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Frontiers in Biology

Front. Biol.    2016, Vol. 11 Issue (3) : 246-255
Combined immunohistochemical and retrograde tracing reveals little evidence of innervation of the rat dentate gyrus by midbrain dopamine neurons
Charlotte M. Ermine1,*(),Jordan L. Wright1,2,Clare L. Parish1,Davor Stanic1,Lachlan H. Thompson1
1. The Florey Institute for Neuroscience and Mental Health, The University of Melbourne, Parkville, Victoria 3010, Australia
2. Current address Wolfson Institute for Biomedical Research, University College London, Gower Street, London WC1E 6BT, UK
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Although the functional neuroanatomy of the midbrain dopamine (mDA) system has been well characterized, the literature regarding its capacity to innervate the hippocampal formation has been inconsistent. The lack of expression of definitive markers for dopaminergic fibers, such as the dopamine transporter, in the hippocampus has complicated studies in this area. Here we have used immunohistochemical techniques to characterize the tyrosine hydroxylase expressing fiber network in the rat hippocampus, combined with retrograde tracing from the dentate gyrus to assess the capacity for afferent innervation by mDA neurons. The results indicate that virtually all tyrosine hydroxylase fibers throughout the hippocampus are of a noradrenergic phenotype, while the overlying cortex contains both dopaminergic and noradrenergic fiber networks. Furthermore, retrograde tracing from the dentate gyrus robustly labels tyrosine hydroxylase-immunoreactive noradrenergic neurons in the locus coeruleus but not mDA neurons.

Keywords noradrenaline      hippocampus      connectivity      DAT      neurogenesis     
Corresponding Author(s): Charlotte M. Ermine   
Just Accepted Date: 25 May 2016   Online First Date: 23 June 2016    Issue Date: 05 July 2016
 Cite this article:   
Charlotte M. Ermine,Jordan L. Wright,Clare L. Parish, et al. Combined immunohistochemical and retrograde tracing reveals little evidence of innervation of the rat dentate gyrus by midbrain dopamine neurons[J]. Front. Biol., 2016, 11(3): 246-255.
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Charlotte M. Ermine
Jordan L. Wright
Clare L. Parish
Davor Stanic
Lachlan H. Thompson
Fig.1  Immunohistochemical detection of dopamine and noradrenaline fibers in the rat dentate gyrus. The dentate gyrus contains a fiber network that can be labeled with antibodies against tyrosine hydroxylase (A) or dopamine-b-hydroxylase (F) but not the dopamine transporter (K). Enlargements of the boxed areas in A and F show that TH (B–E) and DBH (G–J)fiber innervation is particularly dense in and around the sub-granular and granular layers, identified by doublecortin and Prox1 respectively, while there is a complete absence of DAT expression (L–O). Triple-labeling for TH, DBH, and DAT (P-S) shows that all TH+ fibers express DBH, while none co-label with DAT. Scale bars:A, F, and K, 200 µm and B-E, G–J, L–O, and P–S, 50 µm. Abbreviations: DAT, Dopamine transporter; DBH, Dopamine-beta-hydroxylase; DCX, Doublecortin; Prox1, Prospero homeobox1; TH, Tyrosine hydroxylase.
Fig.2  Overview of dopamine and noradrenergic fibers in the septal hippocampus and overlying cortex. A) Photomontage of immunohistochemical detection of TH, DBH, and DAT in the rat hippocampus and cortex. Boxed areas are shown as enlarged views of hippocampal areas with prominent TH fiberlabeling, including the sub-granular zone and molecular layer of the dentate gyrus (B) and CA3 (C) and also the overlying cortex (D) where TH+/DA+ fibers (arrows) were intermingled with TH/DBH fibers. Scale bars: A, 200 µm and B–D, 50 µm. Abbreviations: CA1, Cornus Ammonis 1; CA3, Cornus Ammonis 3; DGmol, Dentate gyrus molecular layer.
Fig.3  Retrograde labeling of midbrain neurons 5–7 days following injection of fluorogold into the dentate gyrus. Immunolabeling of fluorogold in a representative animal at the injection site (A) showing distribution in the dentate gyrus and some labeling in the overlying molecular layer and CA1, and in the midbrain(B), showing retrogradely labeled neurons with TH labeling (green) shown for anatomical reference. (C–E) Merged image and individual color channels showing fluorogold-labeled cells (arrows) interspersed among, but not overlapping with TH+ DA neurons in the VTA. (F) Representative fluorogold labeling at the injection site in one of two animals where fluorogold labeling was found along the injection tract in the cortex. (G–I) In these animals a total of three fluorogold-labeled TH+ DA neurons were identified in the midbrain. Scale bars:A, B, F, 200 µm; C–E and G–I, 50 µm. Abbreviations: FG, Fluorogold; TH, Tyrosine hydroxylase.
Fig.4  Cumulative spatial representation of fluorogold labeling in the midbrain of 10 animals 5–7 days after injection of fluorogold into the dentate gyrus. (A) Schematic overview of three representative coronal levels spanning the A9-A10 cell groups illustrating the location of all 605 fluorogold-labeled cells, indicated as red circles for FG+/TH- cells and yellow circles for FG+/TH+ cells. The black arrow represents the ipsilateral side injected with fluorogold. (B–D) Co-labeling for GABA showed that some of the FG+ neurons innervating the hippocampus were GABAergic projection neurons. Scale bars: B–D, 50 µm. Abbreviations: FG, Fluorogold; fr, fasciculus retroflexus; GABA, g aminobutyric acid; IPC, Interpeduncular nucleus central; MT, Medial terminal nucleus accessory optic tract; SNr, Substantia nigra reticular; VTA, Ventral tegmental area.
Fig.5  Retrograde labeling of noradrenergic neurons in the locus coeruleus 5–7 days after injection of fluorogold into the dentate gyrus. (A–C) Immunolabeling of fluorogold and TH showed extensive overlap in the locus coeruleus (arrowheads) as well as a minor population of FG+/TH- cells (arrows). (D) Schematic overview of three representative coronal levels spanning the locus coeruleus where the location of fluorogold labeled cells are indicated as red circles for FG+/TH- cells and yellow circles for FG+/TH+ cells. The black arrow represents the ipsilateral side injected with fluorogold. Scale bars: A–C, 200 µm. Abbreviations: FG, fluorogold; TH, tyrosine hydroxylase; PCG, pontine central gray; PDTg, posterodorsal tegmental nucleus.
1 Amaral D G, Cowan W M (1980). Subcortical afferents to the hippocampal formation in the monkey. J Comp Neurol, 189(4): 573–591 pmid: 6769979
2 Baker S A, Baker K A, Hagg T (2004). Dopaminergic nigrostriatal projections regulate neural precursor proliferation in the adult mouse subventricular zone. Eur J Neurosci, 20(2): 575–579 pmid: 15233767
3 Ben Abdallah N M, Slomianka L, Vyssotski A L, Lipp H P (2010). Early age-related changes in adult hippocampal neurogenesis in C57 mice. Neurobiol Aging, 31(1): 151–161 pmid: 18455269
4 Bischoff S, Scatton B, Korf J (1979). Biochemical evidence for a transmitter role of dopamine in the rat hippocampus. Brain Res, 165(1): 161–165 pmid: 218689
5 Bjorklund A (1978). Monoaminergic inputs to the hippocampus. In: Symposium, C.F. (ed), Functions of the Septo-Hippocampal System. Elsevier Excerpta Medica North-Holland, Amsterdam
6 Björklund A, Dunnett S B (2007). Dopamine neuron systems in the brain: an update. Trends Neurosci, 30(5): 194–202 pmid: 17408759
7 Borgkvist A, Malmlöf T, Feltmann K, Lindskog M, Schilström B (2012). Dopamine in the hippocampus is cleared by the norepinephrine transporter. Int J Neuropsychopharmacol, 15(4): 531–540
pmid: 21669025
8 Broussard J I, Yang K, Levine A T, Tsetsenis T, Jenson D, Cao F, Garcia I, Arenkiel B R, Zhou F M, De Biasi M, Dani J A (2016). Dopamine regulates aversive contextual learning and associated in vivo synaptic plasticity in the hippocampus. Cell Reports, 14(8): 1930–1939 pmid: 26904943
9 Carr D B, Sesack S R (2000). GABA-containing neurons in the rat ventral tegmental area project to the prefrontal cortex. Synapse, 38(2): 114–123<114::AID-SYN2>3.0.CO;2-R pmid: 11018785
10 Creed M C, Ntamati N R, Tan K R (2014). VTA GABA neurons modulate specific learning behaviors through the control of dopamine and cholinergic systems. Front Behav Neurosci, 8: 8 pmid: 24478655
11 Drapeau E, Mayo W, Aurousseau C, Le Moal M, Piazza P V, Abrous D N (2003). Spatial memory performances of aged rats in the water maze predict levels of hippocampal neurogenesis. Proc Natl Acad Sci USA, 100(24): 14385–14390 pmid: 14614143
12 Dubois A, Savasta M, Curet O, Scatton B (1986). Autoradiographic distribution of the D1 agonist [3H]SKF 38393, in the rat brain and spinal cord. Comparison with the distribution of D2 dopamine receptors. Neuroscience, 19(1): 125–137 pmid: 2946980
13 Emre M (2003). Dementia associated with Parkinson’s disease. Lancet Neurol, 2(4): 229–237 pmid: 12849211
14 Freundlieb N, François C, Tandé D, Oertel W H, Hirsch E C, Höglinger G U (2006). Dopaminergic substantia nigra neurons project topographically organized to the subventricular zone and stimulate precursor cell proliferation in aged primates. J Neurosci, 26(8): 2321–2325 pmid: 16495459
15 Gasbarri A, Sulli A, Packard M G (1997). The dopaminergic mesencephalic projections to the hippocampal formation in the rat. Prog Neuropsychopharmacol Biol Psychiatry, 21(1): 1–22 pmid: 9075256
16 Gasbarri A, Verney C, Innocenzi R, Campana E, Pacitti C (1994). Mesolimbic dopaminergic neurons innervating the hippocampal formation in the rat: a combined retrograde tracing and immunohistochemical study. Brain Res, 668(1-2): 71–79 pmid: 7704620
17 Harley C W (2007). Norepinephrine and the dentate gyrus. Prog Brain Res, 163: 299–318 pmid: 17765726
18 Höglinger G U, Rizk P, Muriel M P, Duyckaerts C, Oertel W H, Caille I, Hirsch E C (2004). Dopamine depletion impairs precursor cell proliferation in Parkinson disease. Nat Neurosci, 7(7): 726–735 pmid: 15195095
19 Ito R, Robbins T W, Pennartz C M, Everitt B J (2008). Functional interaction between the hippocampus and nucleus accumbens shell is necessary for the acquisition of appetitive spatial context conditioning. J Neurosci, 28(27): 6950–6959 pmid: 18596169
20 Kwon O B, Paredes D, Gonzalez C M, Neddens J, Hernandez L, Vullhorst D, Buonanno A (2008). Neuregulin-1 regulates LTP at CA1 hippocampal synapses through activation of dopamine D4 receptors. Proc Natl Acad Sci USA, 105(40): 15587–15592 pmid: 18832154
21 Levy G, Schupf N, Tang M X, Cote L J, Louis E D, Mejia H, Stern Y, Marder K (2002). Combined effect of age and severity on the risk of dementia in Parkinson’s disease. Ann Neurol, 51(6): 722–729 pmid: 12112078
22 Loughlin S E, Foote S L, Bloom F E (1986). Efferent projections of nucleus locus coeruleus: topographic organization of cells of origin demonstrated by three-dimensional reconstruction. Neuroscience, 18(2): 291–306 pmid: 3736860
23 Loy R, Koziell D A, Lindsey J D, Moore R Y (1980). Noradrenergic innervation of the adult rat hippocampal formation. J Comp Neurol, 189(4): 699–710 pmid: 7381046
24 Meibach R C, Siegel A (1977). Efferent connections of the hippocampal formation in the rat. Brain Res, 124(2): 197–224 pmid: 402984
25 Pohle W, Ott T, Müller-Welde P (1984). Identification of neurons of origin providing the dopaminergic innervation of the hippocampus. J Hirnforsch, 25(1): 1–10
pmid: 6725937
26 Regensburger M, Prots I, Winner B (2014). Adult hippocampal neurogenesis in Parkinson’s disease: impact on neuronal survival and plasticity. Neural Plast, 2014: 454696 pmid: 25110593
27 Reymann K, Pohle W, Müller-Welde P, Ott T (1983). Dopaminergic innervation of the hippocampus: evidence for midbrain raphe neurons as the site of origin. Biomed Biochim Acta, 42(10): 1247–1255
pmid: 6202300
28 Rosen Z B, Cheung S, Siegelbaum S A (2015). Midbrain dopamine neurons bidirectionally regulate CA3-CA1 synaptic drive.Nat Neurosci, 18(12): 1763–1771 pmid: 26523642
29 Samuels E R, Szabadi E (2008). Functional neuroanatomy of the noradrenergic locus coeruleus: its roles in the regulation of arousal and autonomic function part I: principles of functional organisation. Curr Neuropharmacol, 6(3): 235–253 pmid: 19506723
30 Sara S J (2009). The locus coeruleus and noradrenergic modulation of cognition. Nat Rev Neurosci, 10(3): 211–223 pmid: 19190638
31 Scatton B, Simon H, Le Moal M, Bischoff S (1980). Origin of dopaminergic innervation of the rat hippocampal formation. Neurosci Lett, 18(2): 125–131 pmid: 7052484
32 Schwab M E, Javoy-Agid F, Agid Y (1978). Labeled wheat germ agglutinin (WGA) as a new, highly sensitive retrograde tracer in the rat brain hippocampal system. Brain Res, 152(1): 145–150 pmid: 79432
33 Schwarz L A, Miyamichi K, Gao X J, Beier K T, Weissbourd B, DeLoach K E, Ren J, Ibanes S, Malenka R C, Kremer E J, Luo L (2015). Viral-genetic tracing of the input-output organization of a central noradrenaline circuit. Nature, 524(7563): 88–92 pmid: 26131933
34 Seib D R, Corsini N S, Ellwanger K, Plaas C, Mateos A, Pitzer C, Niehrs C, Celikel T, Martin-Villalba A (2013). Loss of Dickkopf-1 restores neurogenesis in old age and counteracts cognitive decline. Cell Stem Cell, 12(2): 204–214 pmid: 23395445
35 Simon H, Le Moal M, Calas A (1979). Efferents and afferents of the ventral tegmental-A10 region studied after local injection of [3H]leucine and horseradish peroxidase. Brain Res, 178(1): 17–40 pmid: 91413
36 Small S A, Schobel S A, Buxton R B, Witter M P, Barnes C A (2011). A pathophysiological framework of hippocampal dysfunction in ageing and disease. Nat Rev Neurosci, 12(10): 585–601 pmid: 21897434
37 Smith C C, Greene R W (2012). CNS dopamine transmission mediated by noradrenergic innervation. J Neurosci, 32(18): 6072–6080 pmid: 22553014
38 Spalding K L, Bergmann O, Alkass K, Bernard S, Salehpour M, Huttner H B, Boström E, Westerlund I, Vial C, Buchholz B A, Possnert G, Mash D C, Druid H, Frisén J (2013). Dynamics of hippocampal neurogenesis in adult humans. Cell, 153(6): 1219–1227 pmid: 23746839
39 Sui Y, Horne M K, Stanić D (2012). Reduced proliferation in the adult mouse subventricular zone increases survival of olfactory bulb interneurons. PLoS ONE, 7(2): e31549 pmid: 22363671
40 Suzuki K, Okada K, Wakuda T, Shinmura C, Kameno Y, Iwata K, Takahashi T, Suda S, Matsuzaki H, Iwata Y, Hashimoto K, Mori N (2010). Destruction of dopaminergic neurons in the midbrain by 6-hydroxydopamine decreases hippocampal cell proliferation in rats: reversal by fluoxetine. PLoS ONE, 5(2): e9260 pmid: 20174647
41 Swanson L W (1982). The projections of the ventral tegmental area and adjacent regions: a combined fluorescent retrograde tracer and immunofluorescence study in the rat. Brain Res Bull, 9(1-6): 321–353 pmid: 6816390
42 Szabadi E (2013). Functional neuroanatomy of the central noradrenergic system. J Psychopharmacol, 27(8): 659–693 pmid: 23761387
43 Verney C, Baulac M, Berger B, Alvarez C, Vigny A, Helle K B (1985). Morphological evidence for a dopaminergic terminal field in the hippocampal formation of young and adult rat. Neuroscience, 14(4): 1039–1052 pmid: 2860616
44 Wisman L A, Sahin G, Maingay M, Leanza G, Kirik D (2008). Functional convergence of dopaminergic and cholinergic input is critical for hippocampus-dependent working memory. J Neurosci, 28(31): 7797–7807 pmid: 18667612
45 Wyss J M, Swanson L W, Cowan W M (1979). A study of subcortical afferents to the hippocampal formation in the rat. Neuroscience, 4(4): 463–476 pmid: 107474
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