties orexins to non-image forming visual structures with implications for circadian orexinergic modulation of neurons, which process information on ambient light levels.

Effects of brief inhibition of the ventral tegmental area dopamine neurons on the cocaine seeking during abstinence.

Preclinical studies strongly suggest that cocaine seeking depends on the neuronal activity of the ventral tegmental area (VTA) and phasic dopaminergic (DA) signaling. Notably, VTA pharmacological inactivation or dopamine receptor blockade in the forebrain may induce behavioral inhibition in general and acute aversive states in particular, thus reducing cocaine seeking indirectly. Such artifacts hinder successful translation of these findings in clinical studies and practice. Here, we aimed to evaluate if dynamic VTA manipulations effectively reduce cocaine seeking. We used male tyrosine hydroxylase (TH) IRES-Cre+ rats along with optogenetic tools to inhibit directly and briefly VTA DA neurons during conditioned stimulus (CS)-induced cocaine seeking under extinction conditions. The behavioral effects of optogenetic inhibition were also assessed in the real-time dynamic place aversion, conditioned place aversion, and CS-induced food-seeking tests. We found that brief and nondysphoric/nonsedative pulses of VTA photo-inhibition (1 s every 9 s, ie, for 10% of time) attenuated CS-induced cocaine seeking under extinction conditions in rats expressing archaerhodopsin selectively on the TH+ neurons. Furthermore, direct inhibition of the VTA DA activity reduced CS-induced cocaine seeking 24 hours after photo-modulation. Importantly, such effect appears to be selective for cocaine seeking as similar inhibition of the VTA DA activity had no effect on CS-induced food seeking. Thus, briefly inhibiting VTA DA activity during CS-induced cocaine seeking drastically and selectively reduces seeking without behavioral artifacts such as sedation or dysphoria. Our results point to the therapeutic possibilities of coupling nonpharmacologic treatments with extinction training in reducing cocaine addiction.

Noradrenergic and corticosteroid receptors regulate somatic and motivational symptoms of morphine withdrawal.

Somatic and motivational symptoms accompanying opiate withdrawal are considered one of the major reasons for relapse to opiate-seeking and opiate-taking behaviors. These symptoms are accompanied by the activation of stress-related processes including hypothalamic-pituitary-adrenal axis activity and noradrenergic (NA) signaling. In particular, the NA system plays an important role in the expression of somatic signs of opiate withdrawal, whereas glucocorticoid (GR) and mineralocorticoid receptors (MR) are activated during opiate abstinence. The purpose of our study was to examine the roles of α1-, α2-, and ß-adrenoceptors (ARs) as well as GR and MR, in the formation and expression of physiological and motivational symptoms of morphine withdrawal. We showed that systemic pretreatment with the selective α1-AR antagonist prazosin (0-1 mg/kg), the selective α2-AR antagonist RX821002 (0-2 mg/kg), the selective ß-adrenergic antagonist, propranolol (0-10 mg/kg), or the selective MR antagonist spironolactone (0-50 mg/kg), but not the selective GR antagonist mifepristone (0-40 mg/kg), decreased somatic symptoms of naloxone-precipitated morphine withdrawal in mice chronically treated with morphine. In contrast, only propranolol pretreatment attenuated the dysphoric affective state accompanying naloxone-precipitated morphine withdrawal as assessed in the conditioned place aversion (N-CPA) paradigm. Together, our results demonstrate the important roles of noradrenergic receptors in the modulation of somatic, but not motivational/affective, symptoms of morphine withdrawal. In addition MR but not GR regulates the expression of only somatic symptoms of morphine withdrawal.

Noradrenergic signaling in the VTA modulates cocaine craving.

Exposure to drug-associated cues evokes drug-seeking behavior and is regarded as a major cause of relapse. Conditional stimulus upregulates noradrenaline (NA) system activity, but the drug-seeking behavior depends particularly on phasic dopamine signaling downstream from the ventral tegmental area (VTA). The VTA dopamine-ergic activity is regulated via the signaling of alpha1 -adrenergic and alpha2 -adrenergic receptors (α1 -ARs and α2 -ARs); thus, the impact of the conditional stimulus on drug-seeking behavior might involve NAergic signaling in the VTA. To date, the role of VTA ARs in regulating cocaine seeking was not studied. We found that cocaine seeking under extinction conditions in male Sprague-Dawley rats was attenuated by intra-VTA prazosin or terazosin-two selective α1 -AR antagonists. In contrast, cocaine seeking was facilitated by intra-VTA administration of the selective α1 -AR agonist phenylephrine as well as α2 -AR antagonist RX 821002, whereas the selective ß-AR antagonist propranolol had no effects. In addition, blockade of α1 -AR in the VTA prevented α2 -AR antagonist-induced enhancement of cocaine seeking. Importantly, the potential non-specific effects of the VTA AR blockade on cocaine seeking could be excluded, because none of the AR antagonists influenced sucrose seeking under extinction conditions or locomotor activity in the open field test. These results demonstrate that NAergic signaling potently and selectively regulates cocaine seeking during early cocaine withdrawal via VTA α1 -AR and α2 -AR but not ß-AR. Our findings provide new insight into the NAergic mechanisms that underlie cocaine craving.

Adrenergic Receptor Agonists' Modulation of Dopaminergic and Non-dopaminergic Neurons in the Ventral Tegmental Area.

The ventral tegmental area (VTA) neuronal population consists of dopaminergic (DAergic) and non-DAergic neurons (mainly GABAergic), the activity of which is intertwined with VTA behavioral functions. Both DAergic and GABAergic neurons in the VTA have been shown to express adrenergic receptors (ARs) and respond to AR stimulation. The aim of the present study was to demonstrate the effects of selective AR agonists on DAergic and non-DAergic neuronal activity in the central and lateral parts of the VTA using in vivo electrophysiological recording combined with microiontophoretic drug application in anaesthetized rats. Administration of phenylephrine, a selective α1-AR agonist, while having an inhibitory effect on putative DAergic neurons (11% decrease in firing rate), induced a clear excitatory effect (59% increase in firing rate) on putative non-DAergic neurons. In contrast, application of clonidine, a selective α2-AR agonist, or isoprenaline, a selective ß-adrenergic receptor agonist, did not change the firing rate of either DAergic or non-DAergic neurons but influenced the firing pattern of non-DAergic cells only. Our results suggest that noradrenaline modulates activity of VTA neurons in vivo primarily via α1, but also via ß- and α2-AR to a lesser extent. Furthermore, we show that α1-AR activation has contrasting effects on putative DAergic and non-DAergic neurons. We hypothesize that the phenylephrine-induced inhibition of putative DAergic neurons results from activation of GABAergic terminals present at the site of drug application. Such a mechanism is further supported by the observed α1-AR-induced excitation of putative GABAergic VTA neurons.

Alpha1-adrenergic receptor blockade in the VTA modulates fear memories and stress responses.

Activity of the ventral tegmental area (VTA) and its terminals has been implicated in the Pavlovian associative learning of both stressful and rewarding stimuli. However, the role of the VTA noradrenergic signaling in fear responses remains unclear. We aimed to examine how alpha1-adrenergic receptor (α1-AR) signaling in the VTA affects conditioned fear. The role of α1-AR was assessed using the micro-infusions into the VTA of the selective antagonists (0.1-1µg/0.5µl prazosin and 1µg/0.5µl terazosin) in acquisition and expression of fear memory. In addition, we performed control experiments with α1-AR blockade in the mammillary bodies (MB) - a brain region with α1-AR expression adjacent to the VTA. Intra-VTA but not intra-MB α1-AR blockade prevented formation and retrieval of fear memories. Importantly, local administration of α1-AR antagonists did not influence footshock sensitivity, locomotion or anxiety-like behaviors. Similarly, α1-AR blockade in the VTA had no effects on negative affect measured as number of 22kHz ultrasonic vocalizations during fear conditioning training. We propose that noradrenergic signaling in the VTA via α1-AR regulates formation and retrieval of fear memories but not other behavioral responses to stressful environmental stimuli. It enhances the encoding of environmental stimuli by the VTA to form and retrieve conditioned fear memories and to predict future behavioral outcomes. Our results provide novel insight into the role of the VTA α1-AR signaling in the regulation of stress responsiveness and fear memory.