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. 2018 Jun 15;526(9):1550-1570.
doi: 10.1002/cne.24428. Epub 2018 Apr 6.

Thalamostriatal and Cerebellothalamic Pathways in a Songbird, the Bengalese Finch

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Free PMC article

Thalamostriatal and Cerebellothalamic Pathways in a Songbird, the Bengalese Finch

David A Nicholson et al. J Comp Neurol. .
Free PMC article

Abstract

The thalamostriatal system is a major network in the mammalian brain, originating principally from the intralaminar nuclei of thalamus. Its functions remain unclear, but a subset of these projections provides a pathway through which the cerebellum communicates with the basal ganglia. Both the cerebellum and basal ganglia play crucial roles in motor control. Although songbirds have yielded key insights into the neural basis of vocal learning, it is unknown whether a thalamostriatal system exists in the songbird brain. Thalamic nucleus DLM is an important part of the song system, the network of nuclei required for learning and producing song. DLM receives output from song system basal ganglia nucleus Area X and sits within dorsal thalamus, the proposed avian homolog of the mammalian intralaminar nuclei that also receives projections from the cerebellar nuclei. Using a viral vector that specifically labels presynaptic axon segments, we show in Bengalese finches that dorsal thalamus projects to Area X, the basal ganglia nucleus of the song system, and to surrounding medial striatum. To identify the sources of thalamic input to Area X, we map DLM and cerebellar-recipient dorsal thalamus (DTCbN ). Surprisingly, we find both DLM and dorsal anterior DTCbN adjacent to DLM project to Area X. In contrast, the ventral medial subregion of DTCbN projects to medial striatum outside Area X. Our results suggest the basal ganglia in the song system, like the mammalian basal ganglia, integrate feedback from the thalamic region to which they project as well as thalamic regions that receive cerebellar output.

Keywords: RRID: AB_2174013; RRID: AB_2209751; RRID: AB_2313581; RRID: AB_2313584; RRID: AB_2340613; RRID: AB_2340675; RRID: AB_2340846; RRID: AB_2534132; RRID: AB_2534134; RRID: AB_2536611; basal ganglia; cerebellum; songbird; thalamostriatal.

Conflict of interest statement

Conflict of Interest Statement: The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
The anterior forebrain pathway (AFP) is a thalamocortical-basal ganglia loop in the song system required for learning song. It consists of basal ganglia nucleus Area X (yellow oval), thalamic nucleus DLM (blue oval) and cortical nucleus LMAN (green oval, known song system connections outlined in gray. The rest of the song system is not shown.). Using lentiviral tracing methods, we tested whether thalamus projects to the basal ganglia in a songbird, the Bengalese finch. Specifically we tested whether Area X (yellow oval) receives input from DLM (darker blue dashed arrow) or cerebellar-recipient dorsal thalamus (DTCbN, lighter blue dashed arrows). In order to identify targets of DTCbN, we first mapped out the projections of the cerebellar nuclei (CbN) to dorsal thalamus in detail (red arrow).
Figure 2
Figure 2
Dorsal thalamus in Bengalese finches as shown in parasagittal series. We adopted nomenclature from atlases and previously published literature as described in the Methods section “Nomenclature of Dorsal Thalamus”. Note that by DLM and DMP we mean the thalamic nuclei found only in songbirds which project to cortical nuclei LMAN and MMAN of the song system, respectively, and not the regions in non-songbird species that share the same names. Series of Nissl-stained parasagittal sections moving from medial (a) to lateral (f). Distance lateral is from midline in this figure is sum of the number of sections from midline (determined as described in Methods) multiplied by section thickness, but not adjusted to account for shrinkage due to fixation. Abbreviations: DIP, dorsointermediate posterior nucleus, DLA, dorsolateral anterior nucleus, DLL, dorsolateral lateral nucleus, DLP, Dorsolateral posterior nucleus, DMP, dorsomedial posterior nucleus, Hb, habenula, IC, inferior colliculus, OM, occipto-mesencephalic tract, PC, posterior commissure. Scale bar in a is 500 μm. Left is anterior, dorsal is up.
Figure 3
Figure 3
Dorsal thalamus projects to striatum a, Schematic of experiment. We injected into dorsal thalamus (DT) a 1:1 solution of two lentiviral vectors, one expressing synaptophysin-GFP and the other mCherry, and then looked for label in medial striatum (MSt) which contains basal ganglia nucleus of the song system, Area X. One landmark we used to determine whether sections included MSt was the magnocellular nucleus of the anterior nidopallium (MAN) which is obvious even in unstained tissue. b, Widefield image of injection site from representative case. This injection was outside of thalamic nucleus of the song system DLM, just anterior to the posterior commissure (PC) and in the same plane as auditory thalamus nucleus ovoidalis (Ov) c, Image showing mCherry and synaptophysin-GFP labeled processes in MSt ventral to MAN. Magenta arrows, mCherry signal, green arrows, synaptophysin-GFP signal. PSL, pallial-subpallial lamina, nido, nidopallium outside of MAN. d, Synaptophysin-GFP labeled processes in MSt had varicosities, suggesting they were synapses. Higher-power scan of the area outlined in C with a dashed box. e, f. mCherry labeled processes could also be found in MSt with varicosities, indicating this is the actual morphology of the axons and did not result from ectopic (over)expression of synaptophysin-GFP g. Another example of synaptophysin-GFP labeled processes in MSt. e–g are all in the same section within 500 μm of each other but are enlarged to be easily visible. All sections are parasagittal with anterior to the left and dorsal up. Scale bars: b–c, 500 μm; d–g, 100 μm.
Figure 4
Figure 4. Dorsal thalamus projects to striatum, including Area X
a, Schematic of experiment. As in Fig. 3, we injected into dorsal thalamus a 1:1 solution of two lentiviral vectors, one expressing synaptophysin-GFP and the other mCherry, but in these experiments we labeled Area X immunohistochemically with antibodies against parvalbumin, and then determined whether viral label was found in Area X or medial striatum (MSt) as well as the magnocellular nucleus of the anterior nidopallium (MAN). b, Widefield image of injection site from representative bird. c, Parvalbumin stain to outline Area X (white arrows) from the same bird. d, “Camera lucida” style tracing of GFP and mCherry signal from same section shown in (c). e, Confocal image of area shown in white box in d. Note varicosities suggesting synapses. All sections are parasagittal with anterior to the left and dorsal up. Scale bars in a–d, 500 μm. Scale bar in e, 250 μm.
Figure 5
Figure 5
The lateral cerebellar nucleus (CbL) projects to contralateral dorsal thalamus. a, Schematic (left panel) of experiment showing injection in CbL and widefield image (right) showing injection site. The injection site is shown in Fig. 6, panel b, middle row. b, High-resolution confocal image of axon terminal-like morphology in contralateral dorsal thalamus. This image was taken from the area indicated with a white arrow in i. c–k, representative series of widefield images across dorsal thalamus showing anterograde label from the injection in CbL. c is the most lateral section and k is the most medial. Estimate of distance from midline (e.g. “1.02 lat.”) is given in millimeters, calculated as explained in Methods. Dotted white lines demarcate the areas we considered cerebellar-recipient dorsal thalamus. All sections are parasagittal with anterior to the left and dorsal up. This series is from “bird 2” in Figure 6. All scale bars 500 μm. Abbreviations from c–h: Pt, pretectal nucleus. SpL, lateral spiriform nucleus. ICo, inferior colliculus. DLL, dorsolateral thalamus, lateral part. Rt, nucleus rotundus. Uva, uvaeform nucleus. SpM, medial spiriform nucleus. PC, posterior commissure. Ov, nucleus ovoidalis. DLM, dorsolateral thalamus, medial part.
Figure 6
Figure 6
CbL axon terminals target medial dorsal thalamus adjacent to song system nucleus DLM, but also target more lateral dorsal thalamus. A, Schematic representation of experiment, showing injection site in lateral cerebellar nuclei, and anterograde label in contralateral dorsal thalamus. B, Injection sites. In one case shown (top row), the fluorophore conjugated to the dextran amines was tetramethylrhodamine. In the other cases, the conjugate was fluorescein. The choice of fluorophore did not affect results. C, More medial site in dorsal thalamus with strong label. DLM, dorsolateral thalamus, medial part. Ov, nucleus ovoidalis. PC, posterior commissure. D, More lateral site in dorsal thalamus with strong label. Abbreviations: DLL, dorsolateral thalamus, lateral part. Rt, nucleus rotundus. SpM, medial spiriform nucleus. All sections are parasagittal, left is anterior and up is dorsal. All scale bars 500 μm.
Figure 7
Figure 7
Injections in dorsal thalamus yield retrograde label in the cerebellar nuclei. a, schematic indicating injection site in dorsal thalamus and site of retrograde label in contralateral cerebellar nuclei (CbN) (shown in same plane of cartoon “section”). b, Representative injection site in dorsal thalamus. Parasagittal section. Anterior is left and dorsal is up. DLM, dorsolateral thalamus, medial part. Ov, nucleus ovoidalis. PC, posterior commissure. c, Retrograde label in contralateral lateral cerebellar nucleus (CbL). d, Retrograde label in contralateral intermediate (CbI) and medial (CbM) cerebellar nucleus. All sections are parasagittal with anterior to the left and dorsal up. Scale bar, 500 μm
Figure 8
Figure 8
All of the cerebellar nuclei project to dorsal thalamus. a, Injection sites in dorsal thalamus from 3 birds (magenta, cyan, yellow) arranged from lateral to medial. Note that the 3rd panel from the top corresponds approximately to the injection site shown in figure 5A. DLL, dorsolateral thalamus, lateral part. OM, occipito-mesencephalic tract. DLM, dorsolateral thalamus, medial part. Ov, nucleus ovoidalis. PC, posterior commissure. b, Schematic of retrograde label in the contralateral cerebellar nuclei arranged from lateral to medial.n Colored circles represent retrogradely filled cell bodies. Color of each circle indicates retrograde label from injection site with the same color in a. CbL, lateral cerebellar nucleus. CbI, intermediate cerebellar nucleus. CbM, medial cerebellar nucleus. All sections are parasagittal, left is anterior and up is dorsal. All scale bars 500 μm.
Figure 9
Figure 9
Thalamic song system nucleus DLM is adjacent to but separate from DTCbN. A, Iontophoretic injection of AAV-GFP vector in Area X. Left panel, schematic of injection site. Right panel, representative section showing injection. nido, nidopallium. MAN, magnocellular nucleus of anterior nidopallium. MSt, medial striatum. B–D, Resulting anterograde label of calyceal-like terminals in DLM (dashed white line). Note that label is confined to bright oval as seen in tissue when viewed with DIC filter. DLM, dorsolateral thalamus, medial part. Ov, nucleus ovoidalis. PC, posterior commissure. All sections are parasagittal, left is anterior and up is dorsal. All scale bars: 500 μm
Figure 10
Figure 10
DLM and adjacent DTCbN project to Area X. a–d, case where injection was mostly in DLM. Synaptophysin-GFP label was evident in Area X (for example see white arrowhead in d) e–h, case where injection was in DLM and surrounding DTCbN. Synaptophysin-GFP label was evident in medial striatum (G and H, white arrow) and in Area X (h, white arrowhead). i–l, case where injection was mostly in DTCbN. Although the injection was mainly in DTCbN, we again saw strong synaptophysin-GFP label in Area X (for example k, white arrowhead). a,e,y, Injection sites. Orange region, DLM; cyan region, DTCbN; magenta, cell bodies expressing mCherry. b–d, f–h, j–l. Series of sections from lateral to medial showing Area X, surrounding medial striatum, and overlying nidopallium. Green, GFP signal. Magenta, mCherry signal. Dark gray, no parvalbumin label; gray, some parvalbumin label; light gray, strong parvalbumin label. White arrowheads, examples of GFP signal in Area X; solid white arrow, GFP signal outside Area X in MSt; black arrow with white outline, GFP signal in nidopallium. DLM, dorsolateral thalamus, medial part. MAN, magnocellular nucleus of anterior nidopallium. MSt, medial striatum. nido, nidopallium. Ov, nucleus ovoidalis. PC, posterior commissure. SpM, medial spiriform nucleus. To facilitate comparison across cases, distances from midline for injection sites are based on the map of DLM and DTCbN we created as described in Methods. All sections are parasagittal, left is anterior and up is dorsal. All scale bars 500 μm.
Figure 11
Figure 11
More medial and posterior regions of DTCbN project to medial striatum. a–d, case where injection was mostly in DTCbN and more medial. Strong synaptophysin-GFP label was posterior and/or medial of Area X (d, white arrow). There was almost no GFP label in Area X in this case. e–h, case where injection was in DTCbN but posterior of DLM. Again the strongest Synaptophysin-GFP label was medial of Area X (f, white arrow). i–l, Area X, arrow with white outline in l. a,e,i, Injection sites in DLM and DTCbN for 3 birds. Sections are arranged from medial to lateral reading from left to right. Orange region, DLM; cyan region, DTCbN; magenta, cell bodies expressing mCherry. b–d, f–h, j–l, series of sections from lateral to medial showing Area X, surrounding medial striatum, and overlying nidopallium. Green, GFP signal. Magenta, mCherry signal. Dark gray, no parvalbumin label; gray, some parvalbumin label; light gray, strong parvalbumin label. Arrow with white outline and no fill, GFP signal in Area X; solid white arrow, GFP signal outside Area X in MSt; black arrow with white outline, GFP signal in nidopallium. DLM, dorsolateral thalamus, medial part. MAN, magnocellular nucleus of anterior nidopallium. MSt, medial striatum. nido, nidopallium. Ov, nucleus ovoidalis. PC, posterior commissure. SpM, medial spiriform nucleus. To facilitate comparison across cases, distances from midline for injection sites are based on the map of DLM and DTCbN we created as described in Methods. All sections are parasagittal, left is anterior and up is dorsal. All scale bars 500 μm.
Figure 12
Figure 12. DLM sends a significant projection to Area X
a, representative image of iontophoretic injection in DLM. b, injection site mapped onto borders of DLM and DTCbN as in Figs. 10 and 11, showing that it injection was confined to DLM. c–e, series of consecutive sections showing label from injection throughout Area X and in LMAN fj, Axon segments dotted with varicosities as well as locally-ramifying axon-terminal like morphologies in Area X. Each panel is a confocal image of the area(s) surrounded by a white box in the panel above it. Ov, nucleus ovoidalis, PC, posterior commissure. Scale bars: a, ce 500 μm. fh, 50 μm. j, 100 μm.
Figure 13
Figure 13. Summary of results
We showed that, in the Anterior Forebrain Pathway (AFP) of the song system, the basal ganglia nucleus Area X receives input from the thalamic nucleus, DLM, as well as adjacent subregions of cerebellar-recipient dorsal thalamus (DTCbN) (blue arrows). Hence DLM provides feedback to Area X similar to projections from thalamus to the striatum in mammals, and DTCbN provides a route for output from the cerebellar nuclei (CbN, red arrow) to reach the basal ganglia in the song system through thalamus. More posterior and medial regions of DTCbN project to medial striatum outside of Area X (lighter blue arrow). We also found that DTCbN projects to nidopallium outside of cortical song system nucleus LMAN (lighter blue arrow), implying that DTCbN may communicate both with the song system and with general motor areas outside the song system. Canonical nuclei of the song system are outlined in heavy black lines.

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