The absence of retinal input disrupts the development of cholinergic brainstem projections in the mouse dorsal lateral geniculate nucleus
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The dorsal lateral geniculate nucleus (dLGN) of the mouse has become a model system for understanding thalamic circuit assembly. While the development of retinal projections to dLGN has been a topic of extensive inquiry, how and when nonretinal projections innervate this nucleus remains largely unexplored. In this study, we examined the development of a major nonretinal projection to dLGN, the ascending input arising from cholinergic neurons of the brainstem. To visualize these projections, we used a transgenic mouse line that expresses red fluorescent protein exclusively in cholinergic neurons. To assess whether retinal input regulates the timing and pattern of cholinergic innervation of dLGN, we utilized the math5-null (math5−/−) mouse, which lacks retinofugal projections due to a failure of retinal ganglion cell differentiation.
Cholinergic brainstem innervation of dLGN began at the end of the first postnatal week, increased steadily with age, and reached an adult-like pattern by the end of the first postnatal month. The absence of retinal input led to a disruption in the trajectory, rate, and pattern of cholinergic innervation of dLGN. Anatomical tracing experiments reveal these disruptions were linked to cholinergic projections from parabigeminal nucleus, which normally traverse and reach dLGN through the optic tract.
The late postnatal arrival of cholinergic projections to dLGN and their regulation by retinal signaling provides additional support for the existence of a conserved developmental plan whereby retinal input regulates the timing and sequencing of nonretinal projections to dLGN.
KeywordsRetinogeniculate Dorsal lateral geniculate nucleus Acetylcholine Parabigeminal nucleus Cholinergic tegmentum math5-null
Analysis of variance
Brain-derived neurotrophic factor
Cholera toxin subunit B
Dorsal lateral geniculate nucleus
Direction-selective ganglion cell
Lateral posterior nucleus
Normal goat serum
non-visual sector of thalamic reticular nucleus
Polymerase chain reaction
Retinal ganglion cell
Thalamic reticular nucleus
Visual sector of thalamic reticular nucleus
Ventral lateral geniculate nucleus
Ventral posterolateral nucleus
The dorsal lateral geniculate nucleus (dLGN) of the mouse has become a model system to study the development of thalamic circuits [1, 2, 3, 4]. Much of our present understanding is based on studies focused on the retinogeniculate pathway, the connections between retinal ganglion cells (RGCs) and dLGN neurons. While retinal projections provide the primary excitatory drive for relay neurons of dLGN, the vast majority of input is nonretinal in origin , and acts to modulate the gain of retinogeniculate transmission in a state-dependent manner [6, 7]. The primary sources of nonretinal input to dLGN include glutamatergic neurons of visual cortex layer VI, GABAergic neurons of the thalamic reticular nucleus, and the cholinergic neurons from different brainstem nuclei. Despite the fact that over 90% of all synapses in dLGN arise from nonretinal sources, we know little about how and when these projections innervate dLGN, or how they interact with the arrival and refinement of retinal projections. What little we do know is based on the corticothalamic pathway [8, 9, 10]. Layer VI neurons of the neocortex begin to innervate the dorsal thalamus at perinatal ages, but corticogeniculate innervation occurs largely after the first postnatal week, after the arrival of retinal axons and their refinement into non-overlapping eye-specific domains . Moreover, retinal input orchestrates the timing of corticogeniculate innervation by regulating the levels of aggrecan, a repulsive extracellular matrix molecule . What remains to be tested is whether such sequencing and reliance on retinal input is part of a conserved developmental plan that governs the arrival of other nonretinal inputs to dLGN.
To address this, we examined another major nonretinal projection to dLGN, the ascending cholinergic input from the brainstem. Estimates reveal that about 25% of all synapses in dLGN arise from brainstem cholinergic nuclei . These projections have a substantial influence on retinogeniculate transmission, regulating the firing mode of dLGN neurons , establishing network states during sleep, wakefulness, and arousal [14, 15], as well as modulating visuo-motor interactions . Previous immunohistochemical studies have shown a late postnatal onset for the labeling of acetylcholine synthesizing enzyme (choline acetyltransferase, ChAT) in dLGN, which increases in density over a protracted period of development [17, 18]. However, little is known about the source, trajectory, and pattern of cholinergic innervation in the developing mouse dLGN. In several mammalian species, ascending cholinergic projections to dLGN originate from two distinct brainstem groups, the pedunculopontine and laterodorsal tegmental nuclei (PPTg, & LDTg), as well as the parabigeminal nucleus (PBG) [19, 20, 21]. The projection from PBG is especially notable since these axons course within the optic tract en route to dLGN and superior colliculus [20, 22, 23], raising the possibility that retinal axons participate in the guidance of PBG axons.
Here, we examined the postnatal development of cholinergic input to dLGN and tested whether the absence of retinogeniculate projections affects the timing and patterning of cholinergic innervation. To visualize ascending cholinergic projections, we crossed a ChAT-Cre knock-in mouse line with a Cre-dependent reporter strain (Ai9) to selectively drive expression of the fluorescent protein tdTomato (tdT) in cholinergic neurons [24, 25, 26, 27, 28, 29]. To evaluate the development of cholinergic innervation of dLGN in the absence of retinal projections, we utilized a mutant mouse that lacks math5, a transcription factor necessary for RGC progenitor cell differentiation . Math5−/− mice exhibit a > 95% loss of RGCs, and the surviving RGCs fail to form an optic nerve, thus leaving the brain devoid of retinal input [31, 32, 33, 34]. Furthermore, we used Cre-dependent viral tracing techniques to assess whether the trajectory of optic tract-associated PBG axons to dLGN was disrupted in a brain lacking retinofugal projections.
Materials and methods
All breeding and experimental procedures were approved by the University of Louisville Institutional Animal Care and Use Committee. Transgenic mouse strains ChAT-IRES-Cre (Jackson Labs, stock #006410, strain B6;129S6-Chattm2(cre)Lowl/J), Ai9 (Jackson Labs, stock #007909, strain B6.CgGt(ROSA)26Sortm9(CAG-tdTomato)HZe/J), and math5−/− (on a mixed C57B6/J and 129/SvEV background [9, 30, 32]) of either sex were used for breeding or for experiments. Homozygous ChAT-Cre mice were bred with homozygous Ai9 mice to generate ChAT-Cre x Ai9 offspring. ChAT-Cre+/+ x math5−/− mice were bred with Ai9+/+ x math5−/− mice to generate ChAT-Cre+/− x Ai9+/− x math5−/− offspring.
To genotype for Cre, the following primers were used in polymerase chain reaction reactions (PCR): Cre-F (CCTTCTATCGCCTTCTTGACG), Cre-R (AGATAGATAATGAGAGGCTC), WT-F (GTTTGCAGAAGCGGTGGG), WT-R (AGATAGATAATGAGAGGCTC). PCR amplification was performed in 28 cycles by denaturation at 94 °C for 15 s, annealing at 60 °C for 15 s, and elongation at 72 °C for 10 s. To genotype for math5, the following primers were used: Neo-F (GCCGGCCACAGTCGATGAATC), Neo-R (CATTGAACAAGATGGATTGCA), math5-F (ATGGCGCTCAGCTACATCAT), and math5-R (GGGTCTACCTGGAGCCTAGC). PCR amplification was performed in 35 cycles by denaturation at 94 °C for 30 s, annealing at 59 °C for 30 s, and elongation at 72 °C for 45 s.
To collect brain tissue for analysis, mice were deeply anaesthetized by hypothermia (<P5) or isoflurane vapors, and transcardially perfused with phosphate-buffered saline (PBS, 0.01 M phosphate buffer with 0.9% NaCl) followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer. Brains were postfixed overnight in 4% PFA, then transferred to PBS. A vibratome (Leica VT1000S) was used to make 70 μm-thick sections in the coronal plane. To amplify tdT fluorescence signal and prevent photobleaching during confocal imaging, DsRed (Clontech) antibody was applied using the following procedure: Sections were placed in blocking medium (10% normal goat serum (NGS), and 0.3% Triton X-100 in PBS) for 1 h, and then incubated for 12 h in rabbit anti-DsRed (1:1000) with 1% NGS in PBS. Next, tissue was incubated for 1 h in 1:100 biotinylated goat anti-rabbit IgG antibody (Vector Labs) with 1% NGS in PBS, followed by 1 h in 1:100 streptavidin Alexa Fluor (AF) 546 (Life Technologies) in PBS. All sections were mounted onto gelatin subbed glass slides using ProLong mounting medium containing DAPI (Life Technologies).
Intravitreal binocular eye injections of the anterograde tracer cholera toxin subunit B (CTB) conjugated to Alexa Fluor 488 (Invitrogen) were done in an adult (P60) ChAT-Cre x Ai9 mouse. Under anesthesia (a mixture of ketamine, 120–140 mg/kg, and xylazine, 12–14 mg/kg), the sclera was pierced with a sharp-tipped glass pipette in order to drain excess vitreous. Another pipette, filled with 1% solution of CTB-AF-488 dissolved in distilled water, was inserted into the opening created by the first pipette. A picospritzer attached to the pipette was used to deliver approximately 2–3 μl of the CTB solution into the vitreous of the eye. After a 48-h survival period, brain tissue was harvested using the methods describe above.
Viral tracer injection
Intracranial injections of a Cre-dependent adeno-associated viral tracer Flex-rev-oChIEF-tdTomato (Addgene plasmid #30541, serotype 9) were made in the left PBG of adult (>P60) ChAT-Cre and ChAT-Cre x math5−/− mice. Prior to surgery, mice were deeply anesthetized using a mixture of ketamine/xylazine and head-fixed in a stereotaxic apparatus. An incision was made along the scalp, and a hole was drilled in the skull above the injection site (− 4.2 mm AP, − 1.75 mm ML from Bregma). A Hamilton syringe (World Precision Instruments) was guided by a stereotaxic apparatus, and used to deliver 15 nL of virus into the left PBG. After a 14–17 day incubation period, brain tissue was collected for histology using aforementioned procedures.
Imaging and analysis
To quantify the distribution of virally-labeled PBG arbors, sections containing dLGN were imaged using confocal microscopy, and then binarized using the threshold procedure described above. In each section, two lines were drawn through the middle of dLGN, dividing the nucleus into 4 equal quadrants (dorsomedial, DM; ventromedial, VM; dorsolateral, DL; ventrolateral, VL; Fig.12 inset). ImageJ was used to count the number of fluorescent pixels found in each of the quadrants. Values in each quadrant reflect a percentage of the total fluorescence detected throughout the entire nucleus. Each point depicts the average value taken from 4 to 5 sections of a single hemisphere.
To estimate the number of tdT-labeled PBG neurons in adult WT and math5−/− ChAT-Cre x Ai9 mice, three 20-μm thick sections through the middle of PBG were imaged using confocal microscopy and each tdT-labeled soma was marked and quantified using ImageJ “Cell counter” function. To quantify the total number of PBG neurons labeled by FLEX-AAV-ChIEF-tdT virus, 70 μm-thick coronal sections through the rostro-caudal extent of PBG were used.
To visualize brainstem cholinergic neurons and their projections to dLGN, we crossed ChAT-Cre mice with an Ai9 reporter line [24, 25, 26, 27, 28, 29]. Figure 1a shows the pattern of tdTomato (tdT) labeling in the brainstem in an adult (P60) ChAT-Cre x Ai9 mouse. Somatic labeling of tdT was seen in cholinergic neurons of brainstem nuclei reported to project to dLGN [17, 20, 22, 38], including laterodorsal tegmentum (LDTg, Fig. 1a, left), pedunculopontine tegmentum (PPTg, Fig. 1a, left & right), and parabigeminal nucleus (PBG, Fig. 1a, right). High power views of these nuclei (Fig. 1b) showed that tdT-labeled somata were present in the adult (P60) and at birth (P0), and comparable numbers of neurons were labeled. In adults, labeled projections were evident throughout the dorsal thalamus, including the ventral (vLGN) and dorsal lateral geniculate nuclei (dLGN), lateral posterior nucleus (LP), and in the pretectum (PT) (Fig. 1c, left). At P0, cholinergic innervation was sparse in these regions, and entirely lacking in dLGN (Fig. 1c, right).
We used a ChAT-Cre mouse, crossed to a tdTomato reporter line (Ai9), to visualize and track the development of brainstem cholinergic projections to dLGN. Our results reveal that cholinergic brainstem innervation of mouse dLGN begins at the end of the first postnatal week, and increases slowly with age to reach an adult-like density by the end of the first postnatal month. Initially, cholinergic fibers appear in the dorsolateral region of dLGN just beneath the optic tract, then gradually progress in a ventromedial direction to form a homogeneous plexus of fibers throughout dLGN. This is consistent with earlier studies in cat and mouse, showing a slow but steady increase in ChAT immunoreactivity with postnatal age [17, 18]. Thus, cholinergic innervation of dLGN occurs well after the establishment of the retinogeniculate pathway . Moreover, our results support the view that innervation of dLGN occurs in a sequential manner, with retinal projections arriving before nonretinal ones .
Although corticothalamic and cholinergic brainstem projections begin to arrive in dLGN at roughly the same time (P5), their rate of innervation is vastly different. For example, cortical innervation of dLGN is nearly complete between P9–12 [8, 9], yet brainstem cholinergic innervation is still sparse and restricted largely to the dorsolateral region, only reaching adult-like density of innervation by the end of the first postnatal month. Such timing indicates that the reciprocal connections between the dLGN and visual cortex are established prior to cholinergic brainstem input. The late postnatal onset of cholinergic brainstem innervation is consistent with the maturation of other reticular ascending arousal systems [41, 42, 43, 44]. Overall this suggests that network-wide, state-dependent modulation of sensory information becomes operational well after sensory connections are established.
Our results in math5−/− mice also support earlier studies underscoring the importance of retinal input in regulating the timing of nonretinal innervation of dLGN . The absence of retinal input slowed the rate of cholinergic innervation to dLGN, though the overall degree of innervation measured in adults was preserved. It is important to note that retinal input regulates timing of nonretinal input in a bidirectional manner. Whereas the current study showed a slower rate of cholinergic innervation of dLGN in math5−/− mice, previous studies in the same mutant demonstrate an acceleration of corticogeniculate innervation, suggesting that retinal input regulates nonretinal development through distinct molecular mechanisms. The accelerated arrival of corticogeniculate axons in math5−/− mice results from the disruption of aggrecan, a repellant extracellular matrix molecule that prevents the premature entry of cortical axons into dLGN . While the molecular mechanism underlying retinal regulation of cholinergic innervation in dLGN is unresolved, the disruption in the rate of cholinergic innervation in math5−/− mice may reflect a reduction of trophic support during development. In other regions that receive cholinergic innervation, neurotrophins such as brain-derived neurotropic factor (BDNF) and neurotrophin-3 (NT-3), have been shown to promote the growth of cholinergic neurites [45, 46, 47]. In WT mice, BDNF and NT-3 are anterogradely transported from the retina to dLGN [48, 49]. Therefore, it is possible that a reduction in the levels of these factors may underlie the dystrophic growth of cholinergic fibers observed in math5−/− mutants.
In addition to regulating the rate of cholinergic innervation of dLGN, we demonstrated that retinal input plays a role in establishing the thalamic trajectory of a subset of brainstem cholinergic axons. These axons, which arise from cholinergic neurons of PBG, normally run within the optic tract along the outer border of the thalamus en route to dLGN. However, in math5−/− mice PBG axons are displaced, traveling in a diffuse manner through thalamus. This indicates that during development, PBG axons use the retinal axons of the optic tract as a scaffold to navigate through the thalamus. Such axon-axon interactions are typically mediated by cell-adhesion molecules, which promote growth along the length of an existing axon . Surprisingly, our tracing studies in math5−/− mice revealed that such interactions are not necessary for PBG axons to elongate and reach their appropriate target. We found that PBG axons continued to grow through thalamus and reach dLGN even in the absence of an optic tract. Furthermore, nuclei-specific targeting was preserved in math5−/− mice. PBG axons continued to innervate dLGN (and superior colliculus, unpublished observations) and were not found in neighboring structures within the lateral geniculate complex. In addition, the laterality of the projection did not appear to be affected, as the bulk of PBG arbors targeted the contralateral dLGN in both WT and math5−/− mice. Therefore, it is likely that PBG axons rely on cues expressed within the thalamus to target, innervate, and arborize in dLGN. Future experiments are needed to fully elucidate the specific cues that govern PBG axon guidance.
Though retinal input does not appear to be necessary for nuclei-specific targeting of PBG input, it plays an important role in establishing the appropriate pattern and morphology of arbors within dLGN. In WTs, PBG arbors were consistently found within the dorsomedial pole of the ipsilateral dLGN, adjacent to the optic tract. However in math5−/− mice, this pattern was perturbed, with arbors extending beyond their usual target or perhaps failing to reach the nucleus altogether. Interestingly, in both WT and math5−/− mice, PBG projections to the contralateral dLGN were similar in distribution.
In several mammals, inputs from PBG, superior colliculus, and certain types of retinal ganglion cells converge to terminate in discrete regions of dLGN (e.g., C-laminae of carnivores, koniocellular layers of primates), suggesting the existence of a conserved visual channel involved in the coordination of visuo-motor processing [20, 21, 51, 52, 53]. The rodent dLGN contains a homologous region, known as the dorsolateral shell , which receives input from direction selective retinal ganglion cells (DSGCs), as well as the superficial layers of the superior colliculus [51, 55]. In WT mice, we found an ipsilateral PBG projection to dLGN that targets the dorsolateral shell but in a circumscribed area that appears to represent the upper nasal visual fields [56, 57]. While the presence of retinal input appears to be necessary for the proper targeting of PBG arbors to the ipsilateral dLGN it has little impact if any on the more widespread and diffusely organized contralateral projections. Thus, it is not yet clear how this is accomplished, or whether it involves interactions with other inputs that target the shell, such as those from DSGCs and/or the superior colliculus.
The mouse dLGN has emerged as a useful model system for exploring the development of visual thalamic circuitry. However, relatively little is known about how and when nonretinal connections are formed, or whether their timing of innervation is regulated by retinal input. In this study, we examined the developmental time course of a major ascending nonretinal input to dLGN, cholinergic brainstem nuclei, by using a transgenic mouse line to visualize cholinergic input (ChAT-Cre), and a knockout line devoid of retinal input (math5−/−). We found that the cholinergic innervation has a protracted time course, innervating dLGN over the course of first postnatal month, with the bulk of input arriving well after retinal connections are formed. In math5−/−, we found that the rate of cholinergic innervation is slowed compared to WTs. Furthermore, the routing and pattern of arborization of optic tract-traversing PBG cholinergic axons was altered in the ipsilateral dLGN of math5−/− mice. Our anatomical tracing data demonstrated that while retinal input is not necessary for PBG innervation of dLGN, it plays an important role in the organization of arbors within the nucleus.
We thank Barbara O’Steen for her technical assistance and management of the animal colony, lab members Naomi Charalambakis and Peter Campbell, for assisting in image analysis and a critical reading of the manuscript.
This work was supported by NIH/NEI EY012716 (WG).
Availability of data and materials
All data generated or analyzed for this study are included in the manuscript.
GS and WG were involved in all aspects of the research and writing of the manuscript.
TS participated in some of the experiments and contributed to the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
All experimental procedures were conducted in accordance with protocols approved by the University of Louisville Institutional Animal Care and Use Committee.
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