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.

The indirect amygdala-dorsal striatum pathway mediates conditioned freezing: insights on emotional memory networks.

The dorsal striatum (DS) is involved in various forms of learning and memory such as procedural learning, habit learning, reward-association and emotional learning. We have previously reported that bilateral DS lesions disrupt tone fear conditioning (TFC), but not contextual fear conditioning (CFC) [Ferreira TL, Moreira KM, Ikeda DC, Bueno OFA, Oliveira MGM (2003) Effects of dorsal striatum lesions in tone fear conditioning and contextual fear conditioning. Brain Res 987:17-24]. To further elucidate the participation of DS in emotional learning, in the present study, we investigated the effects of bilateral pretest (postraining) electrolytic DS lesions on TFC. Given the well-acknowledged role of the amygdala in emotional learning, we also examined a possible cooperation between DS and the amygdala in TFC, by using asymmetrical electrolytic lesions, consisting of a unilateral lesion of the central amygdaloid nucleus (CeA) combined to a contralateral DS lesion. The results show that pre-test bilateral DS lesions disrupt TFC responses, suggesting that DS plays a role in the expression of TFC. More importantly, rats with asymmetrical pre-training lesions were impaired in TFC, but not in CFC tasks. This result was confirmed with muscimol asymmetrical microinjections in DS and CeA, which reversibly inactivate these structures. On the other hand, similar pretest lesions as well as unilateral electrolytic lesions of CeA and DS in the same hemisphere did not affect TFC. Possible anatomical substrates underlying the observed effects are proposed. Overall, the present results underscore that other routes, aside from the well-established CeA projections to the periaqueductal gray, may contribute to the acquisition/consolidation of the freezing response associated to a TFC task. It is suggested that CeA may presumably influence DS processing via a synaptic relay on dopaminergic neurons of the substantia nigra compacta and retrorubral nucleus. The present observations are also in line with other studies showing that TFC and CFC responses are mediated by different anatomical networks.

Efferent connections of the rostral linear nucleus of the ventral tegmental area in the rat.

The ventral tegmental area (VTA) is crucially involved in brain reward, motivated behaviors, and drug addiction. This district is functionally heterogeneous, and studying the connections of its different parts may contribute to clarify the structural basis of intra-VTA functional specializations. Here, the efferents of the rostral linear nucleus (RLi), a midline VTA component, were traced in rats with the Phaseolus vulgaris leucoagglutinin (PHA-L) technique. The results show that the RLi heavily innervates the olfactory tubercle (mainly the polymorph layer) and the ventrolateral part of the ventral pallidum, but largely avoids the accumbens. The RLi also sends substantial projections to the magnocellular preoptic nucleus, lateral hypothalamus, central division of the mediodorsal thalamic nucleus, lateral part of the lateral habenula and supraoculomotor region, and light projections to the prefrontal cortex, basolateral amygdala, and dorsal raphe nucleus. A similar set of projections was observed after injections in rostromedial VTA districts adjacent to RLi, but these districts also send major outputs to the lateral ventral striatum. Overall, the data suggest that the RLi is a distinct VTA component in that it projects primarily to pallidal regions of the olfactory tubercle and to their diencephalic targets, the central division of the mediodorsal thalamic nucleus and the lateral part of the lateral habenula. Because the rat RLi reportedly contains a lower density of dopaminergic neurons as compared with most of the VTA, its unusual projections may reflect a non-dopaminergic, putative GABAergic, phenotype, and this distinctive cell population seemingly extends beyond RLi boundaries into the laterally adjacent VTA. By being connected to the central division of the mediodorsal thalamic nucleus (directly and via ventral striatopallidal system) and to the magnocellular preoptic nucleus, the RLi and its surroundings may play a role in olfactory-guided behaviors, which are part of the approach responses associated with appetitive motivational states.

Striatal and central extended amygdala parts of the interstitial nucleus of the posterior limb of the anterior commissure: evidence from tract-tracing techniques in the rat.

The interstitial nucleus of the posterior limb of the anterior commissure (IPAC) lies at the junction of the striatopallidal system and the lateral bed nucleus of the stria terminalis-central amygdaloid nucleus continuum (i.e., the central extended amygdala; EAc). Its efferent connections were investigated in the rat with anterograde (Phaseolus vulgaris leucoagglutinin) and retrograde (Fluoro-Gold and cholera toxin B subunit) tracers and compared with those of the central amygdaloid nucleus. Our anterograde tracing experiments reveal that the projections of the medial IPAC largely reciprocate its afferent connections (Shammah-Lagnado et al. [1999] Neuroscience 94:1097-1123) and are very similar to those of the medial part of the central amygdaloid nucleus. The lateral IPAC, on the other hand, innervates the pallidal complex, substantia nigra and retrorubral field. Local connections are found within medial IPAC and within lateral IPAC, but the two divisions are not interconnected. Our retrograde tracing experiments confirm that IPAC projections to EAc components, parabrachial area, and nucleus of the solitary tract originate chiefly from the medial division, whereas both medial and lateral divisions innervate the retrorubral field. Moreover, in sections processed for choline acetyltransferase, the strong projections from caudal IPACm to the posterior basolateral amygdaloid nucleus and the amygdalopiriform transition area were found to arise chiefly from cholinergic cells. Overall, our results suggest that the medial IPAC is intimately related to the EAc, whereas the lateral IPAC represents a striatal territory.

Supracapsular bed nucleus of the stria terminalis contains central and medial extended amygdala elements: evidence from anterograde and retrograde tracing experiments in the rat.

Neurons that accompany the stria terminalis as it loops over the internal capsule have been termed collectively the supracapsular bed nucleus of the stria terminalis (BSTS). They form two cell columns, a lateral column and a considerably smaller medial column. The lateral column merges rostrally with the lateral bed nucleus of the stria terminalis and caudally with the central amygdaloid nucleus (central extended amygdala components). The medial column is continuous with the medial bed nucleus of the stria terminalis and the medial amygdaloid nucleus (medial extended amygdala districts). The connections of the BSTS were investigated in the rat by placing injections of Phaseolus vulgaris-leucoagglutinin (PHA-L) or retrograde tracers in different parts of the extended amygdala or in structures related to the extended amygdala. BSTS inputs and outputs were identified, respectively, by the presence of varicose fibers and retrogradely labeled neurons within the stria terminalis. The results suggest that the medial-to-lateral compartmentalization of BSTS neurons reflects their close alliance with the medial and central divisions of the extended amygdala. The medial BSTS contains primarily elements that correspond to the posterodorsal part of the medial amygdaloid nucleus and the medial column of the posterior division of the medial bed nucleus of the stria terminalis, and the lateral BSTS contains elements that correspond to the medial and lateral parts of the central amygdaloid nucleus and lateral bed nucleus of the stria terminalis. These results add strong support to the concept of the extended amygdala as a ring-like macrostructure around the internal capsule, and they are of theoretical interest for the understanding of the organization of the basal forebrain.

Cortical afferents to the extended amygdala.

The projections of the cerebral cortex to the extended amygdala were studied in the rat using anterograde and retrograde tract-tracing techniques. Most cortical areas with strong projections to the extended amygdala preferentially targeted either the medial extended amygdala (including the medial amygdalar nucleus, ventromedial substantia innominata, and the medial part of the bed nucleus the stria terminalis) or the central extended amygdala (including the central amygdalar nucleus, dorsolateral substantia innominata, and the lateral part of the bed nucleus of the stria terminalis). Some cortical areas, however, had equal projections to both medial and central portions. The main areas projecting preferentially to the medial extended amygdala were the ventral subiculum, infralimbic cortex, ventral agranular insular area, and the rostral part of the ventrolateral entorhinal area. The main areas projecting preferentially to the central extended amygdala were the prefrontal cortex, viscerosensory and somatosensory portions of the insular cortex, and the amygdalopiriform transitional area. It is suggested that these cortical inputs may be important for cognitive, mnemonic, and affective aspects of emotional and motivated behavior.

Afferent connections of the interstitial nucleus of the posterior limb of the anterior commissure and adjacent amygdalostriatal transition area in the rat.

The interstitial nucleus of the posterior limb of the anterior commissure is, like the striatum, very rich in tyrosine hydroxylase and acetylcholinesterase, but on the basis of most other neurochemical criteria displays features that are typical of the extended amygdala (Alheid, de Olmos and Beltramino, 1995). Its afferent connections were examined in the rat with retrograde (cholera toxin B subunit) and anterograde (Phaseolus vulgaris leucoagglutinin) tracers and compared to those of the neighboring amygdalostriatal transition area and central amygdaloid nucleus. Deposits of cholera toxin B subunit in the interstitial nucleus of the posterior limb of the anterior commissure result in retrograde labeling that is similar to that seen after cholera toxin B subunit injections in the central amygdaloid nucleus. Retrogradely labeled cells are found in insular, infralimbic, prelimbic, piriform, amygdalopiriform transition, entorhinal and perirhinal cortices, as well as in temporal field CA1 of Ammon horn and ventral subiculum, amygdala (nucleus of the lateral olfactory tract, anterior amygdaloid area, anterior cortical, posterolateral cortical, anterior and posterior basomedial, intercalated cells, basolateral and lateral nuclei), and extended amygdala, primarily in its central division. The latter includes the lateral bed nucleus of the stria terminalis, dorsal portions of the sublenticular region, the lateral pocket of the supracapsular bed nucleus of the stria terminalis and the central amygdaloid nucleus. Retrogradely labeled cells are also seen in midline thalamic nuclei, lateral hypothalamus, ventral tegmental area, retrorubral field, dorsal raphe nucleus, pedunculopontine and dorsolateral tegmental nuclei, locus coeruleus and parabrachial area. The central extended amygdala, lateral hypothalamus and parabrachial area display a substantial retrograde labeling only when the injection involves districts of the interstitial nucleus of the posterior limb of the anterior commissure apposed to the pallidum, i.e. its medial part. Our anterograde results confirm that projections from the lateral bed nucleus of the stria terminalis and central amygdaloid nucleus to the interstitial nucleus of the posterior limb of the anterior commissure target its medial part. They also indicate that structures which provide major afferents to the central extended amygdala (the lateral and posterior basolateral amygdaloid nuclei and the amygdalopiriform transition area) innervate chiefly the medial part of the interstitial nucleus of the posterior limb of the anterior commissure and, to a much lesser degree, its lateral part. The piriform cortex, which has well-acknowledged projections to the ventral striatum, innervates only the rostral sector of the interstitial nucleus of the posterior limb of the anterior commissure. Taken together, these data indicate that the medial part of the interstitial nucleus of the posterior limb of the anterior commissure is closely related to the central extended amygdala. Rostral and lateral parts of the interstitial nucleus of the posterior limb of the anterior commissure, on the other hand, appear as transitional territories between the central extended amygdala and ventral striatum. The afferent connections of the zone traditionally termed amygdalostriatal transition area are in general similar to those of the caudate-putamen, which does not receive projections from the central extended amygdala. After cholera toxin B subunit injections in the caudoventral globus pallidus, a dense retrograde labeling is observed in the amygdalostriatal transition area and overlying striatum, but not in the interstitial nucleus of the posterior limb of the anterior commissure. Our results suggest that the interstitial nucleus of the posterior limb of the anterior commissure and the amygdalostriatal transition area are engaged in distinct forebrain circuits; the former is a dopamine-rich territory intimately related to the central ext

Efferent connections of the caudal part of the globus pallidus in the rat.

The efferent connections of the caudal pole of the globus pallidus (GP) were examined in the rat by employing the anterograde axonal transport of Phaseolus vulgaris leucoagglutinin (PHA-L), and the retrograde transport of fluorescent tracers combined with choline acetyltransferase (ChAT) or parvalbumin (PV) immunofluorescence histochemistry. Labeled fibers from the caudal GP distribute to the caudate-putamen, nucleus of the ansa lenticularis, reuniens, reticular thalamic nucleus (mainly its posterior extent), and along a thin strip of the zona incerta adjacent to the cerebral peduncle. The entopeduncular and subthalamic nuclei do not appear to receive input from the caudal GP. Descending fibers from the caudal GP course in the cerebral peduncle and project to posterior thalamic nuclei (the subparafascicular and suprageniculate nuclei, medial division of the medial geniculate nucleus, and posterior intralaminar nucleus/peripeduncular area) and to extensive brainstem territories, including the pars lateralis of the substantia nigra, lateral terminal nucleus of the accessory optic system, nucleus of the brachium of the inferior colliculus, nucleus sagulum, external cortical nucleus of the inferior colliculus, cuneiform nucleus, and periaqueductal gray. In cases with deposits of PHA-L in the ventral part of the caudal GP, labeled fibers in addition distribute to the lateral amygdaloid nucleus, amygdalostriatal transition area, cerebral cortex (mainly perirhinal, temporal, and somatosensory areas) and rostroventral part of the lateral hypothalamus. Following injections of fluorescent tracer centered in the lateral hypothalamus, posterior intralaminar nucleus, substantia nigra, pars lateralis, or lateral terminal nucleus, a substantial number of retrogradely labeled cells is observed in the caudal GP. None of these cells express ChAT immunoreactivity, but, except for the ones projecting to the lateral hypothalamus, a significant proportion is immunoreactive to PV. Our results indicate that caudal GP efferents differ from those of the rostral GP in that they project to extensive brainstem territories and appear to be less intimately related to intrinsic basal ganglia circuits. Moreover, our data suggest a possible participation of the caudal GP in feedback loops involving posterior cortical areas, posterior striatopallidal districts, and posterior thalamic nuclei. Taken as a whole, the projections of the caudal GP suggest a potential role of this pallidal district in visuomotor and auditory processes.

Afferent connections of the parvocellular reticular formation: a horseradish peroxidase study in the rat.

The afferent connections of the parvocellular reticular formation were systematically investigated in the rat with the aid of retrograde and anterograde horseradish peroxidase tracer techniques. The results indicate that the parvocellular reticular formation receives its main input from several territories of the cerebral cortex (namely the first motor, primary somatosensory and granular insular areas), districts of the reticular formation (including its contralateral counterpart, the intermediate reticular nucleus, the nucleus of Probst's bundle, the dorsal paragigantocellular nucleus, the alpha part of the gigantocellular reticular nucleus, the dorsal and ventral reticular nuclei of the medulla, and the mesencephalic reticular formation), the supratrigeminal nucleus and the deep cerebellar nuclei. Moderate to substantial input to the parvocellular reticular formation appears to come from the central amygdaloid nucleus, the parvocellular division of the red nucleus, and the orofacial and gustatory sensory cell groups (comprising the mesencephalic, principal and spinal trigeminal nuclei, and the rostral part of the nucleus of the solitary tract), whereas many other structures, including the substantia innominata, the field H2 of Forel, hypothalamic nuclei, the superior colliculus, the substantia nigra pars reticulata, the retrorubral field and the parabrachial complex, seem to represent relatively modest additional input sources. Some of these projections appear to be topographically distributed within the parvocellular reticular formation. From the present results it appears that the parvocellular reticular formation receives afferents from a restricted group of sensory structures. This finding calls into question the traditional characterization of the parvocellular reticular formation as an intermediate link between the sensory nuclei of the cranial nerves and the medial magnocellular reticular districts, identified as the effector components of the reticular apparatus. Some of the possible physiological correlates of the fiber connections of the parvocellular reticular formation in the context of oral motor behaviors, autonomic regulations, respiratory phenomena and sleep-waking mechanisms are briefly discussed.

Afferent connections of the subthalamic nucleus: a combined retrograde and anterograde horseradish peroxidase study in the rat.

A comprehensive characterization of the afferent connections of the subthalamic nucleus of Luys (STN) is a necessary step in the unraveling of extrapyramidal mechanisms. In the present study, the STN afferents in the rat were systematically investigated with the aid of retrograde and anterograde horseradish peroxidase tracer techniques. The results indicate that, besides a massive input from the dorsal pallidum, the STN receives substantial projections from several districts of the cerebral cortex (the medial division of the prefrontal cortex, the first motor and primary somatosensory areas, and the granular insular territory), the ventral pallidum, the parafascicular nucleus of the thalamus and the pedunculopontine tegmental nucleus, as well as a modest innervation from the dorsal raphe nucleus. In spite of the fact that many additional structures were found to contain retrogradely labeled neurons after tracer injections in the STN, no other projection to the latter nucleus could be effectively established in our anterograde experimental series.

Somatosensory inputs to the subthalamic nucleus: a combined retrograde and anterograde horseradish peroxidase study in the rat.

Previous physiological studies have shown that neurons in the subthalamic nucleus (STN) respond to peripheral somatosensory stimulation. In an attempt to identify anatomical pathways that could mediate such responses, the possible existence of direct projections from somatosensory central territories to the STN was investigated in the rat with the aid of retrograde and anterograde horseradish peroxidase tracer techniques. Our main findings indicate the existence of a hitherto undescribed and relatively substantial direct projection from the primary somatosensory cortex to the ipsilateral STN. The projection appears to originate chiefly from neurons in layer Vb of the rostral half of this cortical area and to terminate basically in the dorsolateral district of the STN. Moreover, our data are compatible with the existence of very sparse direct projections from the spinal trigeminal and dorsal column nuclei to the contralateral STN, but the evidence on this point is hardly conclusive.

Afferent connections of the nuclei reticularis pontis oralis and caudalis: a horseradish peroxidase study in the rat.

The afferent connections of the nuclei reticularis pontis oralis and caudalis were studied experimentally in the rat by the aid of either free horseradish peroxidase or horseradish peroxidase conjugated with wheat germ agglutinin used as retrograde tracers. The results suggest that the nucleus reticularis pontis oralis receives its main input from the zona incerta and field H1 of Forel, the superior colliculus, the central gray substance, and the mesencephalic and magnocellular pontomedullary districts of the reticular formation. Many other structures seem to represent modest additional sources of projections to the nucleus reticularis pontis oralis; these structures include numerous cortical territories, the nucleus basalis, the central amygdaloid nucleus, hypothalamic districts, the anterior pretectal nucleus, the substantia nigra, the cuneiform, the accessory oculomotor and the deep cerebellar nuclei, trigeminal, parabrachial and vestibular sensory cell groups, the nuclei raphe dorsalis and magnus, the locus coeruleus, the dorsolateral tegmental nucleus, and the spinal cord. While the afferentation of the rostral portion of the nucleus reticularis pontis caudalis appears to conform to the general pattern outlined above, some deviations from that pattern emerge when the innervation of the caudal district of the nucleus reticularis pontis caudalis is considered; the most striking of these differences is the fact that both spinal and cerebellar inputs seem to distribute much more heavily to the referred caudal district than to the remaining magnocellular pontine reticular formation. The present results may contribute to the elucidation of the anatomical substrate of the functionally demonstrated involvement of the nuclei reticularis pontis oralis and caudalis in several domains that include the regulation of the sleep-waking cycle and cortical arousal, somatic motor mechanisms and nociceptive behavior.

Afferent connections of the zona incerta: a horseradish peroxidase study in the rat.

Restricted microelectrophoretic injections either of free horseradish peroxidase or of horseradish peroxidase conjugated with wheat germ agglutinin were given to albino rats in order to study the afferent connections of structures of the subthalamic region. The results suggest that the zona incerta receives its main input from several territories of the cerebral cortex, the mesencephalic reticular formation, deep cerebellar nuclei, regions of the sensory trigeminal nuclear complex and the dorsal column nuclei. Substantial input to the zona incerta appears to come from the superior colliculus, the anterior pretectal nucleus and the periaqueductal gray substance, whereas many other structures, among which hypothalamic nuclei, the locus coeruleus, the raphe complex, the parabrachial area and medial districts of the pontomedullary reticular formation, seem to represent relatively modest but consistent additional input sources. The afferentation of neurons in Forel's fields H1 and H2 appears to conform to the general pattern outlined above. As pointed out in the Discussion, the present results provide hodological support for the classic concept according to which the zona incerta can be regarded as a rostral extent of the midbrain reticular core. Some of the possible physiological correlates of the fiber connections of the zona incerta in the context of the sleep-waking cycle, ingestive behaviors, somatic motor mechanisms, visual functions and nociceptive behavior are briefly discussed.

Afferent connections of the mesencephalic reticular formation: a horseradish peroxidase study in the rat.

The afferent connections of the mesencephalic reticular formation were studied experimentally in the rat by the aid of the retrograde horseradish peroxidase tracer technique. The results suggest that the rostral portion of the mesencephalic reticular formation receives its main input from the cerebral cortex, the zona incerta and the fields of Forel, the central gray substance, the nuclei reticularis pontis oralis and caudalis, and the deep cerebellar nuclei. Substantial input to the same territory of the mesencephalic reticular formation appears to come from the superior colliculus, the substantia nigra, the parabrachial area, the spinal trigeminal nucleus, and the nucleus reticularis gigantocellularis, whereas several other brain structures, among which the locus coeruleus and the raphe complex, seem to represent modest but consistent additional input sources. The afferentation of more caudal portions of the mesencephalic reticular formation appears to conform to the general pattern outlined above with only three exceptions; the cerebral cortex, the deep cerebellar nuclei and the spinal trigeminal nucleus seem to be relatively modest sources of projections to these levels. Considering that the mesencephalic reticular formation is a critical structure in the "ascending activating systems" the present results, confirming and extending those of many other investigators, characterize a set of pathways that seem to be an important part of the anatomical substrate of the sleep-walking cycle.

Efferent connections of the olfactory bulb in the opossum (Didelphis marsupialis aurita): a Fink-Heimer study.

The efferent concentrations of the olfactory bulb (OB) in the opossum (Didelphis marsupialis aurita) were studied by the aid of the Fink-Heimer technique. Following lesions restricted to the OB, ipsilateral degenerating fibers entered the lateral olfactory tract and were treated to terminal fields essentially limited to the outer portion of the plexiform layer (sublamina IA) of the following structures: all the subdivisions of the anterior olfactory nucleus, the rostroventral tenia tecta, the full extent of the olfactory tubercle, the nucleus of the lateral olfactory tract, the anterior portion of the medial amygdaloid nucleus, the whole cortical amygdaloid nucleus (in the posteromedial subdivision of this structure the degeneration was very scanty), and the sulcal, piriform, and lateral entorhinal cortices. Some degree of topographical organization in the OB projections was noticed in the rostral portion of the lateral olfactory tract and within the external and lateral subdivisions of the anterior olfactory nucleus. In another series of experiments, when the lesion also involved the accessory olfactory bulb, heavy terminal degeneration occurred along the whole extent of the medial amygdaloid nucleus and in the posteromedial subdivision of the cortical amygdaloid nucleus. These findings indicate that, although very similar to those described in other mammals, the OB efferent connections in the opossum present some peculiarities; namely, the existence of dense terminal fields in the sulcal cortex and in the rostral district of the medial amygdaloid nucleus.