Hypocretin /orexin preferentially activates caudomedial ventral tegmental area dopamine neurons

The hypocretin/orexin (HCRT) neuropeptide system modulates behavioral state and state-dependent processes via actions on multiple neuromodulatory transmitter systems. Recent studies indicate that HCRT selectively increases dopamine (DA) neurotransmission within the prefrontal cortex (PFC) and the shell subregion of the nucleus accumbens (NAs), but not the core subregion of the nucleus accumbens (NAc). The circuitry underlying the differential actions of HCRT across distinct DA systems is unclear. The current study examined whether HCRT preferentially activates PFC- and NAs-projecting relative to NAc-projecting DA neurons within the VTA. One week after infusion of the retrograde tracer fluorogold (FG) into the medial PFC, NAc or NAs, animals received a ventricular infusion of HCRT-1. Subsequent analyses conducted across the rostral-caudal extent of the VTA determined the degree to which: (i) Fos-immunoreactivity (ir) was observed within tyrosine hydroxylase (TH)-ir neurons; (ii) TH-ir was observed within FG-ir neurons; and (iii) Fos-ir was observed within FG-ir neurons. HCRT significantly increased Fos-ir in VTA DA (TH-ir) neurons, primarily in a restricted population of small-to-medium-sized DA neurons located within the caudomedial VTA. Furthermore, within this region of the VTA, PFC- and NAs-projecting TH-ir neurons were more likely to contain Fos-ir than were NAc-projecting TH-ir neurons. These results provide novel evidence that HCRT selectively activates PFC- and NAs-projecting DA neurons within the VTA, and suggest a potential role for HCRT in PFC- and NAs-dependent cognitive and/or affective processes. Moreover, these and other observations suggest that the dysregulation of HCRT-DA interactions could contribute to cognitive/affective dysfunction associated with a variety of behavioral disorders.     

Synapses between corticotropin-releasing factor-containing axon terminals and dopaminergic neurons in the ventral tegmental area are predominantly glutamatergic

Interactions between stress and the mesocorticolimbic dopamine (DA) system have been suggested from behavioral and electrophysiological studies. Because corticotropin-releasing factor (CRF) plays a role in stress responses, we investigated possible interactions between neurons containing CRF and those producing DA in the ventral tegmental area (VTA). We first investigated the cellular distribution of CRF in the VTA by immunolabeling VTA sections with anti-CRF antibodies and analyzing these sections by electron microscopy. We found CRF immunoreactivity present mostly in axon terminals establishing either symmetric or asymmetric synapses with VTA dendrites. We established that nearly all CRF asymmetric synapses are glutamatergic, insofar as the CRF-immunolabeled axon terminals in these synapses coexpressed the vesicular glutamate transporter 2, and that the majority of CRF symmetric synapses are GABAergic, insofar as the CRF-immunolabeled axon terminals in these synapses coexpressed glutamic acid decarboxylase, findings that are of functional importance. We then looked for synaptic interactions between CRF- and DA-containing neurons, by using antibodies against CRF and tyrosine hydroxylase (TH; a marker for DA neurons). We found that most synapses between CRF-immunoreactive axon terminals and TH neurons are asymmetric (in the majority likely to be glutamatergic) and suggest that glutamatergic neurons containing CRF may be part of the neuronal circuitry that mediates stress responses involving the mesocorticolimbic DA system. The presence of CRF synapses in the VTA offers a mechanism for interactions between the stress-associated neuropeptide CRF and the mesocorticolimbic DA system.     

Cholinergic axons in the rat ventral tegmental area synapse preferentially onto mesoaccumbens dopamine neurons

Cholinergic afferents to the ventral tegmental area (VTA) contribute substantially to the regulation of motivated behaviors and the rewarding properties of nicotine. These actions are believed to involve connections with dopamine (DA) neurons projecting to the nucleus accumbens (NAc). However, this direct synaptic link has never been investigated, nor is it known whether cholinergic inputs innervate other populations of DA and gamma-aminobutyric acid (GABA) neurons, including those projecting to the prefrontal cortex (PFC). We addressed these questions by using electron microscopic analysis of retrograde tract-tracing and immunocytochemistry for the vesicular acetylcholine transporter (VAChT) and for tyrosine hydroxylase (TH) and GABA. In tissue labeled for TH, VAChT(+) terminals frequently synapsed onto DA mesoaccumbens neurons but only seldom contacted DA mesoprefrontal cells. In tissue labeled for GABA, one-third of VAChT(+) terminals innervated GABA-labeled dendrites, including both mesoaccumbens and mesoprefrontal populations. VAChT(+) synapses onto DA and mesoaccumbens neurons were more commonly of the asymmetric (presumed excitatory) morphological type, whereas VAChT(+) synapses onto GABA cells were more frequently symmetric (presumed inhibitory or modulatory). These findings suggest that cholinergic inputs to the VTA mediate complex synaptic actions, with a major portion of this effect likely to involve an excitatory influence on DA mesoaccumbens neurons. As such, the results suggest that natural and drug rewards operating through cholinergic afferents to the VTA have a direct synaptic link to the mesoaccumbens DA neurons that modulate approach behaviors.     

In vivo measurement of somatodendritic release of dopamine in the ventral tegmental area

The ventral tegmental area (VTA), the locus of mesolimbic dopamine cell bodies, contains dopamine. Experiments in brain slices have demonstrated that VTA dopamine can be released by local electrical stimulation. Measurements with both push‐pull cannula and microdialysis in intact animals have also obtained evidence for releasable dopamine. Here we demonstrate that dopamine release in the VTA can be evoked by remote stimulations of the medial forebrain bundle (MFB) in the anesthetized rat. In initial experiments, the MFB was electrically stimulated while a carbon‐fiber electrode was lowered to the VTA, with recording by fast‐scan cyclic voltammetry. While release was not observed with the carbon fiber 4–6 mm below dura, a voltammetric response was observed at 6–8 mm below dura, but the voltammogram was poorly defined. At lower depths, in the VTA, dopamine release was evoked. Immunohistochemistry experiments with antibodies for tyrosine hydroxylase (TH) confirmed that dopamine processes were primarily found below 8 mm. Similarly, tissue content determined by liquid chromatography revealed serotonin but not dopamine dorsal to 8 mm with both dopamine and serotonin at lower depths. Evaluation of the VTA signal by pharmacological means showed that it increased with inhibitors of dopamine uptake, but release was not altered by D2 agents. Dopamine release in the VTA was frequency dependent and could be exhausted by stimulations longer than 5 s. Thus, VTA dopamine release can be evoked in vivo by remote stimulations and it resembles release in terminal regions, possessing a similar uptake mechanism and a finite releasable storage pool. Synapse 63:951–960, 2009. © 2009 Wiley‐Liss, Inc.     

Neurotransmitter regulation of dopamine neurons in the ventral tegmental area

Over the last 10 years there has been important progress towards understanding how neurotransmitters regulate dopaminergic output. Reasonable estimates can be made of the synaptic arrangement of afferents to dopamine and non-dopamine cells in the ventral tegmental area (VTA). These models are derived from correlative findings using a variety of techniques. In addition to improved lesioning and pathway-tracing techniques, the capacity to measure mRNA in situ allows the localization of transmitters and receptors to neurons and/or axon terminals in the VTA. The application of intracellular electrophysiology to VTA tissue slices has permitted great strides towards understanding the influence of transmitters on dopamine cell function, as well as towards elucidating relative synaptic organization. Finally, the advent of in vivo dialysis has verified the effects of transmitters on dopamine and γ-aminobutyric acid transmission in the VTA. Although reasonable estimates can be made of a single transmitter's actions under largely pharmacological conditions, our knowledge of how transmitters work in concert in the VTA to regulate the functional state of dopamine cells is only just emerging. The fact that individual transmitters can have seemingly opposite effects on dopaminergic function demonstrates that the actions of neurotransmitters in the VTA are, to some extent, state-dependent. Thus, different transmitters perform similar functions or the same transmitter may perform opposing functions when environmental circumstances are altered. Understanding the dynamic range of a transmitter's action and how this couples in concert with other transmitters to modulate dopamine neurons in the VTA is essential to defining the role of dopamine cells in the etiology and maintenance of neuropsychiatrie disorders. Further, it will permit a more rational exploration of drugs possessing utility in treating disorders involving dopamine transmission.     

Selective dopaminergic lesions of the ventral tegmental area impair preference for sucrose but not for male sexual pheromones in female mice

The role of the meso-accumbens dopaminergic pathway in reward-related behaviours is the subject of intense investigation. In this regard, here we analyse the effects of specific lesions of dopaminergic cells of the ventral tegmental area (VTA) of female mice on two goal-directed behaviours, namely sucrose preference (intake of sucrose solution vs. water) and preference for male sexual pheromones (exploration of male-soiled vs. clean bedding). The results indicate that partial lesions of the VTA that impair neither locomotion nor general exploratory behaviour reduce the preference for sucrose (over a 48-h period) but do not alter the innate attraction that females display for male sexual pheromones (in 5-min tests). This differential effect of the lesions can be interpreted as demonstrating the existence of separate neural mechanisms and circuits for signalling the reward of different natural reinforcers (e.g. sweet taste of sucrose and sexual pheromones). Alternatively, VTA lesions may result in an impaired attribution of incentive salience (which depends on the dopaminergic tegmento-striatal system) of sucrose-predicting cues, thus leading to a long-term decrease in sucrose consumption. By contrast, the same lesions do not affect the unconditioned attraction to male-derived pheromones, which may depend on amygdalo-striatal pathways.     

Exposure of nicotine to ventral tegmental area slices induces glutamatergic synaptic plasticity on dopamine neurons

Nicotine promotes glutamatergic synaptic plasticity in dopaminergic (DA) neurons in the ventral tegmental area (VTA), which is thought to be an important mechanism underlying nicotine reward. However, it is unclear whether exposure of nicotine alone to VTA slice is sufficient to increase glutamatergic synaptic strength on DA neurons and which nicotinic acetylcholine receptor (nAChR) subtype mediates this effect. Here, we report that the incubation of rat VTA slices with 500 nM nicotine induces glutamatergic synaptic plasticity in DA neurons. We measure the ratio of AMPA and NMDA receptor‐mediated currents (AMPA/NMDA) and compare these ratios between nicotine‐treated and ‐untreated slices. Our results demonstrate that the incubation of VTA slices with 500 nM nicotine for 1 h (but not for 10 min) significantly increases the AMPA/NMDA ratio when compared with controls. Preincubation with 10 nM of the α7‐nAChR antagonist, methyllycaconitine (MLA) but not 1 μM α4‐containing nAChR antagonist, dihydro‐β‐erythroidine (DHβE) prevents nicotinic effect, suggesting that α7‐nAChRs are mainly mediated this nicotinic effect. This finding is further supported by the disappearance of this nicotinic effect in nAChR α7 knockout (KO) mice. Furthermore, nicotine reduced paired‐pulse ratio (PPR) of evoked excitatory postsynaptic potential (eEPSP) in the VTA slices prepared from wild‐type (WT) mice but not α7 KO mice. Collectively, these findings suggest that exposure of smoking‐relevant concentrations of nicotine to VTA slices is sufficient to increase glutamatergic synaptic strength on DA neurons and that α7‐nAChRs likely mediate this nicotinic effect through increasing presynaptic release of glutamate. Synapse, 2011. © 2010 Wiley‐Liss, Inc.     

Low doses of ethanol activate dopaminergic neurons in the ventral tegmental area

In unanesthetized rats the intravenous administration of low doses of ethanol (0.125-0.5 g/kg) produced a dose-dependent increase (30-80%) in the firing rate of dopaminergic (DA) neurons in the Ventral Tegmental Area (VTA). In agreement with previous observations, a dose range between 0.5 and 2 g/mg of ethanol was needed to produce comparable stimulant responses in DA neurons of the Substantia Nigra Pars Compacta. However, in anesthetized rats, doses of ethanol up to 1 g/kg failed to activate VTA-DA neurons. The high sensitivity of VTA-DA neurons to ethanol activation suggests that they might be involved in the reinforcing properties of the drug.     

Sex differences and effects of prenatal exposure to excess testosterone on ventral tegmental area dopamine neurons in adult sheep

Prenatal testosterone (T) excess in sheep results in a wide array of reproductive neuroendocrine deficits and alterations in motivated behavior. The ventral tegmental area (VTA) plays a critical role in reward and motivated behaviors and is hypothesised to be targeted by prenatal T. Here we report a sex difference in the number VTA dopamine cells in the adult sheep, with higher numbers of tyrosine hydroxylase (TH)‐immunoreactive (‐ir) cells in males than females. Moreover, prenatal exposure to excess T during either gestational days 30–90 or 60–90 resulted in increased numbers of VTA TH‐ir cells in adult ewes compared to control females. Stereological analysis confirmed significantly greater numbers of neurons in the VTA of males and prenatal T‐treated ewes, which was primarily accounted for by greater numbers of TH‐ir cells. In addition, immunoreactivity for TH in the cells was denser in males and prenatal T‐treated females, suggesting that sex differences and prenatal exposure to excess T affects both numbers of cells expressing TH and the protein levels within dopamine cells. Sex differences were also noted in numbers of TH‐ir cells in the substantia nigra, with more cells in males than females. However, prenatal exposure to excess T did not affect numbers of TH‐ir cells in the substantia nigra, suggesting that this sex difference is organised independently of prenatal actions of T. Together, these results demonstrate sex differences in the sheep VTA dopamine system which are mimicked by prenatal treatment with excess T.     

Relative contributions and mapping of ventral tegmental area dopamine and GABA neurons by projection target in the rat

The ventral tegmental area (VTA) is a heterogeneous midbrain structure that contains dopamine (DA), GABA, and glutamate neurons that project to many different brain regions. Here, we combined retrograde tracing with immunocytochemistry against tyrosine hydroxylase (TH) or glutamate decarboxylase (GAD) to systematically compare the proportion of dopaminergic and GABAergic VTA projections to 10 target nuclei: anterior cingulate, prelimbic, and infralimbic cortex; nucleus accumbens core, medial shell, and lateral shell; anterior and posterior basolateral amygdala; ventral pallidum; and periaqueductal gray. Overall, the non-dopaminergic component predominated VTA efferents, accounting for more than 50% of all projecting neurons to each region except the nucleus accumbens core. In addition, GABA neurons contributed no more than 20% to each projection, with the exception of the projection to the ventrolateral periaqueductal gray, where the GABAergic contribution approached 50%. Therefore, there is likely a significant glutamatergic component to many of the VTA's projections. We also found that VTA cell bodies retrogradely labeled from the various target brain regions had distinct distribution patterns within the VTA, including in the locations of DA and GABA neurons. Despite this patterned organization, VTA neurons comprising these different projections were intermingled and never limited to any one subregion. These anatomical results are consistent with the idea that VTA neurons participate in multiple distinct, parallel circuits that differentially contribute to motivation and reward. While attention has largely focused on VTA DA neurons, a better understanding of VTA subpopulations, especially the contribution of non-DA neurons to projections, will be critical for future work.     

Regulation of mating behaviour in the female rat by gonadotropin-releasing hormone in the ventral tegmental area: effects of selective destruction of the A10 dopamine neurones

Microinfusions of gonadotropin-releasing hormone (GnRH) into the ventral tegmental area (VTA) potentiated lordosis behaviour in oestrogen-primed ovariectomised female rats. Facilitation was observed within 5 min after the infusion and lasted for about 90 min. When GnRH was infused into the VTA of oestrogen-primed animals which were previously subjected to 6-hydroxydopamine treatment (to destroy the A10 dopamine cells), it produced a marked facilitation of lordosis lasting for about 24 h. These results suggest that the A10 dopamine neurones in the VTA may be critically involved in the mechanisms by which GnRH may modulate midbrain circuits involved in the regulation of lordosis behaviour in the female rat. The lesion studies also imply that the A10 dopamine neurones function as inhibitory neurones regulating lordosis behaviour by suppressing the activity of those cells in the VTA which are sensitive to GnRH. Removal of this inhibitory input leads to an exaggerated response.     

Dopaminergic neurons in the rat ventral tegmental area. III. Effects of -and -amphetamine

By use of various histochemical techniques, it was shown that both DA and non-DA cells in the VTA project to the NAc. Of these VTA-NAc output cells, the great majority were DA-containing cells. A small number of non-DA cells were encountered most frequently in the lateral part of the VTA. Correspondingly, two distinct groups of neurons, types I and II, could be identified by antidromic stimulation of the NAc. Several lines of evidence suggest that type I cells are DA-containing neurons. The evidence may be summarized as follows:

1. (1) type I cells had a slow-bursting or regular firing pattern, slow discharge rate and wide spike duration which appears to be identical to the characteristics of DA neurons originally described by Bunney et al.¹⁶;

2. (2) the great majority of these cells could be activated antidromically by stimulation of the NAc;

3. (3) the conduction velocity and absolute refractory period of type I cells are consistent with unmyelinated fine DA fibers;

4. (4) injection of 6-OHDA, but not 5,7-DHT directly in the MFB blocked antidromic responses of these cells;

5. (5) they were extremely sensitive to intravenously administered DA agonist apomorphine (ID₅₀ = 7 μg/kg); and

6. (6) direct fluorescence histochemical examination of serial sections from brains of animals in which type I cells have been identified by antidromic stimulation of the NAc showed that type I cells are most likely catecholamine-containi ng neurons. By contrast, type II cells possessed an entirely different spectrum of physiological characteristics; in addition, they showed no consistent response to apomorphine and their antidromic responses to stimulation of the NAc were not affected by 6-OHDA. It is concluded that (1) VTA output neurons consist of both DA and nonDA neurons, and (2) identified types I and II neurons in the VTA by antidromic stimulation of the NAc are DA and non-DA cells, respectively.

A topographic localization of enkephalin on the dopamine neurons of the rat substantia nigra and ventral tegmental area demonstrated by combined histofluorescence-immunocytochemistry

The concentration of choline acetyltransferase, a specific marker for cholinergic neurons, was determined in the supraoptic nucleus after a variety of lesions. Surgical lesions immediately rostral as well as medial and lateral to the nucleus did not affect the concentration of the enzyme. Only lesions which separated the nucleus from the posterior part of the lateral hypothalamus slightly decreased its concentration in choline acetyltransferase. It is concluded that the bulk of the cholinergic neurons is in the supraoptic nucleus or its immediate vicinity.     

Effects of sustained serotonin reuptake inhibition on the firing of dopamine neurons in the rat ventral tegmental area

Background

Selective serotonin (5-HT) reuptake inhibitors (SSRIs) are efficacious in depression because of their ability to increase 5-HT neurotransmission. However, owing to a purported inhibitory effect of 5-HT on dopamine (DA) neuronal activity in the ventral tegmental area (VTA), this increase in 5-HT transmission might result in a suppression of the firing activity of DA neurons. Since the mesolimbic DA system plays an important role in motivation and reward, a potential decrease in the firing of DA neurons may lead, in some patients, to a lack of adequate response to SSRIs.

Methods

We administered the SSRIs citalopram or escitalopram in rats. We determined DA neuronal activity using in-vivo electrophysiology.

Results

Sustained administration of escitalopram robustly decreased the firing rate and burst activity of DA neurons. There was no difference in the mean number of spontaneously active DA neurons per tract among the 3 groups (citalopram, escitalopram, control). This inhibition was reversed by the selective 5-HT_2C receptor antagonist SB 242084. Citalopram, however, did not alter the overall firing rate but inhibited the burst activity of DA neurons.

Limitations

Our experiments were carried out with the rats under general anesthesia. Therefore, under such conditions the absolute changes produced by SSRIs may heve been different from those occurring in freely moving rats. The exact location of the 5-HT_2C receptors mediating the inhibitory effects of the SSRIs could not be determined in these studies.

Conclusion

The difference between escitalopram and citalopram in their effect on DA neuronal activity may be explained by the higher efficacy of escitalopram as a 5-HT reuptake inhibitor. Since the inhibitory effect of escitalopram on DA neuronal activity is mediated via 5-HT_2C receptors, antagonists of these receptors might be effective adjuncts in SSRI-resistant depression.     

Excitatory and inhibitory responses of dopamine neurons in the ventral tegmental area to nicotine

In the present electrophysiological study the mechanisms by which nicotine activates dopamine neurons in the ventral tegmental area in anesthetized Sprague-Dawley rats were analyzed. Intravenous administration of nicotine caused a dose-dependent increase in firing rate and percentage of spikes fired in bursts of ventral tegmental area dopamine neurons. However, this activation was preceded by an instantaneous but short-lasting inhibition of the firing rate. The excitation of dopamine neurons by nicotine (1.5-400 microg/kg i.v.) was antagonized and even reversed into an inhibitory response by elevated levels (four-fold) of the endogenous glutamate receptor antagonist kynurenic acid, as induced by a potent inhibitor of kynurenine 3-hydroxylase (PNU 156561A, 40 mg/kg, i.v., 5-9 h). The antagonistic action induced by PNU 156561A pretreatment was prevented by administration of D-cycloserine (128 mg/kg, i.v., 5 min). Administration of the GABA(B)-receptor antagonist CGP 35348 (200 mg/kg, i.v., 3 min) facilitated the nicotine-induced increase in burst firing activity of dopamine neurons and antagonized the short-lasting decrease in firing rate by nicotine. The results of the present study show that nicotine produces both inhibition and excitation of ventral tegmental area dopamine neurons, actions that appear to be related to the release of GABA and glutamate, respectively. Whereas the excitatory action of nicotine may be associated with motivational processes underlying learning and cognitive behavior, the inhibitory action of the drug may play a more prominent role in the situation of a profound dysregulation of the mesocorticolimbic dopamine system and may help to explain the high prevalence of tobacco-smoking in schizophrenics.     

GIRK2 expression in dopamine neurons of the substantia nigra and ventral tegmental area

G-protein-regulated inward-rectifier potassium channel 2 (GIRK2) is reported to be expressed only within certain dopamine neurons of the substantia nigra (SN), although very limited data are available in humans. We examined the localization of GIRK2 in the SN and adjacent ventral tegmental area (VTA) of humans and mice by using either neuromelanin pigment or immunolabeling with tyrosine hydroxylase (TH) or calbindin. GIRK2 immunoreactivity was found in nearly every human pigmented neuron or mouse TH-immunoreactive neuron in both the SN and VTA, although considerable variability in the intensity of GIRK2 staining was observed. The relative intensity of GIRK2 immunoreactivity in TH-immunoreactive neurons was determined; in both species nearly all SN TH-immunoreactive neurons had strong GIRK2 immunoreactivity compared with only 50-60% of VTA neurons. Most paranigral VTA neurons also contained calbindin immunoreactivity, and approximately 25% of these and nearby VTA neurons also had strong GIRK2 immunoreactivity. These data show that high amounts of GIRK2 protein are found in most SN neurons as well as in a proportion of nearby VTA neurons. The single previous human study may have been compromised by the fixation method used and the postmortem delay of their controls, whereas other studies suggesting that GIRK2 is located only in limited neuronal groups within the SN have erroneously included VTA regions as part of the SN. In particular, the dorsal layer of dopamine neurons directly underneath the red nucleus is considered a VTA region in humans but is commonly considered the dorsal tier of the SN in laboratory species.     

Modulation of singing-related activity in the songbird ventral tegmental area by social context

Successful reproduction depends critically on social interactions. To understand the neural mechanisms underlying such interactions, the study of courtship singing of songbirds has many advantages. Male zebra finches produce a similar song during courtship of a female and while alone. However, singing-related neural activity in the anterior forebrain pathway (AFP), a basal ganglia-forebrain circuit, is markedly dependent on the social context in which singing occurs. Thus, the AFP should receive a signal of social context from outside the song system. Here, we have begun to investigate the neural source of such a signal by recording from neurons in the ventral tegmental area (VTA), which provides dopaminergic input to Area X, a striatal nucleus of the AFP. The level of activity of most VTA neurons we recorded (32/35) was clearly modulated during singing, especially when males sang to a female bird. Modulation of the level of activity could occur in the presence of a female without singing, but typically was further increased when males sang to the female. In addition, activity of some neurons was patterned in relation to song elements, and appeared related to motor output. These results suggest that VTA activity could carry signals related to motivational aspects of singing, as well as more primary sensory and motor signals.     

The behavioural effects of enkephalin analogues injected into the ventral tegmental area and globus pallidus

Two stable analogues of enkephalind-Ala²-Met⁵-enkephalinamide (DAMA) andd-Ala²,d-Leu⁵-enkephalin (DADLE) injected bilaterally into the ventral tegmental area (VTA) induced locomotion, characterized by bursts of morphine-like activity. The response of DADLE was blocked by systematic α-flupenthixol (0.2 mg/kg) and naloxone (1 mg/kg). At higher doses of enkephalins in the VTA, stereotypy behaviour involving gnawing, became apparent. When injected bilaterally into the globus pallidus (GP), both analogues induced a dose-dependent increase in locomotor activity but stereotypy was not observed. The locomotor behaviour induced by DADLE was blocked by naloxone but not by α-flupenthixol. These results suggest that naloxone-sensitive opiate receptors modulate, at a number of different sites, the neural pathways involved in the expression of behavioural activity.