Glutamatergic afferents of the ventral tegmental area in the rat - PubMed
- ️Mon Jan 01 2007
Micrographs illustrating the binding of probes complementary to VGLUT1, VGLUT2, and VGLUT3 mRNA mostly in distinct parts of the brain. A, VGLUT1 mRNA was predominantly expressed by neurons in cortex [e.g., in the infralimbic (IL) and piriform (Pir) areas and anterior olfactory nucleus (AON)], whereas most subcortical structures were devoid of VGLUT1 mRNA binding, as apparent in the caudate–putamen (CPu), accumbens (Acb), and olfactory tubercle (OT). TT, Taenia tecta. B, In contrast, VGLUT2 mRNA was mainly expressed by neurons in subcortical structures, such as, for example, the mediodorsal thalamic nucleus (MD), lateral hypothalamic area (LH), and entopeduncular nucleus (EPN). C, VGLUT3 mRNA (arrows) was present in discrete cell populations [e.g., in the accumbens core (Acbc) and shell (Acbsh)]. ac, Anterior commissure; cc, corpus callosum; lv, lateral ventricle; ic, internal capsule. Scale bar: (in A) A, 500 μm; B, 400 μm; C, 200 μm.
A, B, Micrographs illustrating injections of the retrograde tracer WGA-apo HRP-gold extending through the rostral (A, B) and caudal (A′, B′) VTA. Note that the dense black core of the tracer deposit is surrounded by a less dense halo (B, B′). The sections were subjected to silver enhancement to better visualize the injection sites (black). In addition, sections of case 05179 (A, A′) were processed to exhibit tyrosine hydroxylase immunoreactivity and cresyl violet counterstaining to delineate the VTA and surrounding structures. Sections of case 06056 (B, B′) were processed for nonisotopic in situ hybridization with a probe against VGLUT2 mRNA. Scale bar: (in A) A–B′, 1 mm. C, C′, Schematic representations of all of the tracer injections into the VTA. Note that all extend from the rostral (C) to caudal (C′) VTA. IP, Interpeduncular nucleus; ml, medial lemniscus; SNr, substantia nigra reticulata.
A–I, Schematic representations showing the distribution of VGLUT1 mRNA-positive, VTA-projecting neurons (red triangles) and retrogradely labeled neurons lacking detectable VGLUT1 mRNA (blue dots). Each triangle or dot reflects one neuron. Note that few retrogradely labeled neurons were not positive for VGLUT1 mRNA (see also insets in C, D). Most double-labeled neurons were situated in the medial prefrontal cortex [e.g., in the prelimbic (PrL) and infralimbic (IL) cortices (D–E)]. To simplify the diagram, retrogradely labeled neurons were plotted only in the areas expressing VGLUT1 mRNA-positive neurons. [For complete retrograde labeling after tracer injections into the VTA, see Geisler and Zahm (2005, 2006b)]. J–O, VGLUT1mRNA was abundantly expressed by neurons in the cingulate cortex (J), an area that also contained numerous VTA-projecting neurons (K, shown in dark field). At higher magnifications [M, N (an enlargement of M)], the retrograde labeling (black puncta) can be clearly recognized in neurons positive for VGLUT1 mRNA (purple). The dorsal peduncular cortex (E, F, L), situated lateral to the taenia tecta (TT) and dorsomedial to the lateral septum (LS), also contained many double-labeled neurons (O, an enlargement of L). Arrows indicate double-labeled neurons; arrows with asterisks indicate the same double-labeled neurons in M and N. DP, Dorsal peduncular cortex; AI, agranular insular cortex; ac, anterior commissure; BLA, basolateral amygdala; cc, corpus callosum; Cg, cingulate cortex; Cl, claustrum; CPu, caudate–putamen; LO, lateral orbital cortex; MHb, medial habenula; MO, medial orbital cortex; VO, ventral orbital cortex. Scale bar: (in J) J, K, 300 μm; L, 200 μm; M, 30 μm; N, O, 10 μm.
Schematic representations and photomicrographs illustrating the distribution of VGLUT2 mRNA-positive, VTA-projecting neurons in the forebrain. A–H, Each dark gray dot represents one double-labeled neuron. The parts of the sections illustrated by photomicrographs are indicated by rectangles on the corresponding drawings. The injection site is illustrated in Figures 2 and 6F–H (case 06151). To give an impression of double labeling relative to neurons exhibiting only retrograde label, retrogradely labeled, probe-negative neurons are shown in E (light gray triangles). VGLUT2 mRNA-positive, VTA-projecting neurons were widely distributed throughout the brain. Relatively few neurons per structure were detected, however. AI, AII, Strongly VGLUT2 mRNA-positive neurons were scattered throughout the ventral endopiriform area. The accumulation of silver grains in some of these neurons was among the densest observed in this study (compare AIII with HIII, Fig. 6DII,DIII,KII,KIII). H, Density and staining intensity of VGLUT2 mRNA-positive neurons in the medial (e.g., paraventricular and ventromedial hypothalamic nucleus) and lateral hypothalamus (HII is an enlargement of HI. The fornix serves as a reference mark). An example of a double-labeled neuron is given in HIII (arrow, enlargement of HII). Black arrows in A and H point to double-labeled neurons. The open arrow in AI points to the VGLUT2 mRNA-positive neuron lacking the retrograde label shown in AII. ac, Anterior commissure; Acb, accumbens; BST, bed nucleus of stria terminalis; CPu, caudate–putamen; cc, corpus callosum; DB, diagonal band of Broca; f, fornix; GP, globus pallidus; HDB, horizontal limb of DB; ic, internal capsule; LA, lateroanterior hypothalamic nucleus; LH, lateral hypothalamic area; LPO, lateral preoptic area; lv, lateral ventricle; MPA, medial preoptic area; MS, medial septum; ot, optic tract; Pa, paraventricular hypothalamic nucleus; SLEA, sublenticular extended amygdala; SLSI, sublenticular substantia innominata; SFi, septofimbrial nucleus; VEn, ventral endopiriform nucleus; VMH, ventromedial hypothalamic nucleus; VP, ventral pallidum; ZI, zona incerta; III, third ventricle. Scale bars: AI, HII, 200 μm; HI, 300 μm; AII, AIII, HIII, 20 μm.
VGLUT2 mRNA-positive, VTA-projecting neurons in the ventral pallidum (VP). A–C and D–F illustrate rostral and caudal parts of the VP, respectively. A and D are micrographs of sections immunoreacted to exhibit neuronal nitric oxide synthase (NOS) to delineate the VP and surrounding structures. B and E illustrate sections adjacent to those shown in A and D, respectively, which were subjected to silver enhancement to visualize the retrograde tracer and probe against VGLUT2 mRNA. B, E, G–I, Numerous densely packed VGLUT2 mRNA-positive neurons were observed in the rostral VP (B, G, H), whereas fewer, but darkly stained, VGLUT2 mRNA neurons were observed further caudal in the VP (E, I). A–F, VGLUT2 mRNA-positive neurons were also present in the subcommissural extended amygdala (scEA), horizontal limb of diagonal band of Broca (HDB), and medial (MPA) and lateral (LPO) preoptic area (D, E). C and F are schematic representations of B and E, respectively, illustrating retrogradely labeled neurons (gray triangles) and VGLUT2 mRNA-positive, VTA-projecting neurons (black dots). Note that although many VGLUT2 mRNA-positive neurons (B, E) and retrogradely labeled neurons (gray triangles) were present in the basal forebrain, relatively few neurons contained both markers (black dots). G and H are enlargements of the boxed areas in B; I is an enlargement of the boxed area in E. Black arrows point to double-labeled neurons. Black arrows with asterisks point to the double-labeled neurons that are enlarged in the insets. Open arrows point to retrogradely labeled neurons. d, Dorsal; l, lateral; v, ventral; m, medial; ac, anterior commissure; CPu, caudate–putamen; vBST, ventral bed nucleus of stria terminalis. Scale bar: (in A) A–F, 300 μm; G–I, 20 μm.
A–Q, Continuation of the series of sections shown in Figure 4 illustrating the distribution of VGLUT2 mRNA-positive, VTA-projecting neurons in the caudal forebrain and brainstem. Symbols and formatting are as in Figure 4. The injection site is illustrated in F–H. Note that the center of the injection site (G) is surrounded by a less dense halo of silver enhancement product (F, G, hatched area). A, J, To give an impression of double labeling relative to neurons exhibiting only retrograde label, retrogradely labeled, probe-negative neurons are shown (light gray triangles). VGLUT2 mRNA-positive, VTA-projecting neurons were widely distributed throughout the forebrain and brainstem. D, Numerous VGLUT2 mRNA-positive neurons were present in the lateral habenula (DI), of which a moderate number contained retrograde labeling (DII, DIII). K, The median raphe contained scattered, strongly VGLUT2 mRNA-positive neurons (KI), some of which were retrogradely labeled (KII, KIII). Black arrows in D and K point to double-labeled neurons. aq, Aqueduct; BLA, basolateral amygdala; cbl, cerebellum; CG, central gray; (Figure legend continues) Cn, cuneiform nucleus; CPu, caudate–putamen; CS, superior colliculus; DpMe, deep field of mesencephalic reticular formation; DR, dorsal raphe; f, fornix; fr, fasciculus retroflexus; GP, globus pallidus; ic, internal capsule; IP, interpeduncular nucleus; LDTg, laterodorsal tegmental nucleus; LH, lateral hypothalamic area; LHb, lateral habenula; MHb, medial habenula; MM, mammillary body; MR, median raphe; ot, optic tract; Pa, paraventricular hypothalamic nucleus; PAG, periaqueductal gray; PB, parabrachial nucleus; Pf, parafascicular nucleus; PH, posterior hypothalamic nucleus; PnC, caudal field of pontine reticular formation; PnO, oral field of pontine reticular formation; Pr, prehypoglossal nucleus; PPTg, pedunculopontine tegmental nucleus; R, red nucleus; RIP, raphe interpositus; scp, superior cerbellar peduncle; SNr, substantia nigra reticulata; STN, subthalamic nucleus; SUM, suparmammillary nucleus; TC, tuber cinereum; VEn, ventral endopiriform nucleus; VMH, ventromedial hypothalamic nucleus; xscp, decussatio of scp; ZI, zona incerta; III, third ventricle; 4v, fourth ventricle; 7n, facial nerve. Scale bar: (in D) DI, 300 μm; KI, 200 μm; DII, DIII, KII, KIII, 20 μm.
Micrographs illustrating sections through rostral (A), middle (B), and caudal (C, D) levels of the pedunculopontine tegmental nucleus (PPTg). A–C, Sections immunoreacted for neuronal nitric oxide synthase, which identifies the PPTg and laterodorsal tegmental nucleus (LDTg). D, A section, adjacent to that in C, subjected to nonisotopic in situ hybridization against VGLUT2 mRNA and silver intensification. The positions of VGLUT2 mRNA-positive (A, B, blue diamonds) and VGLUT2–VTA-projecting (A–D, red dots) neurons were plotted onto the sections shown in A–C from appropriately processed adjacent sections, such as that shown in D (some VGLUT2 mRNA-positive neurons are indicated by arrows) after tracer injection in the VTA. A, Note that relatively few VGLUT2 mRNA-positive neurons are found rostrally and these are located predominantly in the vicinity of the PPTg. B, D, In contrast, numerous VGLUT2 mRNA-positive neurons are scattered within the reticular formation more caudally. C, Only after having superimposed the plots of double-labeled neurons onto adjacent sections reacted for neuronal nitric oxide synthase immunoreactivity did it become apparent that double-labeled neurons are localized in the dissipated (dp) and compact (cp) parts of the PPTg and in the LDTg, periaqueductal gray (PAG), and cuneiform nucleus (Cn). The encircled red dot in D indicates a double-labeled neuron enlarged in the inset. The same double-labeled neuron, when plotted onto the adjacent section immunoreacted for neuronal nitric oxide synthase, occupies the compact part of the PPTg (C, arrow). Note in D that only a small proportion of VGLUT2 mRNA-positive neurons (some are indicated by arrows) are also retrogradely labeled (red dots). Not shown are retrogradely labeled neurons that do not contain VGLUT2 mRNA. aq, Aqueduct; DpMe, deep field of mesencephalic reticular formation; DR, dorsal raphe; ml, medial lemniscus; xscp, decussation of the superior cerebellar peduncle. Scale bar: (in A) A, B, 200 μm; C, D, 250 μm.
D–G, VGLUT3 mRNA-positive, VTA-projecting neurons (black circles) were localized predominantly in the dorsal (DR) and median (MR) raphe nuclei. A, B, Occasional double-labeled neurons were present in the medial division of the anterior bed nucleus of the stria terminalis (BSTMA; A) and lateral habenula (LHb; B). C, H, In addition, few double-labeled neurons were observed in the caudal interpeduncular nucleus (IP; C) and pontine central gray (Cg; H). Note that although many neurons in the MR were retrogradely labeled (gray triangles), only relatively few of them were also positive for VGLUT3 mRNA (black dots). In contrast, many double-labeled neurons were present in the dorsal raphe. [To simplify the presentation, retrogradely labeled neurons were plotted only in the raphe nuclei and central gray. For complete retrograde labeling after tracer injections into the VTA, see Geisler and Zahm (2005, 2006b)]. ac, Anterior commissure; cp, cerebral peduncle; CPu, caudate–putamen; DTg, dorsal tegmental nucleus; LC, locus ceruleus; LDTg, laterodorsal tegmental nucleus; ml, medial lemnisus; Mo5, motor nucleus of fifth cranial nerve; PAG, periaqueductal gray; PMnR, paramedian raphe; Pn, pontine nuclei; PPTg, pedunculopontine tegmental nucleus; scp, superior cerebellar peduncle; VTg, ventral tegmental nucleus; xscp, decussation of the superior cerebellar peduncle.
A–C, VGLUT3 mRNA was strongly expressed in neurons of the median raphe (MnR), whereas no signal was detected in the adjacent deep field of the mesencephalic reticular formation (DpME). Although many retrogradely labeled neurons were present in the median raphe (open arrows), only a few of them expressed both markers (black arrows). C is an enlargement of B, illustrating the double-labeled and retrogradely labeled neurons that are marked in B with arrows indicated by asterisks. Scale bars: A, 100 μm; B, 20 μm; C, 10 μm.
Summary diagram depicting glutamatergic projections to the VTA. The brain areas containing VGLUT1 (dark gray), VGLUT2 (light gray), or VGLUT3 (slate) mRNA-positive, VTA-projecting neurons are indicated separately. The sizes of ovals reflect the hypothetical proportion of the VGLUT–VTA-projecting neurons in those structures relative to all VGLUT mRNA-positive, VTA-projecting neurons. To arrive at these figures, the averages of VGLUT1, VGLUT2, and VGLUT3 mRNA-positive, VTA-projecting neurons of all cases (n = 12 rats) were added and taken as the total number of neurons, and the proportions of the VGLUT in every structure were calculated from it. The right side of the figure is ipsilateral to the injections. Cl/Den, Claustrum/dorsal endopiriform area; CG, central gray; Cn, cuneiform nucleus; DR, dorsal raphe; LH, lateral hypothalamic area; LHb, lateral habenula; LPO, lateral preoptic area; MH, medial hypothalamus; MPA, medial preoptic area; MR, median raphe; MS/DB, medial septum/diagonal band complex; PB, parabrachial nucleus; PFC*, prefrontal cortex, prelimbic cortex not included; PPTg/LDTg, pedunculopontine and laterodorsal tegmental nuclei; PrL, prelimbic cortex; RF, reticular formation; SLSI, sublenticular substantia innominata; VEn, ventral endopiriform area; VP, ventral pallidum.