Opioid receptor subtypes associated with ventral tegmental facilitation and periaqueductal gray inhibition of feeding

Eating was induced in sated animals by lateral hypothalamic electrical stimulation following central microinjections of mu- (morphine), delta-([D-Pen2,D-Pen5]enkephalin) or kappa-(U-50,488H) receptor agonists, or saline. With stimulation intensity fixed at a moderate level, time to eat 3 45-mg food pellets decreased with increases in stimulation frequency, approaching an asymptote near 7 s at ca. 70 Hz. Ventral tegmental injections (8 but not 0.8 nmol) of each of the 3 drugs reduced the minimum frequency required to produce eating of 3 pellets within 20 s and reduced the frequency at which asymptotic performance was produced; the drugs were equally effective at these doses. Naloxone (2 mg/kg) reversed the effects of each drug; naloxone was slightly more effective against morphine than against DPDPE or U-50,488H. These data suggest that all 3 receptor classes may contribute to the ventral tegmental facilitation of feeding. Periaqueductal gray injections (16 but not 1.6 nmol) of morphine had the opposite effect; they increased the stimulation frequency required to cause eating of 3 pellets in 20 s, and decreased the speed of eating across all stimulation frequencies. Periaqueductal gray injections of the delta- and kappa-agonists were each without effect. These data indicate that the periaqueductal gray inhibition of feeding is mediated solely by mu-receptors and their associated periaqueductal gray circuitry.     

Opposite effects of ventral tegmental and periaqueductal gray morphine injections on lateral hypothalamic stimulation-induced feeding

Eating was induced in sated animals by lateral hypothalamic electrical stimulation following central microinjections of morphine or saline. With stimulation intensity fixed at a moderate level, time to eat 3 food pellets of 45 mg decreased with increases in stimulation frequency, approaching an asymptote near 7 s at approximately 70 Hz. Ventral tegmental area morphine injections (0.8-8 nmol) reduced the minimum frequency required to produce eating of 3 pellets within 20 s and reduced the frequency at which asymptotic performance was produced; it did not substantially change the speed of eating at which performance approached asymptote. Periaqueductal gray morphine injections (1.6-16 nmol) had the opposite effect; they increased the stimulation frequency required to meet either the 20 s (maximum) or the 7 s (asymptotic) response criterion. Since the ventral tegmental injections did not alter the absolute level of asymptotic performance, the changes in eating threshold associated with these injections are assumed to reflect motivational influences of morphine.     

Responses of ventral tegmental area GABA neurons to brain stimulation reward

Dopamine neurons in the ventral tegmental area (VTA) have been implicated in rewarded behaviors, including intracranial self-stimulation (ICSS). We demonstrate, in unrestrained rats, that the discharge activity of a homogeneous population of presumed VTA GABA neurons, implicated in cortical arousal, increases before ICSS of the medial forebrain bundle (MFB). These findings suggest that VTA GABA neurons may be involved in the attentive processes related to brain stimulation reward (BSR).     

Blockade of orexin-1 receptors in the ventral tegmental area could attenuate the lateral hypothalamic stimulation-induced potentiation of rewarding properties of morphine

The orexins (hypocretins) are lateral hypothalamic (LH) neuropeptides that have been implicated in a variety of behaviors ranging from feeding to sleep and arousal. Evidence from animal models suggests a role for orexins in reward processing and drug addiction. In the present study, we investigated the direct effect of an orexin antagonist in the ventral tegmental area (VTA) on acquisition and expression of morphine conditioned place preference (CPP) induced by concurrent stimulation of the LH. Eighty-one adult male Wistar rats weighing 220–280 g were unilaterally implanted by two separate cannulae into the LH and VTA. The CPP paradigm was done; conditioning score and locomotor activity were recorded by Ethovision software. The animals received SB334867 as a selective orexin-1 receptor antagonist (0.1, 1 and 10 nmol/0.3 μl DMSO) in the VTA, just 5 min prior to intra-LH administration of ineffective dose of carbachol as a cholinergic agonist (62.5 nmol/0.5 μl saline) that stimulates orexin neurons in the LH and ineffective dose of morphine (1 mg/kg, subcutaneously) concurrently during conditioning phase (acquisition experiments) or post-conditioning phase (expression experiments). Data showed that the blockade of orexin-1 receptors in the VTA could inhibit the acquisition (development) but not expression of LH stimulation-induced morphine CPP in the rats. Our findings suggest that the orexinergic projections from the LH to the VTA are involved in the development of the LH stimulation-induced potentiation of morphine rewarding properties and orexin-1 receptors in the VTA have a substantial role in this phenomenon.     

Brain reward circuitry: Four circuit elements “wired” in apparent series

Activation of a variety of anatomically distinct sites in the central nervous system can produce rewarding states. Four central reward phenomena are amphetamine injections into nucleus accumbens, morphine injections into the ventral tegmental area, electrical stimulation of the ventral tegmental area, and electrical stimulation of the lateral hypothalamic medial forebrain bundle. Current evidence suggests that these four rewarding events trigger activity in elements of a common reward circuit and that the elements are connected in series. The four partially identified elements in this circuit are

1. (1) descending, fast-recovering, short refractory period fibers of the medial forebrain bundle,

2. (2) separate, opioid peptide-containing afferents to the ventral tegmental area,

3. (3) the dopaminergic cells projecting from the ventral tegmental area to nucleus accumbens, and

4. (4) the dopaminoceptive cells of nucleus accumbens.

The substrate for brain-stimulation reward in the lateral preoptic area. II. Connections to the ventral tegmental area

This experiment investigated the existence of a direct anatomical connection between lateral preoptic and ventral tegmental areas that mediate brain stimulation reward using the behavioral adaptation of the collision test. This test is a double-pulse, two-electrode technique based on the axonal conduction failure that occurs when two separate sites in the same axon bundle are concurrently stimulated. This anatomical arrangement is inferred from the shape of the function relating the effectiveness of double-pulse stimulation to the interval between pulses. In this study, nine rats with a total of 44 pairs of sites were examined. In two pairs only was there a profile suggestive of an axonal collision effect, while the double-pulse effectiveness curve consistent with the properties of transynaptic collision was apparent for a single pair of sites; the remaining 93% were associated with relatively flat effectiveness curves. While electrode misalignment could be responsible for these results, there was adequate sampling to suggest that the preponderance of first stage signals that give rise to the rewarding effects mediated by the lateral preoptic and ventral tegmental areas do not travel along the same fiber bundle. However, stimulation applied to both sites concurrently produces a summation that is roughly 40% greater than stimulation at either site alone, suggesting reasonable integration of the reward signals generated by lateral preoptic and ventral tegmental area stimulation.     

Effects of continuous naloxone administration on ventral tegmental self-stimulation

Continuous subcutaneous administration of naloxone (3 mg/kg/h), shifted ventral tegmental self-stimulation rate-frequency curves to the right, without suppressing behavioral performance. In addition this chronic blockade of opioid receptors altered μ binding parameters in the hippocampus and olfactory tubercle of naloxone-treated animals. These findings speak to the role opioid peptides play in the mediation of brain stimulation reward.     

Influence of ventral tegmental area (A10 region) on flight behaviour elicited by hypothalamic stimulation in the cat

The influence of the ventral tegmental area (VTA) (dopaminergic A10 group neurons) on flight behaviour, induced by hypothalamic stimulation, was studied in the cat. Co-stimulation of hypothalamus and VTA (ipsi- or contralateral) induced an increase of the flight latency. Slow-motion analysis of flight behaviour showed that this increase was due to the augmentation of the fixation latency (FL) whereas the upright latency (UL) was not modified. Sulpiride injection (50 mg/kg i.p.) provoked the disappearance of VTA effect without affecting the basal behavioural sequence. The results show that DA A10 group neurons increase the attentive component of the flight reaction, suggesting a possible influence of the DA A10 system on the mechanisms modulating focused attention in the animal.     

Brain stimulation reward in the lateral hypothalamic medial forebrain bundle: Mapping of boundaries and homogeneity

The boundaries and relative fiber concentration of the brain stimulation reward (BSR) sustaining system coursing through the lateral hypothalamic medial forebrain bundle (MFB) were mapped using a dorso-ventral moveable electrode. High response rates for BSR were found in a region extending dorso-ventrally from the zona incerta (ZI) to the base of the brain and medio-laterally from the fornix to the medial tip of the internal capsule (IC). Self-stimulation associated with perifornical area and self-stimulation associated with the tip of the internal capsule were mixed with aversion and forced movements, respectively. Current intensity threshold variations suggest: (i) that the reward system has a well-defined dorsal boundary ventral to the ZI, and (ii) that the core of the MFB contains a relatively higher concentration of reward relevant fibers than do its lateral, medial, dorsal and ventral components. No evidence was seen of independent mid-lateral and far-lateral MFB systems, though independent BSR sites in the dorsomedial and ventromedial hypothalamus were seen.     

Sexual motivation: involvement of endogenous opioids in the ventral tegmental area

The sexual motivation and performance of male rats were observed in a bilevel testing chamber after bilateral infusion of 40 pmol β-endorphin, 2.75 nmol naloxone or saline into the ventral tegmental area for four succeeding, weekly tests. In the 5 min prior to introduction of the female rat, the male rat explores the bilevel testing chamber. It was previously shown that the increase over tests of these anticipatory level changes is sexually motivated and a response to olfactory stimuli. Naloxone infusion into the VTA prevented the increase of anticipatory level changes. β-Endorphin failed to affect the anticipatory level-changing behavior. The sexual performance was unaffected by naloxone or β-endorphin treatment, but the number of ejaculating rats decreased with repeated testing after naloxone treatment. It is concluded that endogenous opioid systems in the ventral tegmental area contribute to the stimulation of sexual motivation and/or reward, presumably by stimulating the mesolimbic dopamine system in response to sex-related olfactory stimuli.     

Neuroanatomical boundaries of the reward-relevant opiate-receptor field in the ventral tegmental area as mapped by the conditioned place preference method in rats

The conditioned place preference produced by morphine microinjected into the ventral tegmental area was studied in rats. Cannula placements were varied along the rostrocaudal plane to determine the approximate anatomical focus of morphine's rewarding effect. Microinjections within a 1.4-mm range produced a significant change in place preference suggesting that morphine injected into this zone is rewarding. Injection sites rostral and caudal to this zone were ineffective as were injections ventral to this region. The approximate anatomical boundaries of the reward-relevant opiate-receptor field within the ventral tegmental area correspond well with the distribution of the A10 dopamine-containing cell bodies.     

Electrical stimulation of the ventral tegmental area and catecholamine metabolism in discrete regions of the rat brain

Data are presented which show a different pattern of dopamine and noradrenaline utilization in terminal regions of the A9 and A10 dopaminergic systems and in terminal regions of the dorsal noradrenergic bundle after electrical stimulation of the ventral tegmental area and an adjacent area. At the sites of the electrodes an enhanced turnover of noradrenaline was found. These results are discussed relating the location of the electrode sites and the location of the dopaminergic cell bodies A9 and A10 and catecholaminergic fibers passing through the MFB innervating forebrain and limbic structures. It is concluded that activation of parts of the A10 dopaminergic system and the A6 noradrenergic system is correlated with intracranial selfstimulation.     

TPH2 in the ventral tegmental area of the male rat brain

This study surveyed the distribution of tryptophan hydroxylase 2 (TPH2) mRNA, protein, and enzymatic activity throughout the male Sprague-Dawley rat brain.TPH2 is the genetic isoform of TPH that catalyzes the rate-limiting step in serotonin biosynthesis within the central nervous system. Although cell bodies of serotonergic neurons are located mainly in the raphe, serotonin-containing axons innervate many regions of the brain. In the present study, we assessed the levels of mRNA, protein expression, and enzyme activity of TPH2 in the rat raphe, ventral tegmental area (VTA), substantia nigra, hippocampus, cerebellum, dorsal striatum, nucleus accumbens, amygdala, and medial prefrontal cortex to more fully understand the distribution of this enzyme throughout the central nervous system. The pineal gland was used as a control tissue that expresses TPH1 (the peripheral enzyme), but not TPH2. As expected, the raphe showed the highest brain TPH2 activity and protein expression. In the contrast to other reports, however, the VTA followed the raphe as the region with the second-highest amount of TPH2 activity, mRNA and protein expression. There were significantly lower TPH activities and levels of TPH2 protein in the other regions. In addition, TPH2 immunocytochemistry demonstrated the presence of TPH-positive cell bodies within the VTA. The results of this study indicate that TPH2 and serotonergic signaling may play an important role in the mesolimbic/mesocortical reward pathway.     

A comparison of the effects of electrical stimulation of the lateral and ventromedial hypothalamus on the activity of neurons in ventral tegmental area and substantia nigra

Extracellular unit recordings were obtained from neurons in the ventral tegmental area (VTA), the substantia nigra, zona compacta (SNC) and zona reticulata (SNR) of adult female albino rats anaesthetized with urethane and chloral hydrate. Neurons were divided into two types based on their electrophysiological characteristics; Type I neurons had long duration action potentials (>2.6 msec) and slow discharge rates and Type II neurons had shorter duration action potentials and a wider range of discharge rates. Both types of neurons were found in the VTA and SNC, but there were only Type II neurons in the SNR. The effects of single pulse stimuli delivered to the ipsilateral ventromedial (VMH) or lateral (LH) hypothalamic areas on activities of the two types of neurons were investigated. Only a small portion of neurons in the VTA and SNC responded to VMH stimulation, but in contrast a majority of the two types of neurons in the VTA and SNC responded to LH stimulation. Most neurons in the SNR did not respond to VMH or to LH stimulation. Type II neurons in the VTA and SNC were predominantly suppressed by LH stimulation with short onset latencies (<6 msec), indicating the possibility of monosynaptic mediation. However Type I neurons in the VTA and SNC were activated and suppressed and the onset latencies of these responses were relatively longer. The high proportion of neurons of VTA and SNC responding to electrical stimulation of LH is consistent with anatomical evidence. Suppression and activation of Type I neurons in VTA and SNC suggest that the LH exerts modulatory influences on these neurons of the midbrain.     

Increased ipsilateral expression of Fos following lateral hypothalamic self-stimulation

Immunohistochemical labeling of Fos protein was used to visualize neurons activated by rewarding stimulation of the lateral hypothalamic level of the medial forebrain bundle (MFB). Following training and stabilization of performance, seven rats were allowed to self-stimulate for l h prior to anesthesia and perfusion. Brains were then processed for immunohistochemistry. Two control subjects were trained and tested in an identical manner except that the stimulator was disconnected during the final l h test. Among the structures showing a greater density of labeled neurons on the stimulated side of the brains of the experimental subjects were the septum, lateral preoptic area (LPO), medial preoptic area, bed nucleus of the stria terminalis, substantia innominata (SI), and the lateral hypothalamus (LH). Several of these structures, the LPO, SI, and LH, have been implicated in MFB self-stimulation by the results of psychophysical, electrophysiological, and lesion studies.     

Chronic food restriction and weight loss produce opioid facilitation of perifornical hypothalamic self-stimulation

Electrical stimulation frequency thresholds for lateral hypothalamic (LH) self-stimulation were monitored throughout a 3 week period of food restriction and a subsequent 3 week period of re-feeding. Rats with electrodes placed in the perifornical LH were sensitive to this dietary manipulation as evidenced by a high positive correlation between body weight and self-stimulation threshold. Rats with electrodes in the zona incerta/subincertal region or ventral hypothalamus displayed little or no change in threshold. Lateral ventricular injection of naltrexone (200.0 nM) reversed the decline in threshold that was otherwise present during food restriction in rats with perifornical placements. Naltrexone had no effect on thresholds of rats with placements outside the perifornical region. These findings suggest that food restriction and weight loss activate an opioid mechanism that facilitates perifornical LH self-stimulation. The documented association of perifornical LH with the phenomenon of stimulation-induced feeding, and the reciprocal connections between this region and gustatory structures, supports the hypothesis that facilitation of self-stimulation by food restriction is related to the natural phenomenon of positive alliesthesia (i.e. the hunger-dependency of food reward).     

Increased food intake after opioid microinjections into nucleus accumbens and ventral tegmental area of rat

The nucleus accumbens (ACC) and ventral tegmental area (VTA), two areas believed to subserve reinforcement and increases in locomotor activity produced by opioid microinjections, were examined for their involvement in opioid-produced changes in ingestive behavior. Opioids were infused bilaterally, and food and water intakes were measured for 1 h thereafter. Different morphine doses were administered and, with placements in globus pallidus and lateral ventricles as controls for diffusion, it was found that only ACC and VTA microinjections (0.1-10 nmol) produced dose-related increases in food intake. In both the ACC and VTA low doses of morphine also produced increases in water intake while in ACC high doses produced a decrease. Administration of morphine and an enkephalin analogue (Tyr-D-Met-Gly-Phe(4-NO2)-Pro-NH2) at different depths in the ACC indicated that the increase in food intake occurred at a site separate from that of the decrease in water intake. Using levorphanol, dextrorphan and morphine mixed with naloxone, it was shown that the effects were due to activation of opioid receptors. Additional experiments demonstrated that food intake is increased by ACC morphine under different levels of deprivation, with different times of testing and with availabilities of various goal objects in addition to food. The effect also did not appear to undergo development of tolerance or sensitization. It was concluded that there are sites in the ACC and VTA where increased activity of endogenous opioid peptide systems reliably increase food intake and it was hypothesized that these sites may contribute to changes in ingestive behavior after systemic morphine administration. Also, together with other effects produced by opioid microinjections into the ACC and VTA, the present findings suggest that increased opioid activity in these areas produce a pattern of behaviors similar to that produced in normal animals by food conditioned stimuli.     

Facilitatory effect of unilateral lesion of the ventral tegmental area on locomotor response to stimulation of the contralateral ventral tegmental area: involvement of GABAergic transmission

It was found previously that in the rat, unilateral electrolytic lesion of the ventral tegmental area (VTA) facilitated feeding induced by electrical stimulation of the homologous VTA tissue in the contralateral hemisphere. In the present work, VTA stimulation-induced locomotor response was tested in male Wistar rats using a latency to move/stimulation frequency curve shift paradigm in order to check for functional generality of the "contralateral facilitation effect" and also with the aim of elaborating an easy and reliable behavioral model to study this phenomenon. In a further step, the hypothesis was tested that enhancement of function of the intact VTA results from elimination of tonic GABAergic influence derived normally from the lesioned VTA. GABA(A) (bicuculline, doses 0, 0.5 and 5.0 ng) and GABA(B) (phaclofen, doses 0, 500 and 1000 ng) receptors antagonists, and for comparison, a GABA(A) receptor agonist (muscimol, doses 0, 12.5, 25. 0 and 50.0 ng), were injected unilaterally to VTA and their effect on locomotor response elicited by electrical stimulation of the contralateral VTA was tested in a latency/frequency paradigm. It was found that similar to feeding, locomotor response evoked by unilateral electrical stimulation of the VTA was facilitated after contralateral VTA lesion which manifested as a decrease of the locomotion threshold and a leftward shift of the function relating latency to move to stimulation frequency. The effect was immediate, long-lasting and specific to the VTA destruction; lesions outside the VTA area caused gradual impairment of the locomotor response to stimulation. The facilitatory effect of the electrolytic lesion could be replicated by bicuculline, which significantly facilitated stimulation-induced behavior. Phaclofen exerted slight facilitating influence only at a low dose. No effect of muscimol on the locomotion threshold was found. We conclude that "the contralateral facilitation effect" at the level of VTA reflects the interhemispheric regulation of activity of the dopaminergic (DA) cells in which GABA(A)-mediated interhemispheric communication plays a significant role.     

Analysis of opioid receptor subtype antagonist effects upon mu opioid agonist-induced feeding elicited from the ventral tegmental area of rats

The present study examined opioid receptor(s) mediation of feeding elicited by mu opioid agonists in the ventral tegmental area using general or selective opioid antagonist pretreatment. Naltrexone as well as equimolar doses of selective mu and kappa, but not delta opioid antagonists in the ventral tegmental area significantly reduced mu agonist-induced feeding, indicating a pivotal role for these receptor subtypes in the full expression of this response.