The monkey and rat tissue to be examined under the light microscope was subject to microwave antigen retrieval prior to immunostaining in order to optimize the immunolabeling. The monkey and rat parasagittal sections used for mapping were immunoreacted together to maximize the comparability of the data; the remaining rat tissue was immunoreacted in independent experiments using the same protocol. The coronal sections used for electron microscopy were incubated in a cryoprotectant solution (25% sucrose and 3% glycerol in 0.05 M phosphate buffer) for 50 min and then rapidly freeze-thawed by immersion in liquid nitrogen to enhance the penetration of immunoreagents. Thereafter, these sections underwent the same immunohistochemical procedure as the tissue for light microscopy, save for the complete omission of Triton X-100 in the incubations. The primary antibody was MAB369, a rat monoclonal supplied by Chemicon International (Temecula, CA). This antibody corresponds to the DAT/Nt antibody generated against human DAT N-terminus aminoacids 1–66 by Ciliax et al. (1995), who demonstrated its specificity using immunoblot analysis, immunoprecipitation, preadsorption followed by immunoblotting and immunohistochemistry, and several immunohistochemical assays. MAB369 was used at a 1:1000 dilution. The secondary antiserum was AP164B, a biotinylated rabbit anti-rat antiserum, from the same company, used at a 1:400 dilution. The sections were incubated in Vectastain Elite avidin/biotin complex from Vector Labs (Burligame, CA) prior to peroxidase development. The sensitive glucose oxidase–diaminobenzidine–nickel method (Shu et al. 1988) was used to develop the rat and monkey sections to be examined under the light microscope. A standard diaminobenzidine protocol (0.05% diaminobenzidine and 0.003% H₂O₂) was used to develop the monkey sections to be processed for ultrastructural analysis.

The different densities of the dopamine innervation in the primate thalamic nuclei are expressed in Figure 6 by different intensities of red. In light of the dopamine innervation in the thalamic nuclei and the tight connections between thalamus and cortex, it follows that the cortical regions most influenced by thalamic dopamine are the prefrontal and motor cortices, which themselves receive a strong direct dopamine innervation (Williams and Goldman-Rakic 1993; Lewis et al. 2001), and dorsal portions of the posterior parietal cortex (Fig. 6; see review of primate thalamo-cortical connections of the nuclei with high or moderate dopamine innervation in Table 1 of García-Cabezas et al. 2007). The cingulate cortex and large territories of parieto-occipito-temporal association cortex would be moderately influenced by thalamic dopamine while the primary sensory areas would be the least influenced (Fig. 6). The present data provide, however, an exception to this general scheme: the primary gustatory cortex and the nearby primary somatosensory cortex where the inner mouth mucosa is represented (located in the frontal operculum and adjacent anterior parietal cortex—Krubitzer et al. 1995; Hirata et al. 2005; Miyamoto et al. 2006; Smits et al. 2007), receive connections from thalamic nuclei that appear moderately innervated by DAT-expressing axons, specifically, from VPMpc and the ventral part of VPM (Figs 1 and ​and44).

In marked contrast with the prominent presence of DAT-expressing axons in the monkey thalamus, the rat thalamus holds only limited clusters of axons and scattered axons that express DAT (Fig. 2). Our findings of DAT-positive axon clusters in the Pv and MD nuclei, and in the Hl, are consistent with the reported presence of dopamine-ir fibers in these nuclei (Groenewegen 1988) and with changes described in rat MD neuron membrane properties following D2 receptor stimulation (Lavin and Grace 1998). In the present study we also found substantial DAT immunolabeling in the rat R nucleus and ZI, and clusters of DAT-ir axons in the VM and VL ventral nuclei, and scattered DAT-ir axons in Pt, AV, CL, and Pf. The presence of DAT-ir axons in the rat Pv, Hl and R concurs with tract-tracing studies reporting projections to these nuclei from dopaminergic neurons of the fore-and/or midbrain (Takada et al. 1990; Li et al. 1993; Anaya-Martinez et al. 2006). However, we did not observe DAT-ir axons in the GL, where dopamine-ir axons have been reported (Papadopoulos and Parnavelas 1990), and a number of studies have shown that delivery there of dopamine or dopamine-receptor agonists has an effect on neuronal activity (Albrecht et al. 1996; Govindaiah and Cox 2005; Munsch et al. 2005). It is possible that dopaminergic axons in the GL nucleus, if present, are devoid of DAT or express it below the threshold level for our methods. It should be borne in mind that not all dopaminergic neurons express DAT (Sánchez-González et al. 2005); that, among those expressing it, levels are variable (Haber et al. 1995; Sánchez-González et al. 2005); and that some dopaminergic cell groups, specifically A8 through A11, hold a mixed population of cell bodies that, being dopaminergic, are either DAT-positive or DAT-negative (Table 1 in Sánchez-González et al., 2005). Tract-tracing data in the monkey show that the thalamus receives projections from both dopaminergic DAT-positive and DAT-negative neurons. In the midline nuclei, for example, the majority of the dopaminergic axons come from hypothalamic and lateral parabrachial dopaminergic cell groups that do not express DAT; the non-midline nuclei, which make up most of the thalamic volume, however, receive the bulk of their afferent dopaminergic input from DAT-expressing cell groups (Sánchez-González et al. 2005). It is possible, then, that the rat GL nucleus receives dopaminergic input from neurons that do not express DAT. Examination of the origin of the dopaminergic axons targeting the rat thalamic nuclei, including GL, would help to settle if the latter receives a dopamine innervation. In addition, it would help to clarify if the origin of the dopamine innervation of the rat thalamus comes from such a diverse collection of dopaminergic cell groups as in the monkey, which includes groups that express DAT, as in the mesencephalon, and those that are devoid of DAT expression, as in the hypothalamus (Sánchez-González et al. 2005).

The observed expansion in extent and density of the dopamine innervation in the primate versus the rodent dorsal thalamus deserves comment. The general layout of dopaminergic cell groups is comparable in the primate and rodent brains although dopaminergic neurons are more widely distributed in the primate than in the rodent mesencephalon (Kitahama et al. 1994; Verney 1999). The general distribution of dopaminergic terminals in the striatum is also similar (Reiner 1994; Ciliax et al. 1995; Ciliax et al. 1999). In the cerebral cortex, however, the patterns of regional and laminar distribution of dopaminergic axons differ significantly between primates and rodents. In rats, dopamine innervation is present mainly in the prefrontal, anterior cingulate, insular, piriform, entorhinal, and perirhinal cortices, with the posterior cingulate, motor, parietal, and temporal cortices being lightly innervated. Dopaminergic terminals in the rat cortex are distributed in deep layers with the exception of the anterior cingulate and entorhinal cortices where they are present in deep and superficial layers (Berger et al. 1991; Ciliax et al. 1995). In primates the dopamine innervation in the cerebral cortex is expanded and differently distributed: it targets all cortical areas, is densest in the motor cortices, and is present in the deep and superficial layers with the molecular layer being the most densely innervated in all areas (Berger et al. 1991; Williams and Goldman-Rakic 1993; Ciliax et al. 1999; Lewis et al. 2001). Thus, it appears that the major differences in dopamine innervation between the rodent and primate brains are in the cerebral cortex and dorsal thalamus (present report and García-Cabezas et al. 2007).

Moreover, there seems to be a correlation between the relative densities of dopaminergic axons in the primate cortex and thalamus, with cortical regions heavily innervated by dopamine connected with densely innervated thalamic nuclei (i.e., motor, prefrontal, and posterior parietal areas; ventral motor, MD, and LP thalamic nuclei), and poorly innervated cortical regions linked with dopamine poor thalamic nuclei (e.g., primary visual cortex; GL nucleus). Thus, the wider and denser dopamine innervation in the primate dorsal thalamus as compared with the rodent may parallel the expansion of the mesocortical dopaminergic system noted by Berger and Gaspar (1994) in relation to encephalization.

In the relatively large number of labeled axons examined here we were able to identify 6 synaptic junctions, 5 asymmetric and one symmetric, and their corresponding postsynaptic profiles. In all cases, the latter showed the features of presynaptic dendrites on a thalamic interneuron. Identification of synapses and postsynaptic profiles in catecholaminergic axons of the monkey thalamus has been elusive. We have identified them in only 2% of the DAT-immunolabeled axons, mostly within the population of axons that were cut longitudinally. Despite the low numbers, the present data represent the largest number of synaptic contacts identified to date in thalamic dopaminergic axons. In previous studies only 3 (Liu and Jones 1991) and 2 (Melchitzky et al. 2006) synaptic profiles were observed, all in tyrosine hydroxylase immunolabeled axons; and hence, they were only putatively dopaminergic. Among these 5 synapses the postsynaptic element was identified in only 3 cases: it was a dendritic shaft in 2 (Liu and Jones 1991; Melchitzky et al. 2006) and a presynaptic dendrite in the third case (Melchitzky et al. 2006). Apart from the number of axons analyzed, which should be high enough to provide a sizable number of synaptic forms, a main reason for the paucity of data on synapses and postsynaptic targets of catecholaminergic fibers in the monkey thalamus may lie in the plane of section. In our material synaptic specializations appeared at a mean distance of about 0.5 μm from the DAT immunoprecipitate; for this reason axons cut longitudinally are more likely to display synaptic specializations. In the monkey MD, most DAT-ir axons follow a rostro-caudal course (Fig. 1) and thus the coronal plane of section used so far (Melchitzky et al. 2006; present study) renders mostly transversely cut axons (76% of the total DAT-ir axons in our material); the parasagittal plane of section should render more longitudinally cut axons and would thus be more appropriate for the analysis of dopaminergic synapses. Another technical means to refine and increase the identification of synaptic profiles in dopaminergic axons would be the use of serial sections. Future exhaustive ultrastructural analyses may uncover further details of the morphological relationships between dopaminergic axons and their targets in the monkey thalamus.

The subcellular location of the DAT immunoprecipitate, and the identification in the present study of the interneurons as the predominant postsynaptic element in the monkey MD are worth consideration. In most axons (82%) the DAT immunoprecipitate was apposed to the membrane, and when synapses were identified, the immunoprecipitate was located far from them (Fig. 5D). This suggests that in the macaque MD synaptically released dopamine may diffuse for considerable time and distance before being re-uptaken by DAT. This possibility is compatible with a “volume” or “extrasynaptic” transmission mode in the thalamic dopaminergic system comparable to that proposed for the mesostriatal and mesocortical systems (Pickel et al. 1996; Zoli et al. 1998; Lewis et al. 2001). The plausible existence of volume transmission would mean that the released dopamine may spread farther than the postsynaptic neurons, thus reaching projection neurons.

The gift of monkey tissue by Dr Uwe Ilg and Dr Peter Thier, University of Tübingen, Germany, is gratefully acknowledged. We thank Gemma De la Fuente, Begoña Rodríguez Menéndez, Rosa Sánchez Lozano, Covadonga Aguado Ballano, and Francisco Urbano for qualified technical assistance.

This manuscript was prepared following a presentation at the Kavli Symposium in Memoriam of Patricia S. Goldman-Rakic held in New Haven, CT, USA, in May 2006. Carmen Cavada wishes to express her most sincere gratitude to Patricia Goldman-Rakic: Pat's mentorship at Yale University in the 1980s was decisive for my scientific career. Pat exposed me to the nonhuman primate brain; it was a privileged and exciting experience. Pat's influence is still having a protracted and extensive effect on the collaborators that have worked and are now working in my group, always with a focus on the primate brain. The professional collaboration between Pat and myself mingled and continued with a growing friendship. I value most what she taught me through her grace and attitudes: that each person has his talents, and that it is wise to discover, nurture and focus on them. It was a blessing to enjoy Pat's mentorship and amity. This article is dedicated to her memory. Conflict of Interest: None declared.