Is the cuneiform nucleus a critical component of the mesencephalic locomotor region? An examination of the effects of excitotoxic lesions of the cuneiform nucleus on spontaneous and nucleus accumbens induced locomotion

The cuneiform nucleus and the pedunculopontine tegmental nucleus have both been suggested as possible sites for the mesencephalic locomotor region (MLR), an area from which controlled stepping on a treadmill can be elicited following electrical or chemical stimulation in a decerebrate animal. It has been shown that excitotoxic lesions of the pedunculopontine tegmental nucleus impair neither spontaneous locomotion nor locomotion induced by stimulation of the nucleus accumbens. Excitotoxic lesions of the cuneiform nucleus have not previously been investigated. Rats received either bilateral ibotenate or sham lesions of the cuneiform nucleus combined with bilateral implantation of guide cannulae aimed at the nucleus accumbens. On recovery from surgery spontaneous locomotion was tested, followed by accumbens-stimulated locomotion. For nucleus accumbens stimulation, each rat received bilateral microinjection of each of three doses of d-amphetamine (10.0, 20.0 and 30.0 μg) and a vehicle only injection. Locomotor activity was recorded following the injection. In comparison to the sham-lesioned group, the ibotenate-lesioned group showed no differences in either spontaneous or amphetamine-induced locomotor activity. These results suggest that, like the pedunculopontine tegmental nucleus, the cuneiform nucleus is not involved in the direct mediation of spontaneous or accumbens-induced locomotion, and thus is very unlikely to be the anatomical substrate of the MLR. The role of the cuneiform nucleus in other types of behavioural control is discussed.     

The mesencephalic locomotor region (MLR) in the rat

These studies demonstrate the presence of the MLR in the rat brain. Controlled locomotion on a treadmill could be induced by low level stimulation (< 50 μA) of an area in the posterior midbrain following a precollicular-prenigral brainstem transection. This area included the lateral part of the cuneiform nucleus and anterior as well as posterior portions of the pedunculopontine nucleus. In addition, the presence of a subthalamic locomotor region in the fields of Forel was determined in rats after prethalamic transections.     

Activity in the mesencephalic locomotor region during locomotion

The activity of single neurons in the mesencephalic locomotor region (MLR) was recorded extracellularly in cats during spontaneous locomotion on a treadmill. Although stimulation of the MLR is required to induce locomotion on a treadmill after a precollicular-postmamillary brain stem transection in the cat, spontaneous locomotion may occur after a precollicular-premamillary transection. The activity of flexor and extensor muscles of each limb also was recorded by EMG. Nearly 50% of the MLR neurons exhibited rhythmic firing patterns during locomotion. In about one-half of those cells, unit firing patterns could be correlated with the EMG activity in one or more muscles by using spike-triggered averaging. Single MLR neurons were found to be correlated to EMG activity in a single limb, and others were related to the EMG from muscles in two limbs or in all four limbs. Passive movement or stoppage of the limb(s) did not abolish rhythmicity in these neurons. In addition, somatosensory stimulation did not appear to affect the firing patterns of MLR neurons. Averaged EMGs of correlated forelimb muscles revealed a postspike mean latency of 7.1 ms. These measurements agreed well with reports of a 1- to 1.5-ms delay in MLR projections to reticulospinal neurons and a 5- to 6-ms delay (postspike) in reticulospinal activity correlated to EMGs during locomotion. These findings suggest that (a) MLR neurons are rhythmically active during locomotion, (b) the activity of MLR neurons can be correlated with that of EMGs in one or more limbs, (c) rhythmicity in MLR neurons may be independent of phasic sensory input, and (d) the downstream influence of the MLR may be relayed, at least in part, via reticulospinal neurons.     

Autoradiographic demonstration of the projections from the mesencephalic locomotor region

An autoradiographic tracing technique was used to examine the projections of the classically defined mesencephalic locomotor region (MRL). Injections of [3H]proline and [3H]leucine were made into sites in the caudal mesencephalon which can be stimulated to produce locomotion. The injection sites were confined to the cuneiform nucleus (stereotaxic coordinates P2.0, L4.0, H-1.0). Descending projections were primarily ipsilateral to the gigantocellular and magnocellular reticular formation of the pons and medulla, the dorsal tegmental reticular nucleus, and the nucleus raphe magnus. Some sparse contralateral projections were also observed within the magnocellular and gigantocellular reticular formation. Direct axonal connections with the spinal cord were not consistently observed. Ascending projections were observed to the subthalamic nucleus, caudal hypothalamic nuclei, the centrum medianum nucleus of the thalamus, the ventral tegmental area of Tsai, the superior colliculus, and the periaqueductal gray region. The ascending projections were also ipsilateral, with sparse contralateral labeling confined to areas which received ipsilateral projections. Projections to the contralateral cuneiform nucleus were also consistently observed. The results, when compared to those of another study, suggest that the classical MLR is anatomically distinct from the more medial sites in the mesencephalon which can also induce locomotion.     

Chemical activation of the mesecephalic locomotor region

Electrical stimulation of the mesencephalic locomotor region (MLR) in the precollicular-postmammillary transected cat is known to induce controlled locomotion on a treadmill. We have been able to induce and block locomotion in this preparation by using localized infusions of transmitters and their agonists and antagonists. Infusions of the GABA antagonists bicuculline and picrotoxin into the MLR elicit locomotion at low concentration (5 mM). Applications of muscimol (5 mM) or GABA (0.5 M) were found to block chemically-induced locomotion, as well as electrically-elicited and spontaneous walking. Priming infusions of Diazepam amplified the blockage of locomotion by GABA. On the other hand, applications of strychnine (10 mM) were ineffective in inducing stepping, as were infusions of the excitatory agents glutamic acid, acetylcholine and norepinephrine. These findings suggest that the MLR is under inhibitory GABAergic input. The substantia nigra is the only known afferent to the MLR located posterior to the brainstem transection, and is a likely source for this input. A model is proposed to account for our results, as well as those of others, and it provides a working hypothesis for the neurochemical events occurring in brainstem centers which modulate locomotor events.     

Evidence that projections from substantia innominata to zona incerta and mesencephalic locomotor region contribute to locomotor activity

A series of anatomical, electrophysiological and behavioral experiments was carried out in the rat to investigate the possible functional significance of a recently demonstrated neural pathway from the substantia innominata of the subpallidal forebrain to the mesencephalic locomotor region. Following injections of the anterogradely transported lectin PHA into the substantia innominata labeled fibers with terminal boutons were observed in the zona incerta, dorsal to the medial part of the subthalamic nucleus, and some appeared to continue on to the pedunculopontine nucleus. Electrophysiological recordings of action potentials were made from neurons in the substantia innominata and some of these neurons were activated antidromically by single-pulse stimulation of the zona incerta and/or by single-pulse stimulation of the pedunculopontine nucleus as well. Neurons in the zona incerta responded orthodromically to stimulation of the substantia innominata. Locomotor activity was initiated by injecting picrotoxin, a GABA antagonist, unilaterally into the substantia innominata through chronic cannulae, as reported previously. This picrotoxin-initiated locomotor activity was reduced significantly when procaine (a neuronal blocker) was injected into the ipsilateral zona incerta. Injecting procaine into the contralateral zona incerta had little or no effect on the picrotoxin-initiated locomotor activity. Taken together these observations suggest the tentative working hypothesis that projections from the substantia innominata to the zona incerta as well as the pedunculopontine nucleus may contribute to the locomotor component of adaptive behaviors resulting from limbic forebrain integrative activities, an hypothesis that can now be investigated further.     

Hippocampal modulation of locomotor activity induced by focal activation of postsynaptic dopamine receptors in the core of the nucleus accumbens

The locomotor effects of intra-NAcc injection of dopamine receptor agonists following discrete lesion or inhibition of the DH or the VH have been poorly investigated using only the indirect dopamine receptor agonist amphetamine. In the present study, we investigated how lidocaine in the DH or the VH modulated hyperlocomotion induced by focal injection into the NAcc core of the selective D1-like receptor agonist, SKF 38393, or coinjection of SKF 38393, and the selective D2-like receptor agonist, LY 171555; the latter pharmacological condition being required for the full expression of the postsynaptic effects of D2-like receptor agonists, and recognized to produce a locomotor response mainly mediated by D2-like postsynaptic receptors. Rats were given the D1-like receptor agonist SKF 38393 alone or in combination with the D2-like receptor agonist LY 171555 into the NAcc core, and lidocaine into the DH or the VH. Then, locomotor activity was recorded. Focal injection into the NAcc core of SKF 38393 alone or in combination with LY 171555 resulted in an increase of locomotor activity. Administration of lidocaine into the DH further potentiated the increase in locomotor activity induced by activation of D1-like receptors or co-activation of D1-like and D2-like receptors in the NAcc core. Administration of lidocaine into the VH also potentiated the increase in locomotor activity induced by D1-like receptor activation, but decreased that produced by co-activation of D1-like and D2-like receptors in the NAcc core. Taken together, these results suggest that under lidocaine-free conditions the DH may exert a tonic inhibitory modulation on hyperlocomotion mediated by D1-like and D2-like postsynaptic receptors in the NAcc core, while the VH may exert a tonic inhibitory on hyperlocomotion mediated by D1-like receptors and a tonic facilitatory control on hyperlocomotion mediated by D2-like postsynaptic receptors.     

Connections of the mesencephalic locomotor region (MLR) I. Substantia nigra afferents

These studies were designed to determine whether or not substantia nigra (SN) neurons project to the mesencephalic locomotor region (MLR). An attempt was made to activate SN neurons antidromically from the same site which induced locomotion on a treadmill following a precollicular-postmammillary transection in the same animal. Less than 10% of posterior SN neurons were activated antidromically from the physiologically-identified MLR. These results support previous anatomical findings describing a sparse projection from the SN to the MLR [5]. Locomotion on a treadmill was elicited at low current strengths (20–50 μA) from an area around the cuneiform nucleus in the posterior mesencephalon. This area included the lateral central gray, mesencephalic trigeminal root, dorsal brachium conjunctivum and nucleus tegmenti pedunculopontinus (NTPP) and perhaps anterodorsal locus coeruleus. Stimulation of the area just described induced a complete stepping cycle with a flexion phase and a three-part extension phase. Stimulation of the posterior SN produced spastic locomotion on a treadmill at higher current strengths (70 μA) in cats with a precollicular-postmammillary transection.     

Group III metabotropic glutamate receptors and D1-like and D2-like dopamine receptors interact in the rat nucleus accumbens to influence locomotor activity

Evidence for functional interactions between metabotropic glutamate (mGlu) receptors and dopamine (DA) neurotransmission is now clearly established. In the present study, we investigated interactions between group III mGlu receptors and D1- and D2-like receptors in the nucleus accumbens (NAcc). Administration, into the NAcc, of the selective group III mGlu receptor agonist, AP4, resulted in an increase in locomotor activity, which was blocked by pretreatment with the group III mGlu receptor antagonist, MPPG. In addition, pretreatment with AP4 further blocked the increase in motor activity induced by the D1-like receptor agonist, SKF 38393, but potentiated the locomotor responses induced by either the D2-like receptor agonist, quinpirole, or coinfusion of SKF 38393 and quinpirole. MPPG reversed the effects of AP4 on the motor responses induced by D1-like and/or D2-like receptor activation. These results confirm that glutamate transmission may control DA-dependent locomotor function through mGlu receptors and further indicate that group III mGlu receptors oppose the behavioural response produced by D1-like receptor activation and favour those produced by D2-like receptor activation.     

Septal lesions enhance hyperactivity induced either by amphetamine or lesions of the nucleus accumbens septi

Spontaneous locomotor activity was measured in 4 groups of rats subjected to either septal, accumbens, combined septal-accumbens or sham operations. Accumbens but not septal lesions increased activity above control levels and the combined septal-accumbens lesion markedly increased activity levels above that produced by the accumbens lesion. Thus, septal lesions appeared to exaggerate the activity changes produced by the accumbens lesions. Furthermore, both septal and combined septal-accumbens lesions enhanced the hyperactivity induced by 0.25, 0.5 and 1.0 mg/kg d-amphetamine-HCl but attenuated the hyperactivity induced by 0.25, 0.5 and 1.0 mg/kg scopolamine-HCl. Rats with accumbens lesions did not differ from controls in their response to amphetamine or scopolamine. Thus, septal lesions modified both lesion and drug-induced changes in locomotor activity.     

Connections of the mesencephalic locomotor region (MLR) III. Intracellular recordings

The responses of neurons in the area of the cat mesencephalic locomotor region (MLR) following stimulation of the entopeduncular nucleus (EN) were recorded intracellularly. At the end of each experiment a precollicular-postmamillary brainstem transection was performed and stimulation of the recording site(s) was employed to induce locomotion on a treadmill. This procedure was assumed to establish that intracellularly studied cells in the vicinity of a locomotion-inducing site were MLR neurons. About 10% of MLR neurons were found to respond to stimulation of the EN at short latencies. Stimulation of MLR efferent pathways was used to identify output neurons by antidromic activation. Very few MLR output neurons were found to receive EN projections (i.e. to respond at short latency following EN stimulation). These experiments support previous results describing a sparse projection from the EN to the MLR. This projection appears to be functionally varied (EPSP, IPSP and EPSP-IPSP responses were observed in MLR neurons following EN stimulation) and to exert its major influence on interneurons, not on output neurons, of the MLR.     

Increased locomotor response to amphetamine induced by the repeated administration of the selective kappa-opioid receptor agonist U-69593

Acute administration of kappa opioid receptor (KOR) agonists decreases both dopamine (DA) extracellular levels in the nucleus accumbens (NAc) and locomotor activity. Opposing to its acute effects, recent studies show that chronic administration of KOR agonists potentiates both stimulated DA release and induced locomotor activity. Since KOR agonists have been considered as potential treatment for stimulant dependence, the effects of their repeated administration on psychostimulant actions are of major concern. The present study was undertaken to investigate the in vivo effect of repeated administration of the KOR agonist U-69593 on DA extracellular levels in the NAc and on the locomotor activity challenged with amphetamine. Rats were injected once daily with the selective KOR agonist U-69593 or vehicle for four consecutive days. One-day after the last U-69593 injection, microdialysis studies assessing extracellular DA levels in the NAc and locomotor activity challenged with amphetamine were conducted. Microdialysis studies revealed that preexposure to U-69593 had no effect on basal DA levels but significantly augmented amphetamine-induced DA extracellular levels. Accordingly, amphetamine-induced locomotor activity was also significantly potentiated in U-69593 preexposed rats. These results suggest that long-term effect of KOR activation results in facilitation of amphetamine-induced DA extracellular levels in the NAc accompanied by sensitization of amphetamine-induced increase in locomotor activity.     

Presynaptic modulation of dopamine-induced locomotor activity by oxotremorine in nucleus accumbens of the rat

Summary Behavioural studies were carried out to determine whether central cholinergic mechanisms regulate dopaminergic activity by presynaptic mechanisms as suggested by recentin vitro studies. Bilateral injections of a muscarinic receptor agonist, oxotremorine (4g), into the nucleus accumbens were found to enhance dopamine-induced locomotor activity. In rats deprived of presynaptic terminals by pretreatment with intraaccumbens injections of 6-hydroxydopamine, oxotremorine did not enhance dopamine-induced locomotor activity but on the contrary produced a marked reduction of locomotor activity. Although dopamine induced locomotor activity could occur in 6-hydroxydopamine treated animals, the muscarinic regulation was dependent on the presence of the intact dopamine terminal and hence a presynaptic receptor may be involved.     

Decrease of locomotor activity by injections of carbachol into the anterior hypothalamic/preoptic area of the rat

Cholinergic elements in forebrain structures are implicated in locomotion but their role is still unclear. In the present study, the effects of intracerebrally injected carbachol or atropine on spontaneous locomotion and rearing activity were investigated. Effective injection sites were found in the area between frontal planes 5.3 and 6.3 mm from interaural plane and between the ventricle wall and lateral plane 1.1 mm from the midline which corresponds to the medial anterior hypothalamic/preoptic area. Injections of 1.0 micrograms of carbachol into this area decreased locomotor activity and rearing to one-third of the control level during the first 5 min of recording. These reductions of locomotion and rearing were dose-dependent and reversed by 1.5 microgram of atropine. Atropine alone, at this dose, had no effect on locomotion but higher doses (20.0-60.0 micrograms) of atropine produced a dose-dependent increase of locomotion. A comparison of the injection sites with recent maps of the cholinergic system indicates that muscarinic cholinoceptive, presumably non-cholinergic, cells throughout the medial AH/POA might be associated with a decrease of locomotor activity caused by intracerebral injections of carbachol.     

Effects of ventro-medial mesencephalic tegmentum (VMT) stimulation on the spontaneous activity of nucleus accumbens neurones: Influence of the dopamine system

The effects of VMT-stimulation (100-500 microA, 0.6 ms; 1 Hz) on the spontaneous activity of neurones in the nucleus accumbens were analyzed in ketamine-anaesthetized rats. On spontaneously active cells (firing greater than 0.5 spikes/s), 3 types of responses were observed: either inhibition (36%), excitation (5%) or a composite sequence of excitation followed by inhibition (12%). Moreover, 14% of silent nucleus accumbens neurones were excited by single pulse VMT-stimulation. Finally, 3% of nucleus accumbens neurones recorded were driven antidromically by VMT-stimulation. Destruction of dopamine (DA) projections by 6-hydroxydopamine prevented the inhibitory responses to VMT stimulation in the great majority of cells studied, without affecting the excitatory responses. After systemic administration of haloperidol or sulpiride, the inhibitory responses to VMT stimulation were attenuated markedly, whilst the excitatory responses were, however, maintained. These results suggest that the inhibitory, but not the excitatory, effects of VMT-stimulation on nucleus accumbens neurones may be mediated by an activation of the mesolimbic DA system.     

Amphetamine elevates nucleus accumbens dopamine via an action potential‐dependent mechanism that is modulated by endocannabinoids

The reinforcing effects of abused drugs are mediated by their ability to elevate nucleus accumbens dopamine. Amphetamine (AMPH) was historically thought to increase dopamine by an action potential‐independent, non‐exocytotic type of release called efflux, involving reversal of dopamine transporter function and driven by vesicular dopamine depletion. Growing evidence suggests that AMPH also acts by an action potential‐dependent mechanism. Indeed, fast‐scan cyclic voltammetry demonstrates that AMPH activates dopamine transients, reward‐related phasic signals generated by burst firing of dopamine neurons and dependent on intact vesicular dopamine. Not established for AMPH but indicating a shared mechanism, endocannabinoids facilitate this activation of dopamine transients by broad classes of abused drugs. Here, using fast‐scan cyclic voltammetry coupled to pharmacological manipulations in awake rats, we investigated the action potential and endocannabinoid dependence of AMPH‐induced elevations in nucleus accumbens dopamine. AMPH increased the frequency, amplitude and duration of transients, which were observed riding on top of slower dopamine increases. Surprisingly, silencing dopamine neuron firing abolished all AMPH‐induced dopamine elevations, identifying an action potential‐dependent origin. Blocking cannabinoid type 1 receptors prevented AMPH from increasing transient frequency, similar to reported effects on other abused drugs, but not from increasing transient duration and inhibiting dopamine uptake. Thus, AMPH elevates nucleus accumbens dopamine by eliciting transients via cannabinoid type 1 receptors and promoting the summation of temporally coincident transients, made more numerous, larger and wider by AMPH. Collectively, these findings are inconsistent with AMPH eliciting action potential‐independent dopamine efflux and vesicular dopamine depletion, and support endocannabinoids facilitating phasic dopamine signalling as a common action in drug reinforcement.     

Involvement of neuronal cell bodies of the mesencephalic locomotor region in the initiation of locomotor activity of freely behaving rats

The locomotor activity of freely-moving rats was increased substantially by injections of L-sodium glutamate or of picrotoxin, a GABA antagonist, into the region of the tegmental pedunculopontine nucleus. The onset of hyper-motility was more rapid with L-glutamate than with picrotoxin and the duration shorter. Locomotor activity from injecting amphetamine unilaterally into the nucleus accumbens was reduced by injections of GABA into the ipsilateral pedunculopontine nucleus. These observations provide additional evidence implicating neurons of the MLR and possibly GABA synaptic inputs to these neurons in locomotor activity and suggest that they may mediate indirect inputs from the nucleus accumbens.     

6-Hydroxydopamine lesions of nucleus accumbens core abolish amphetamine-induced conditioned activity

Environmental cues associated with drug experiences appear to play a critical role in drug dependence. We have previously reported that dopamine-depleting lesions of the nucleus accumbens medial shell inhibit amphetamine-conditioned place preference. Here, we examined the effects of analogous lesions on amphetamine-conditioned locomotor activity. Bilateral core, but not medial shell, lesions attenuated unconditioned locomotion and abolished the conditioned locomotor response. Taken with our previous results, these findings confirm a role for accumbens core in amphetamine-induced locomotor activity and suggest that the role of medial shell DA transmission in conditioned place preference is related to reward processing rather than conditioning in general.     

The effects of 6-hydroxydopamine lesions of the nucleus accumbens and caudate nucleus of rats on feeding in a novel environment

Open field activity and feeding behavior were studied in separate groups of rats 1 or 6 weeks after infusions of the neurotoxin, 6-hydroxydopamine (6-OHDA) into the nucleus accumbens or caudate nucleus. These infusions resulted in a specific destruction of the catecholamine innervation of the injected area. When compared with rats which received infusions of vehicle alone, both lesions resulted in a relative reduction in locomotor activity 1 week, but not 6 weeks, after the operation. Both lesions also resulted in a more rapid onset of feeding from wire baskets containing familiar laboratory chow. Rats with lesions of the nucleus accumbens also fed for longer, consumed more food, and fed in longer bouts than did sham-lesioned animals, but rats with lesions of the caudate nucleus did not. These data show changes in feeding behaviour produced by 6-OHDA infusions into the dopamine terminal fields which were long lasting in comparison with the locomotor impairments, and did not arise as a result of locomotor impairment. The differences in feeding behaviour may result from more general changes in behaviour such as an impairment in the reactivity to novel environmental stimuli or switching between types of behaviour.