Response of ventral pallidal neurons to amygdala stimulation and its modulation by dopamine projections to nucleus accumbens

It has been shown that the nucleus accumbens receives input from the amygdala and that mesolimbic dopaminergic projection from the ventral tegmental area (VTA) modulates the response of accumbens neurons to amygdala input. Since the nucleus accumbens projects to the ventral pallidum, the purpose of this study was to investigate, using electrophysiological techniques, whether or not the nucleus accumbens relays the projection from the amygdala to the ventral pallidum and whether or not the mesolimbic dopamine projection interacts with this pathway. Extracellular single-unit recordings were obtained from the ventral pallidum of urethan-anesthetized rats, and the responses of these neurons to electrical stimulation of the amygdala were investigated. Of 392 neurons tested, 36% were inhibited and 11% were excited following amygdala stimulation. Latency of onset of inhibitory responses showed a bimodal distribution with peaks in the ranges of 4-6 ms and 16-18 ms, respectively. Fifty-four percent of inhibitory responses with latencies greater than 12 ms were attenuated by 1) injection of procaine hydrochloride into the nucleus accumbens, or 2) injection of d-amphetamine into the nucleus accumbens, or 3) stimulation of VTA with a train of 10 pulses (10 Hz) prior to stimulation of amygdala. Acute administration of haloperidol intraperitoneally or injection of 6-hydroxydopamine into the ipsilateral VTA, 2 days prior to the recording experiment, reduced the attenuating effects of intra-accumbens injection of d-amphetamine and VTA conditioning stimulations on the inhibitory response of ventral pallidal neurons to amygdala stimulation. These results support the hypothesis that the nucleus accumbens provides a link between the amygdala and the ventral pallidum. Since the amygdala is a limbic structure and the ventral pallidum has possible connections with the extrapyramidal motor system, it is suggested that the amygdala to nucleus accumbens to ventral pallidum projection may be a bridge between the limbic and motor systems. We also suggest that this relay of output from the amygdala to the ventral pallidum via the nucleus accumbens is under the modulating influence of the mesolimbic dopamine projection from the ventral tegmental area.     

The ventral striatopallidal parts of the basal ganglia in the rat--II. Compartmentation of ventral pallidal efferents

This paper describes the results of experiments designed to address whether neuron populations giving rise to different ventral pallidal efferent projections are segregated in a manner concordant with the recently described immunohistochemical compartmentation of ventral pallidum. The retrograde transport of horseradish peroxidase conjugated to wheatgerm agglutinin following injections in the ventral tegmental area of Tsai, medial substantia nigra and subthalamic nucleus was charted in relationship to the patterns of immunohistochemical staining in the forebrain following incubation of sections in antisera against substance P, neurotensin or leucine-enkephalin. In some cases the retrograde labeling and immunohistochemical protocols were combined in the same experiment. As a supplement, the electron microscope was utilized to investigate the ultrastructure and synaptic input of projecting cells making up populations of ventral pallidum neurons that project to different efferent targets. The results indicated that the immunocytochemical compartmentation of ventral pallidum observed in our earlier study is reflected in the organization of neurons from which ventral pallidal efferents originate. Thus, axons destined to terminate in the medial parts of substantia nigra and subthalamic nucleus project from neurons located in the lateral, neurotensin immunoreactivity-deficient part of ventral pallidum and the globus pallidus. Fibers en route to the ventral tegmental area originate in neurotensin immunoreactivity-rich medial parts of the ventral pallidum as part of a large cluster of retrogradely labeled neurons that invades several forebrain structures, including the bed nucleus of the stria terminalis, preoptic regions, and the nuclei of the diagonal band. The electron microscopic results provided additional evidence distinguishing the medial and lateral compartments of ventral pallidum. Whereas projection neurons located in both compartments display similar cytologic features, the density of synaptic input to retrogradely labeled perikarya and proximal dendrites following injections in the subthalamic nucleus is significantly greater than that seen following injections in the ventral tegmental area. Although no attempt was made to examine more distal parts of labeled dendrites in the present study, the observation that most dendritic profiles in the medial part of ventral pallidum were less contacted by boutons than their counterparts in the lateral district suggests that the level of innervation of projection neurons is generally lesser in medial ventral pallidum. This hypothesis is further supported by the presence in the medial ventral pallidum of significant numbers of "glial blockades," a cytologic configuration that is clearly capable of preventing functional interactions and is rarely observed in the lateral ventral pallidum...     

Projections of the substantia nigra, ventral tegmental area and amygdala to the pallidum in dog brain

Using the method based on HRP retrograde axonal transport organization of projections of substantia nigra, tegmental ventral field and amygdala on pallidum was studied. Neuronal fibres from all dopaminergic portions of substantia nigra and tegmental ventral field were found to project on both structures of dog dorsal pallidum (globus pallidus and entopeduncular nucleus). Ventral pallidum receives projectional axons only from neurons of basal nucleus of amygdala and tegmental ventral field.     

Stereological estimates of dopaminergic, GABAergic and glutamatergic neurons in the ventral tegmental area, substantia nigra and retrorubral field in the rat

Midbrain dopamine neurons in the ventral tegmental area, substantia nigra and retrorubral field play key roles in reward processing, learning and memory, and movement. Within these midbrain regions and admixed with the dopamine neurons, are also substantial populations of GABAergic neurons that regulate dopamine neuron activity and have projection targets similar to those of dopamine neurons. Additionally, there is a small group of putative glutamatergic neurons within the ventral tegmental area whose function remains unclear. Although dopamine neurons have been intensively studied and quantified, there is little quantitative information regarding the GABAergic and glutamatergic neurons. We therefore used unbiased stereological methods to estimate the number of dopaminergic, GABAergic and glutamatergic cells in these regions in the rat. Neurons were identified using a combination of immunohistochemistry (tyrosine hydroxylase) and in situ hybridization (glutamic acid decarboxylase mRNA and vesicular glutamate transporter 2 mRNA). In substantia nigra pars compacta 29% of cells were glutamic acid decarboxylase mRNA-positive, 58% in the retrorubral field and 35% in the ventral tegmental area. There were further differences in the relative sizes of the GABAergic populations in subnuclei of the ventral tegmental area. Thus, glutamic acid decarboxylase mRNA-positive neurons represented 12% of cells in the interfascicular nucleus, 30% in the parabrachial nucleus, and 45% in the parainterfascicular nucleus. Vesicular glutamate transporter 2 mRNA-positive neurons were present in the ventral tegmental area, but not substantia nigra or retrorubral field. They were mainly confined to the rostro-medial region of the ventral tegmental area, and represented approximately 2-3% of the total neurons counted ( approximately 1600 cells). These results demonstrate that GABAergic and glutamatergic neurons represent large proportions of the neurons in what are traditionally considered as dopamine nuclei and that there are considerable heterogeneities in the proportions of cell types in the different dopaminergic midbrain regions.     

Dopaminergic and non-dopaminergic neurons in the ventral tegmental area of the rat project, respectively, to the cerebellar cortex and deep cerebellar nuclei.

It has been suggested recently that dopamine in the cerebellum not only acts as a precursor for noradrenaline in afferent fibers supplied by locus coeruleus neurons, but also subserves an independent transmitter role in a separate neural system. The present study was initiated to investigate the possible sources for dopaminergic innervation of the cerebellum. Employing anterograde and retrograde axonal tracing with cholera toxin and a combination of fluorescent retrograde axonal tracing with Fluoro-Gold and tyrosine hydroxylase immunofluorescence histochemistry, we found in the rat that the ventral tegmental area, containing the A10 dopaminergic cell group, sends projection fibers to the cerebellum bilaterally with a slight contralateral predominance. The projections from the ventral tegmental area to the cerebellum were segregated into the dopaminergic one to the cerebellar cortex and the non-dopaminergic one to the deep cerebellar nuclei. Dopaminergic fibers projecting from the ventral tegmental area to the cerebellar cortex terminated mainly in the granular layer, additionally in the Purkinje cell layer, but not at all in the molecular layer. They were distributed predominantly in the crus I ansiform lobule and paraflocculus, and to a lesser extent in the crus II ansiform lobule. On the other hand, non-dopaminergic fibers projecting from the ventral tegmental area to the deep cerebellar nuclei were seen to terminate mainly in the lateral nucleus, to a lesser extent in the interpositus nucleus, but not at all in the medial nucleus. The ventral tegmental area was also observed to receive projection fibers from the lateral and interpositus cerebellar nuclei bilaterally with a contralateral predominance. The projections from the ventral tegmental area to the cerebellum revealed in the present study might exert limbic influences upon the cerebro-cerebellar loops subserving the execution and co-ordination of voluntary movements.     

Neurons of origin of the neurotensinergic plexus enmeshing the ventral tegmental area in rat: retrograde labeling and in situ hybridization combined

The morphological and physiological substrates that underlie the mutual regulatory interactions of neurotensin and dopamine in the rat mesotelencephalic projections and related structures remain to be fully described. A salient candidate for neurotensinergic effects on the mesotelencephalic dopamine projection is the dense plexus of neurotensin immunoreactive axons that enmeshes the ventral tegmental area and substantia nigra, but the locations of the neurons that give rise to this plexus have not been identified and its systemic context remains obscure. To address this, Fluoro-Gold and the cholera toxin β subunit, retrogradely transported axonal tracers, were injected into the ventral tegmental area of rats and the brains were processed to demonstrate neurons that contained both retrograde tracer immunoreactivity and a probe against neurotensin/neuromedin N messenger RNA. Substantial numbers of double-labeled neurons were observed in the rostral part of the lateral septum, and in a region centered on the shared boundaries of the bed nucleus of stria terminalis, ventromedial ventral pallidum, diagonal band of Broca, lateral preoptic area and rostral lateral hypothalamus. A few double-labeled neurons were also observed in the dorsal raphe nucleus and adjacent periaqueductal gray. Despite the administration of haloperidol and D-amphetamine to elicit and enhance neurotensin/neuromedin N messenger RNA expression in striatum, including the nucleus accumbens and olfactory tubercle, no double-labeled neurons were observed there.These results identify a novel brain substrate for control of midbrain dopamine levels, which affect reward mechanisms and motivation.     

A comparison of ventral tegmental neurons projecting to optic flow regions of the inferior olive vs. the hippocampal formation

The ventral tegmental area (catecholaminergic group A10) is a midbrain region characterized by concentrated dopaminergic immunoreactivity. Previous studies in pigeons show that the ventral tegmental area provides a robust projection to the hippocampal formation and to the medial column of the inferior olive. However, the distribution, morphology, and neurochemical content of the neurons that constitute these projections have not been resolved. In this study, we used a combination of retrograde tracing techniques and immunofluorohistochemistry to address these issues. Retrograde tracers were used to demonstrate that the distribution of ventral tegmental area neurons projecting to the hippocampus and the inferior olive overlap in the caudo-ventral ventral tegmental area. The hippocampus- and inferior olive-projecting ventral tegmental area neurons could not be distinguished based on morphology: most neurons had small- to medium-sized multipolar or fusiform soma. Double-labeling with fluorescent retrograde tracers revealed that the hippocampus- and medial column of the inferior olive-projecting neurons were found intermingled in the ventral tegmental area, but no cells were double labeled; i.e. individual ventral tegmental area neurons do not project to both the hippocampal formation and medial column of the inferior olive. Finally, we found that a minority (8.2%) of ventral tegmental area neurons providing input to the hippocampus were tyrosine hydroxylase-immunoreactive, whereas none of the inferior olive-projecting neurons were tyrosine hydroxylase positive. Combined, our findings show that the projections to the hippocampus and olivocerebellar pathway arise from intermixed subpopulations of ventral tegmental area neurons with indistinguishable morphology but only the hippocampal projection involves dopaminergic neurons. We suggest that equivalent projections from the ventral tegmental area to the hippocampal formation and inferior olive exist in mammals and discuss their potential role in the processing of optic flow and the analysis of self-motion.     

Chemical organization of projection neurons in the rat accumbens nucleus and olfactory tubercle

Projection neurons in the ventral striatum, the accumbens nucleus and olfactory tubercle, were examined by combining the retrograde tracing method and immunocytochemistry with antibodies against C-terminals of the preprodynorphin (PPD), preproenkephalin (PPE), preprotachykinin A (PPTA) and preprotachykinin B (PPTB). When the retrograde tracer was injected into the ventral pallidum, about 60% and 40% of retrogradely labeled neurons in the accumbens nucleus were immunoreactive for PPD and PPE, respectively. In contrast, all accumbens nucleus neurons projecting to the ventral mesencephalic regions including the substantia nigra and ventral tegmental area were immunopositive for PPD but not for PPE. Although no olfactory tubercle neurons projected fibers to the mesencephalic regions, 60% and 40% of olfactory tubercle neurons projecting to the ventrolateral portion of the ventral pallidum were immunoreactive for PPD and PPE, respectively, as were the accumbens nucleus neurons. About 70% of accumbens nucleus and olfactory tubercle neurons projecting to the ventral pallidum and all accumbens nucleus neurons projecting to the ventral mesencephalic regions showed PPTA immunoreactivity. A small population (2-12%) of accumbens neurons projecting to the ventral pallidum and mesencephalic regions displayed immunoreactivity for PPTB. Compared with the dorsal striatopallidal projection neurons that were reported to mostly express PPE, it was characteristic of the ventral striatum that only the smaller population (about 40%) of ventral striatopallidal projection neurons expressed PPE. This suggests that the ventral striatopallidal projection system is less specialized than the dorsal striatopallidal system in terms of peptide production, or that the ventral pallidum should be compared with a combined region of the globus pallidus and entopeduncular nucleus in the dorsal system.     

Dissociated high-purity dopaminergic neuron cultures from the substantia nigra and the ventral tegmental area of the postnatal rat.

We have developed dissociated primary neuronal cultures obtained from the substantia nigra and from the ventral tegmental area of postnatal rats (two to three days old). After making brain slices, the regions of the substantia nigra and the ventral tegmental area were separately dissected. The removed fragments of brain tissue were dissociated and cultured on a glial feeder layer. Double immunocytochemical labeling for tyrosine hydroxylase and GABA on cultures grown for two to three weeks showed the presence of 42% dopaminergic and 39% GABAergic neurons in substantia nigra cultures, whereas in ventral tegmental area cultures there were 65% dopaminergic and 21% GABAergic neurons. The dopaminergic neurons were characterized by thick and straight primary processes dividing into several branches. Varicosities were found mainly on distal parts of the processes. In contrast, GABAergic neurons possessed highly branched thick and thin primary processes with intensive arborization and numerous varicosities. Co-existence of dopamine and cholecystokinin was found in about 70% of dopaminergic neurons from the substantia nigra and in about 35% of dopaminergic neurons from the ventral tegmental area. Physiological properties of these cultured dopaminergic neurons were investigated with the whole-cell version of the patch-clamp method. After each physiological experiment, immunocytochemical labeling confirmed that the cell was dopaminergic. Properties of single action potentials, with an action potential height of 92 mV and duration of 1.6 ms, were similar to those reported for dopaminergic neurons in brain slices. The neurons showed a high resting potential, and no spontaneous firing of action potentials. Constant current depolarizations elicited trains of action potentials. In the majority of cells, the train stopped firing within a few seconds, while in some cells it lasted indefinitely. When the cell was hyperpolarized, the voltage response started to decline slowly (sag), indicating the presence of hyperpolarization-activated currents (time-dependent inward rectification). These results show that by using our culture method it is possible to obtain separate dissociated cultures of the substantia nigra and the ventral tegmental area from newborn rats. Because they are rich in functional dopaminergic neurons, these cultures will be a useful tool for studying various properties of dopaminergic neurons.     

In vivo modulation of ventral tegmental area dopamine and glutamate efflux by local GABA(B) receptors is altered after repeated amphetamine treatment

The activity of dopamine neurons in the ventral tegmental area is modulated by excitatory (glutamatergic) and inhibitory (GABAergic) afferents. GABA, released by intrinsic neurons and by projection neurons originating in the nucleus accumbens and other regions, inhibits dopamine neurons via activation of GABA(A) and GABA(B) receptor subtypes. Using in vivo microdialysis in freely moving rats, we investigated the role of ventral tegmental area GABA(B) receptors in modulating levels of dopamine and glutamate within the ventral tegmental area, both in naive rats and in rats treated repeatedly with saline or amphetamine (5 mg/kg i.p., for 5 days). In naive rats, administration of a potent and selective GABA(B) receptor antagonist (CGP 55845A) into the ventral tegmental area elicited a concentration-dependent increase in dopamine levels, but did not alter glutamate levels. In rats tested 3 days after discontinuing repeated amphetamine administration, 50 microM CGP 55845A increased dopamine levels to a greater extent than in saline controls. This difference was no longer present in rats tested 10-14 days after discontinuing repeated amphetamine injections. CGP 55845A (50 microM) had no effect on glutamate levels in the ventral tegmental area of saline-treated rats. However, it produced a robust increase in glutamate levels in rats tested 3 days, but not 10-14 days, after discontinuing repeated amphetamine injections.These results suggest that somatodendritic dopamine release is normally under strong tonic inhibitory control by GABA(B) receptors. Repeated amphetamine administration enhances GABA(B) receptor transmission in the ventral tegmental area during the early withdrawal period, increasing inhibitory tone on both dopamine and glutamate levels. This is the first demonstration, in an intact animal, that drugs of abuse alter GABA(B) receptor transmission in the ventral tegmental area.     

Stimulation of the pedunculopontine tegmental nucleus in the rat produces burst firing in A9 dopaminergic neurons.

Stimulation of the medial prefrontal cortex in the rat produces events in midbrain dopaminergic neurons which resemble natural bursts, and which are closely time-locked to the stimulation, albeit with a very long latency. As a consequence, we have previously argued that such bursts are polysynaptically generated via more proximal excitatory amino acidergic afferents, arising, for example, from the pedunculopontine tegmental nucleus. In the present study, single-pulse electrical stimulation applied to this nucleus (and other sites in the rostral pons) was found to elicit responses in the majority of substantia nigra (A9) dopaminergic neurons. Responses usually consisted of long-latency, long-duration excitations or inhibition-excitations. Thirty-seven percent of responses (currents combined) elicited by stimulation of the pedunculopontine tegmental nucleus contained time-locked bursts, the bursts being embedded in the long-duration excitatory phases of excitation and inhibition-excitation responses. Stimulation sites located within 0.5 mm of the pedunculopontine tegmental nucleus were also effective at eliciting time-locked bursts (although less so than sites located in the nucleus itself), whereas more distal sites were virtually ineffective. For responses containing time-locked bursts, a higher percentage of stimulations produced a burst when the response was elicited from within the pedunculopontine tegmental nucleus than when it was elicited from outside: the bursts themselves having a very long latency (median of 96.2 ms; shorter than that of medial prefrontal cortex-induced bursts). Finally, although there was no difference in the distribution within the substantia nigra pars compacta of cells which exhibited time-locked bursting and those which did not, stimulation-induced bursts were elicited more frequently in dopaminergic neurons which were classified as "bursting" on the basis of their basal activity. The pedunculopontine tegmental nucleus appears to be a critical locus in the rostral pons for the elicitation of time-locked bursts in A9 dopaminergic neurons. Since time-locked bursts were more often elicited from cells which exhibited bursting under basal conditions, this suggests that rostral pontine sites, in particular the pedunculopontine tegmental nucleus, may play a role in the natural burst activity of dopaminergic neurons. Given that bursts in dopaminergic neurons are generated in response to primary and secondary reinforcers, the projection from the pedunculopontine tegmental nucleus could be one means by which motivationally relevant information (arising, for example, from the medial prefrontal cortex) reaches these cells.     

Effects of cerebral lesions on binding sites for calcitonin and calcitonin gene-related peptide in the rat nucleus accumbens and ventral tegmental area.

Binding site densities of [125I]-labelled salmon calcitonin and human calcitonin gene-related peptide were investigated in the rat nucleus accumbens and ventral tegmental area by means of quantitative autoradiography following selective brain lesions. [125I]salmon calcitonin and [125I]human calcitonin gene-related peptide binding sites were highly concentrated in the accumbens, whereas the ventral tegmental area only contained [125I]salmon calcitonin binding sites. Unilateral injection of 6-hydroxydopamine into the ventral tegmental area did not alter [125I]salmon calcitonin and [125I]human calcitonin gene-related peptide binding site densities in the ipsilateral accumbens, while it produced a significant decrease in [125I]salmon calcitonin binding sites in the lesioned ventral tegmental area (-50%). In contrast, following unilateral injection of quinolinic acid into the accumbens, the densities of [125I]salmon calcitonin and [125I]human calcitonin gene-related peptide binding sites were significantly decreased in the lesioned accumbens (-57% and -56%, respectively), while [125I]salmon calcitonin binding site densities were not modified in the ipsilateral ventral tegmental area. The present study clearly suggests that [125I]salmon calcitonin and [125I]human calcitonin gene-related peptide binding sites are located on intrinsic neurons but not on the dopaminergic nerve terminals in the accumbens. Moreover, a certain proportion of [125I]salmon calcitonin binding sites could be present on dopaminergic cell bodies in the ventral tegmental area.     

GABA(A) agents injected into the ventral pallidum differentially affect dopaminergic pivoting and cholinergic circling elicited from the shell of the nucleus accumbens

The ability of GABA(A) receptors in the ventral pallidum to modulate shell-specific behavior was studied. Injections of the non-selective acetylcholine receptor agonist, carbachol (5 microg), into the shell of the nucleus accumbens elicited contraversive circling, namely turning marked by normal stepping; in contrast, injections of a mixture of dopamine D(1) (SKF 38393, 5 microg) and D(2) (quinpirole, 10 microg) receptor agonists into this brain structure elicited contraversive pivoting, namely turning marked by abnormal hindlimb stepping. Unilateral injections of the GABA(A) receptor agonist muscimol (10, 25 and 50 ng) into the ventral pallidum dose-dependently mimicked shell-specific circling, especially when given at a level +8.6mm anterior to the interaural line; this effect was GABA(A) receptor specific, because it was prevented by the GABA(A) receptor antagonist bicuculline (150 ng). Unilateral pallidal injections of a dose of muscimol that was ineffective per se (10 ng) abolished contraversive pivoting elicited by shell injections of dopamine receptor agonists; instead, it elicited moderate ipsiversive pivoting. Pallidal injections of bicuculline (150 ng) replaced the contraversive pivoting elicited by dopamine receptor agonist with ipsiversive circling. In contrast, unilateral pallidal injections of 10 ng muscimol (anterior +8.6mm level) suppressed the contraversive circling elicited by shell injections of carbachol; instead, it elicited moderate ipsiversive pivoting. Pallidal injections of bicuculline (150 ng) produced short-lasting ipsiversive circling that was followed by contraversive pivoting.We conclude that the ventromedial portion of the ventral pallidum contains GABA(A) receptors that are crucial for the transmission of information from the shell of the nucleus accumbens via the ventral pallidum towards other brain structures; this holds especially for information about shell-specific circling elicited by carbachol. The same portion of the ventral pallidum also contains GABA(A) receptors that control the transfer of information from the nucleus accumbens towards structures outside the ventral pallidum; this holds especially for information about shell-specific pivoting elicited by dopaminergic agonists.     

Projections from the intracerebellar nuclei to the ventral midbrain tegmentum in the rat

The present study was undertaken to provide anatomical evidence, in the rat, for a direct projection from the cerebellum towards structures, other than the red nucleus, which belong to the ventral midbrain tegmentum, by using the retrograde as well as the anterograde horseradish peroxidase transport method. Following unilateral injection in the ventral midbrain tegmentum of horseradish peroxidase, free or conjugated to wheat germ agglutinin, sparing the red nucleus, retrogradely labeled neurons were found in the contralateral cerebellar lateral nucleus and, at lower density, in the interpositus nucleus. No labeled neurons were found in the fastigial nucleus of either side. Anterogradely labeled axons from lectin coupled horseradish peroxidase injection sites in the lateral and interpositus nuclei reached the contralateral ventral midbrain tegmentum. Terminal labeling was observed in the entire red nucleus as well as in the lateral division of the ventral tegmental area of Tsai, in the dorsal region of the substantia nigra pars compacta, and in the medial part of the retrorubral field. No terminal labeling was found in the caudal linear nucleus, interfascicular nucleus, peripeduncular nucleus, rostral linear nucleus of the raphe, substantia nigra pars lateralis and the substantia nigra pars reticulata. Terminal labeling was also not observed in the ventral midbrain tegmentum following horseradish peroxidase injection in lateral and interpositus nuclei of rats pretreated with kainic acid. In conclusion, it is noteworthy that, besides the red nucleus, the sole structures of ventral midbrain tegmentum receiving cerebellar efferents are those with a higher density of dopaminergic cells.     

Organization of ventral tegmental area projections to the ventral tegmental area-nigral complex in the rat

The ventral tegmental area (VTA) is a nodal link in reward circuitry. Based on its striatal output, it has been subdivided in a caudomedial part which targets the ventromedial striatum, and a lateral part which targets the ventrolateral striatum [Ikemoto S (2007) Dopamine reward circuitry: two projection systems from the ventral midbrain to the nucleus accumbens-olfactory tubercle complex. Brain Res Rev 56:27-78]. Whether these two VTA parts are interconnected and to what extent the VTA innervates the substantia nigra compacta (SNc) and retrorubral nucleus (RR) are critical issues for understanding information processing in the basal ganglia. Here, VTA projections to the VTA-nigral complex were examined in rats, using Phaseolus vulgaris leucoagglutinin (PHA-L) as anterograde tracer. The results show that the dorsolateral VTA projects to itself, as well as to the dorsal tier of the SNc and RR, largely avoiding the caudomedial VTA. The ventrolateral VTA innervates mainly the interfascicular nucleus. The components of the caudomedial VTA (the interfascicular, paranigral and caudal linear nuclei) are connected with each other. In addition, the caudomedial VTA (especially the paranigral and caudal linear nuclei) innervates the lateral VTA, and, to a lesser degree, the SNc and RR. The caudal pole of the VTA sends robust, bilateral projections to virtually all the VTA-nigral complex, which terminate in the dorsal and ventral tiers. Modest inputs from the medial supramammillary nucleus to ventromedial parts of the VTA-nigral complex were also identified. In double-immunostained sections, PHA-L-labeled varicosities were sometimes found apposed to tyrosine hydroxylase-positive neurons in the ventral mesencephalon. Overall, the results underscore that VTA projections to the VTA-nigral complex are substantial and topically organized. In general, these projections, like the spiralated striato-nigro-striatal loops, display a medial-to-lateral organization. This anatomical arrangement conceivably permits the ventromedial striatum to influence the activity of the lateral striatum. The caudal pole of the VTA appears to be a critical site for a global recruitment of the mesotelencephalic system.     

Glutamatergic axons from the lateral habenula mainly terminate on GABAergic neurons of the ventral midbrain

The concept of cortical-subcortical loops emphasizes the importance of the basal ganglia for motor, psychomotor, and emotional cortical functions. These loops are bidirectionally controlled by the midbrain dopaminergic system, predominantly but not exclusively at the level of the striatum including the accumbens nucleus. Successful behaviors increase the activities of the mesostriatal (arising in the complex part of the substantia nigra) and mesolimbic (arising in the ventral tegmental area, VTA) neurons, thereby reinforcing the corresponding actions. In contrast, unsuccessful behaviors result in an increased activation of the lateral habenular complex (LHb), thereby decreasing the activities of mesolimbic neurons. Correspondingly, electrical stimulation of the LHb effectively blocks neuronal activity in the VTA. Whether this block is due to an inhibitory projection from the LHb to the VTA, or whether axons from excitatory LHb neurons target inhibitory neurons within the VTA, is presently not known. Here we show, using in situ hybridization and immunocytochemical double labeling at the light and electron microscopic level, that GABAergic neurons are scarce in the LHb and that glutamatergic axons from the LHb mostly target GABAergic neurons in the VTA and the mesopontine rostromedial tegmental nucleus (RMTg), also known as tail of the VTA (tVTA). These data explain the inhibitory effect of LHb activation on the VTA. In addition, however, a small number of LHb terminals in the VTA actually contacts dopaminergic neurons. The biological importance of these terminals requires further investigation.     

Biochemical evidence for γ-aminobutyrate containing fibres from the nucleus accumbens to the substantia nigra and ventral tegmental area in the rat

Glutamate decarboxylase activity, a specific marker for γ-aminobutyrate-containing neurons, has been analysed in microdissected samples from rat mesencephalon following unilateral electrocoagulations of the nucleus accumbens. This lesion resulted in a consistent decrease of 50% in the enzyme activity in the rostromedial substantia nigra, and a slight, but insignificant decrease (?15%) in the medial parts of the caudal pars compacta of the substantia nigra. No change was found in the lateral pars compacta or the central pars reticulata. In the ventral tegmental area, the highest activity was found in the rostromedial part, adjacent to the mammillary body. At this level, a significant decrease of 20% was found in the ventral tegmental area on the lesioned side. In contrast, the activities in the medial accessory optic nucleus and the caudal ventral tegmental area adjacent to the interpenduncular nucleus were unchanged.The results indicate that the nucleus accumbens sends γ-aminobutyrate-containing fibres to the rostromedial substantia nigra and to the rostral ventral tegmental area. The caudal ventral tegmental area, the lateral pars compacta and the central pars reticulata do not receive measurable amounts of such fibres.     

Anterograde tracer and field potential analysis of the neocortical layer I projection from nucleus ventralis medialis of the thalamus in cat

The projection of the ventromedial nucleus of the thalamus to the neocortex was studied in cat by means of anterograde and retrograde transport of horseradish peroxidase, by the depth profile of evoked thalamocortical field potentials, and by superfusion of the cortex with manganese to block transmitter release. Horseradish peroxidase injected into the ventromedial nucleus was anterogradely transported to the outer third of layer I in the neocortex, extending from the depth of the cruciate sulcus anterior to the olfactory bulb and tract. The region of projection from the ventromedial nucleus extended mediolaterally from the medial wall of the proreus gyrus to the ventral tip of the coronal gyrus. Horseradish peroxidase injections or applications in these areas of the neocortex resulted in the retrograde labeling of neurons in the ventromedial nucleus. Injections in many other cortical areas did not result in labeled neurons in this nucleus. Stimulation of the ventromedial nucleus with single pulses elicited surface-negative waves in the medial part of the precruciate region that had superficial isoelectric points. Superfusion of the precruciate area with manganese resulted in the suppression of the ventromedial-evoked wave, whereas control extracellular waves in deeper layers were unaffected. An additional additional finding was that horseradish peroxidase injections in the ventromedial nucleus led to a dense reciprocal retrograde labeling of neurons in layer VI of that part of the cortex to which the ventromedial nucleus projects. We conclude that, in cat, (1) the ventromedial nucleus projects to layer I of the cerebral cortex anterior to the cruciate sulcus and receives a dense reciprocal projection from layer VI; (2) stimulation of neurons in the ventromedial nucleus causes depolarization of structures in layer I and these neurons are responsible for recruiting responses in the anterior cortex.     

Ascending projections of presumed dopamine-containing neurons in the ventral tegmentum of the rat as demonstrated by horseradish peroxidase

The projections of presumed dopamine-containing neurons in the zona compacta of the substantia nigra and the ventral tegmental area were examined by stereotaxic injections of horseradish peroxidase into diverse cortical and subcortical regions which are known to include dopamine-containing terminals. Neurons in the lateral half of the substantia nigra pars compacta were labelled after injections into the caudolateral aspect of the caudate-putamen, while neurons in the medial part of the substantia nigra pars compacta and lateral aspect of the ventral tegmental area projected to the anteromedial portion of the caudate putamen. Injections of horseradish peroxidase into the amygdala resulted in the appearance of reactive neurons in the anterior portion of the ventral tegmental area, but the more caudally located entorhinal cortex received projections from the posterior half of the ventral tegmental area. Injections of horseradish peroxidase into the frontal cortex, anterior to the genu, produced scattered labelled cells in the rostral half of the ventral tegmental area, whereas more posterior injections into the cingulate cortex resulted in the appearance of reactive cells which were confined to the medial one-quarter of the substantia nigra pars compacta. The near-midline structure, the lateral septum, was innervated by neurons with cell bodies primarily in the medial half of the ventral tegmental area. Injections of horseradish peroxidase into the nucleus accumbens, which contains very high levels of dopamine, resulted in the appearance of many labelled neurons throughout the ventral tegmental area and some reactive neurons in the medial part of the substantia nigra pars compacta. A few labelled cells were also occasionally observed in the contralateral ventral tegmental area after accumbens injections.These results suggest that although there is considerable overlap, and that the same subdivisions within the substantia nigra pars compacta and the ventral tegmental area appear to innervate diverse regions of the forebrain, there also exists a general topographical organization with respect to the projections of these neurons.Injections of horseradish peroxidase into some of the forebrain regions also resulted in the appearance of reactive cells in mesencephalic nuclei not known to contain dopaminergic perikarya. For example, labelled cells were observed in the supramamillary nucleus after injections into the frontal cortex, entorhinal cortex, accumbens and lateral septum. Injections into the amygdala produced reactive cells in the suprageniculate nucleus, the peripeduncular nucleus, and the magnocellular nucleus of the medial geniculate. These latter results are discussed with reference to the possibility that such pathways may mediate the responsiveness of cells in the amygdala to a wide range of sensory stimuli.