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Nuclei-specific Differences in Nerve Terminal Distribution, Morphology, and Development in Mouse Visual Thalamus

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Nuclei-specific Differences in Nerve Terminal Distribution, Morphology, and Development in Mouse Visual Thalamus

Sarah Hammer et al. Neural Dev.

Abstract

Background: Mouse visual thalamus has emerged as a powerful model for understanding the mechanisms underlying neural circuit formation and function. Three distinct nuclei within mouse thalamus receive retinal input, the dorsal lateral geniculate nucleus (dLGN), the ventral lateral geniculate nucleus (vLGN), and the intergeniculate nucleus (IGL). However, in each of these nuclei, retinal inputs are vastly outnumbered by nonretinal inputs that arise from cortical and subcortical sources. Although retinal and nonretinal terminals associated within dLGN circuitry have been well characterized, we know little about nerve terminal organization, distribution and development in other nuclei of mouse visual thalamus.

Results: Immunolabeling specific subsets of synapses with antibodies against vesicle-associated neurotransmitter transporters or neurotransmitter synthesizing enzymes revealed significant differences in the composition, distribution and morphology of nonretinal terminals in dLGN, vLGN and IGL. For example, inhibitory terminals are more densely packed in vLGN, and cortical terminals are more densely distributed in dLGN. Overall, synaptic terminal density appears least dense in IGL. Similar nuclei-specific differences were observed for retinal terminals using immunolabeling, genetic labeling, axonal tracing and serial block face scanning electron microscopy: retinal terminals are smaller, less morphologically complex, and more densely distributed in vLGN than in dLGN. Since glutamatergic terminal size often correlates with synaptic function, we used in vitro whole cell recordings and optic tract stimulation in acutely prepared thalamic slices to reveal that excitatory postsynaptic currents (EPSCs) are considerably smaller in vLGN and show distinct responses following paired stimuli. Finally, anterograde labeling of retinal terminals throughout early postnatal development revealed that anatomical differences in retinal nerve terminal structure are not observable as synapses initially formed, but rather developed as retinogeniculate circuits mature.

Conclusions: Taken together, these results reveal nuclei-specific differences in nerve terminal composition, distribution, and morphology in mouse visual thalamus. These results raise intriguing questions about the different functions of these nuclei in processing light-derived information, as well as differences in the mechanisms that underlie their unique, nuclei-specific development.

Figures

Figure 1
Figure 1
Distribution of inhibitory and modulatory nerve terminals in subnuclei of mouse visual thalamus. A,B. Confocal images of immunohistochemistry (IHC) for GAD67 (A) and VAChT (B) in coronal sections of adult mouse LGN. Outlines of the dorsal lateral geniculate nucleus (dLGN) and the external division of the ventral lateral geniculate nucleus (vLGN) are depicted with white dots. d, dLGN; ve, external division of vLGN; vi, internal division of vLGN; i, IGL. White boxes depict regions enlarged in C,D,I, and J. C,D. High magnification images of GAD67-immunoreactivity in dLGN (C) and vLGN (D) from the regions boxed in A. E,F. High magnification images of GAD65-immunoreactivity in dLGN (E) and vLGN (F). Note the lack of GAD65-immunoreactivity in dLGN. G,H. High magnification images of VGAT-immunoreactivity in dLGN (G) and vLGN (H). High magnification images of VAChT-immunoreactivity in dLGN (I) and vLGN (J) from the regions boxed in B. All images are maximum projection confocal images. Scale bar in A = 200 μm for A,B and in J = 25 μm for C-J.
Figure 2
Figure 2
Distribution of excitatory nerve terminals in subnuclei of mouse visual thalamus. A,B: Confocal images of immunohistochemistry (IHC) for VGluT1(A) and VGluT2 (B) in coronal sections of adult mouse lateral geniculate nucleus (LGN). d, dorsal lateral geniculate nucleus (dLGN); ve, external division of ventral lateral geniculate nucleus (vLGN); vi, internal division of vLGN; i, intergeniculate nucleus (IGL). C,D: High magnification images of VGluT1-immunoreactivity in dLGN (C) and vLGN (D) from the regions boxed in A. E,F. High magnification images of Green Fluorescent Protein (GFP) distribution in dLGN (E) and vLGN (F) of adult Golli-tau-gfp transgenic mice. GFP was detected by GFP-immunostaining. Layer VI cortical neurons are selectively labeled with tau-GFP in these transgenic mice. G,H. A single optical section of a confocal image of GFP (G,H)- and VGluT2 (H)-immunoreactivity in the dLGN of an adult Golli-tau-gfp. GFP-positive cortical axon arbors densely populate the dLGN neuropil. Regions devoid of GFP-immunoreactivity in dLGN are occupied by cell bodies (asterisks), VGluT2-positive terminals (arrowheads) or blood vessels (not labeled here). I,J. High magnification images of VGluT2-immunoreactivity in dLGN (I) and vLGN (J) from the regions boxed in B. Note the difference in VGluT2-positive terminal size in dLGN and vLGN. K,L. High magnification images of VGluT2 (green) and VAChT (magenta)-containing nerve terminals in dLGN (K) and vLGN (L). VGluT2-positive terminals in dLGN are not only larger than those in vLGN, but are dramatically larger than other types of terminals in dLGN. M,N. To demonstrate that VGluT2-positive terminals originate from retinal ganglion cells, we assessed their distribution in LGN of adult math5-/- mutants, which lack retinogeniculate projections. Few, if any, nerve terminals appeared to contain VGluT2 in these mutants. All images are maximum projection confocal images except G,H. Scale bar in A = 200 μm for A,B and in N = 25 μm for C-N.
Figure 3
Figure 3
Genetic labeling of retinal terminals in subnuclei of mouse visual thalamus. A. Retinal projections (magenta) were labeled by crossing Rosa- tdt reporter mice with Math5-cre driver mice. Few (if any) cells in the thalamus express tdTomato (tdT) in Math5-cre; Rosa-tdt transgenic reporter mice. Ipsilateral retinal projections (green) were co-labeled anterogradely by intraocular injection of AlexaFluor488-conjugated cholera toxin subunit B (CTB) in the ipsilateral eye. Outlines of dorsal lateral geniculate nucleus (dLGN) and the external division of ventral lateral geniculate nucleus (vLGN) are depicted with white dots. d, dLGN; ve, external division of vLGN; vi, internal division of vLGN; i, IGL. White boxes depict regions enlarged in B and C. B,C. High magnification images of tdTomato (tdT; magenta) labeled retinal projections and CTB-labeled ipsilateral retinal projections. D, E. High magnification images of regions of dLGN (D) and vLGN (E) highlighted by the white boxes in B and C, respectively. Arrows highlight tdT-containing retinal terminals in dLGN and vLGN. All images are maximum projection confocal images. F. Relative tdT-labeled retinal terminal size (compared to retinal terminals in dLGN) was quantified in single optical sections of dLGN, vLGN and IGL. Terminal sizes in IGL and vLGN were statistical smaller than those in dLGN (P <0.001 by Neuman-Keuls Test), but were not statistically different from each other (P = 0.23). Scale bar in A = 200 μm, in C = 25 μm for B,C, in E = 10 μm for D,E.
Figure 4
Figure 4
Anterograde labeling of retinal terminals in subnuclei of mouse visual thalamus. A. Retinal projections in P35 wild-type mice were labeled by intraocular injection of fluorescently conjugated cholera toxin subunit B (CTB). Left eyes were injected with Alexa Fluor 555 CTB (magenta) and right eyes were injected with Alexa Fluor 488 CTB (green). LGN from right hemispheres are shown. ‘Contra’ denotes projections originating from the contralateral retina and ‘ipsi’ denotes projections originating from the ipsilateral retina. Outlines of dorsal lateral geniculate nucleus (dLGN) and the external division of ventral lateral geniculate nucleus (vLGN) are depicted with white dots. d, dLGN; ve, external division of vLGN; i, IGL. White boxes depict regions enlarged in B and C. B,C. High magnification images of CTB-labeled retinal terminals in dLGN (B) and vLGN (C). Note the punctate appearance of retinal projections in dLGN and the denser and diffuse labeling of retinal terminals in vLGN. D,E. High magnification image of ipsilateral retinal projections from B,C depict differences in retinal terminal size in dLGN (D) and vLGN (E). F,G. Immunolabeling CTB-labeled tissue with antibodies against VGluT2 demonstrated that CTB was enriched at synaptic sites in LGN and confirmed that VGluT2-positive terminals were derived from retinal projections. All images are maximum projection confocal images. Scale bar in A = 200 μm, in C = 25 μm for B,C, in E = 10 μm for D,E, and in G =10 μm for F,G.
Figure 5
Figure 5
Laminar-specific differences in retinal terminal morphology in ventral lateral geniculate nucleus (vLGN). A-C. Retinal projections in P35 wild-type mice were labeled by intraocular injection of fluorescently conjugated cholera toxin subunit B (CTB). Left eyes were injected with Alexa Fluor 555 CTB (magenta) and right eyes were injected with Alexa Fluor 488 CTB (green). ‘Contra’ denotes projections originating from the contralateral retina and ‘ipsi’ denotes projections originating from the ipsilateral retina. Three sections of lateral geniculate nucleus (LGN) from different rostral to caudal regions of the right hemispheres are shown. In more caudal sections, a small, lateral region of vLGN emerges that contains retinal terminals that appear to share characteristics of dorsal lateral geniculate nucleus (dLGN) retinal terminal morphology (arrows). Insets in B,C show high magnification images of this regions, which we term the lateral shell of caudal vLGN (lcvLGN). ‘Contra’ denotes projections originating from the contralateral retina and ‘ipsi’ denotes projections originating from the ipsilateral retina. Outlines of dLGN and the external division of vLGN are depicted with white dots. d, dLGN; ve, external division of vLGN; vi, internal division of vLGN; i, IGL. D-G. High magnification images of CTB-labeled retinal terminals in dLGN (D), external division of the vLGN (E), IGL (F), and lcvLGN (G). All images are maximum projection confocal images. H. Relative CTB-labeled retinal terminal areas (compared to retinal terminals in dLGN) were quantified in single optical sections of dLGN, vLGN, IGL and lcvLGN. Retinal terminal sizes in IGL, vLGN and lcvLGN were statistical smaller than those in dLGN (P <0.0001 by Neuman-Keuls Test). CTB-labeled terminal sizes in IGL and vLGN were also significantly smaller than those in lcvLGN (P <0.02 by Neuman-Keuls Test), but were not statistically different from each other (P = 0.48). Scale bar in A = 200 μm for A-C and in G = 25 μm for D-G.
Figure 6
Figure 6
Anterograde labeling of retinal terminals in other retino-recipient nuclei. A. Retinal projections in P35 wild-type mice were labeled by intraocular injection of fluorescently conjugated cholera toxin subunit B (CTB). Left eyes were injected with Alexa Fluor 555 CTB (magenta) and right eyes were injected with Alexa Fluor 488 CTB (green). Lateral geniculate nucleus (LGN) from the right hemispheres is shown. ‘Contra’ denotes projections originating from the contralateral retina and ‘ipsi’ denotes projections originating from the ipsilateral retina. Confocal images were acquired from the dorsal lateral geniculate nucleus (dLGN), intergeniculate nucleus (IGL), ventral lateral geniculate nucleus (vLGN), suprachiasmatic nucleus (SCN), olivary pretectal nucleus (OPN), and superior colliculus (SC). IGL is outlined by white dots. B. High magnification images of both contralateral and ipsilateral retinal projections to each region are shown (note - contralateral and ipsilateral panels are not all from the same image). Scale bar in A = 200 μm and in B = 20 μm.
Figure 7
Figure 7
Ultrastructural analysis of retinal terminals in subnuclei of mouse visual thalamus. Retinal terminals were identified in serial block face scanning electron microscopy (SBFSEM) micrographs based upon their pale mitochondria and round, abundant synaptic vesicles. A,B. Retinal terminals in P42 dorsal lateral geniculate nucleus (dLGN). A’ shows non-pseudo-colored image depicted in A. C,D. Retinal terminals in P42 ventral lateral geniculate nucleus (vLGN). Each terminal and axon traced was given a unique color; therefore terminals labeled the same color belong to the same axonal arbor. Retinal terminals in dLGN contain a large number of processes from dendrites that extend into the nerve terminals (compare arrows in A,B with those in D). E. Retinal terminals in dLGN are statistically larger than those in vLGN (P <0.000001 by Student’s t-test; n = 3 datasets containing a total of 111 terminals in dLGN and 148 in vLGN). F. Quantification of the percent of terminals traced that contain postsynaptic intrusions (see arrows in A,B,D,G, and J). G-I. SBFSEM micrographs demonstrate the complex nature of terminal-dendrite interactions in dLGN. A single retinal terminal is labeled in pink and its dendritic partner is labeled in blue. In G’ and G”, arrows highlight dendritic projections into the retinal terminal. H and H’ depict 3D reconstructions of the retinal terminal and dendrite labeled in G. I,I’ represents a high magnification image of the terminal-dendrite interface in H. The retinal terminal has been made translucent in H’ and I’. 3 finger-like dendritic protrusions that invade a single terminal bouton are highlighted with arrows in I’. J-N. Multiple SBFSEM micrographs through two retinogeniculate synapses demonstrate the less complex nature of terminal-dendrite interactions in vLGN. K and M depict 3D reconstructions of the retinal terminals and dendrites traced in J and L respectively. Arrows in J and K’ indicate a small dendritic protrusion that extends into the retinal terminal. In M, 4 terminals from the same axon contact the purple dendrite and none contain dendritic protrusions. N shows a high magnification, rotated image of the terminal and dendrite in indicated by the arrow in M. Note the absence of dendritic protrusions into this terminal bouton. Retinal terminals and axons been made translucent in K’,M’ and N’. Scale bar in A = 1.5 μm for A-D, G, J, and L.
Figure 8
Figure 8
Ultrastructural analysis of numbers of terminals per retinal axon in subnuclei of mouse visual thalamus. A-J. Serial block face scanning electron microscope (SBFSEM) micrographs show multiple terminal boutons and axons from a single retinal axon in dLGN (A-E) and vLGN (F-J). K, L. 3D reconstructions of axonal arbors from A-E (see K) and F-J (see L). These reconstructions are at the same scale and represent entire axonal arbors from dorsal lateral geniculate nucleus (dLGN) and ventral lateral geniculate nucleus (vLGN) datasets that were 40 μm by 40 μm by 15 μm. M. Numbers of terminal boutons were counted in each axon traced in dLGN and vLGN (n = 3 datasets per tissue regions; 59 axons in dLGN, 44 axons in vLGN). Retinal axons in dLGN datasets contain fewer terminal boutons than those in vLGN. N. Quantitation of the mean numbers of terminal boutons per axon in dLGN and vLGN. Axons traced in dLGN contain a significantly smaller number of terminal boutons than those in vLGN (P <0.00005 by Student t-test). Scale bar in A = 1 μm for A-J and in L = 1.5 μm for K,L.
Figure 9
Figure 9
Glutamatergic synaptic responses evoked by optic tract stimulation in subnuclei of mouse visual thalamus. A. Examples of synaptic responses in P35 dorsal lateral geniculate nucleus (dLGN) and ventral lateral geniculate nucleus (vLGN) neurons. Synaptic responses in dLGN neurons show all-or-none and larger amplitude excitatory postsynaptic currents (EPSC), whereas vLGN neurons show graded responses with smaller amplitude EPSCs. B. Population data reveal that EPSC amplitudes were smaller in vLGN and progressively increasing stimulus intensity increased peak amplitudes of EPSCs in vLGN but not dLGN. This suggests that while dLGN relay neurons are innervated by only one or two retinal axons, vLGN cells receive smaller inputs from larger numbers of retinal axons. C. Differences in pair-pulse depression (PPD) were observed at retinogeniculate synapses in dLGN and vLGN. An example of synaptic responses recorded in dLGN and vLGN neurons following a train of stimuli with a 25 ms interstimulus interval is shown in C. Examples of current traces showing differences in pair-pulse depression at retinogeniculate synapses in dLGN and vLGN. Synaptic responses recorded in dLGN and vLGN neurons following a train of stimuli with a 50 ms interstimulus interval. D. Average peak EPSC amplitudes in dLGN and vLGN following trains of stimuli (20 Hz, 10 pulses). E. The paired pulse ratio was plotted using the peak EPSC amplitudes following the first and second stimuli. Thus, in addition to exhibiting weaker postsynaptic responses, retinogeniculate synapses in vLGN show weaker paired pulse depression compared to those in dLGN. F. Examples of reconstructions of biocytin-filled relay neurons in dLGN and vLGN.
Figure 10
Figure 10
Anterograde labeling of retinal terminals in developing subnuclei of mouse visual thalamus. A. Retinal projections in P5 and P14 wild-type mice were labeled by intraocular injection of fluorescently conjugated cholera toxin subunit B (CTB). Left eyes were injected with Alexa Fluor 555 CTB (magenta) and right eyes were injected with Alexa Fluor 488 CTB (green). LGN from right hemispheres are shown. ‘Contra’ denotes projections originating from the contralateral retina and ‘ipsi’ denotes projections originating from the ipsilateral retina. Outlines of dorsal lateral geniculate nucleus (dLGN) and the external division of ventral lateral geniculate nucleus (vLGN) are depicted with white dots. d, dLGN; ve, external division of vLGN; i, IGL. B. High magnification images of CTB-labeled retinal terminals in dLGN and vLGN at P5 and P14. C. CTB-labeled retinal terminal areas were quantified in single optical sections of dLGN and vLGN at P5 and P14. Retinal terminal sizes in vLGN and dLGN were not statistically different from each other at P5 but statistically different at P14 (P <0.0001 by the Tukey Kramer test for differences between means). All images are maximum projection confocal images. Scale bar in A = 200 μm and in B = 15 μm.

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