Morphology, projection pattern, and neurochemical identity of Cajal's "centrifugal neurons": the cells of origin of the tectoventrogeniculate pathway in pigeon (Columba livia) and chicken (Gallus gallus)

Abstract

The nucleus geniculatus lateralis pars ventralis (GLv) is a prominent retinal target in all amniotes. In birds, it is in receipt of a dense and topographically organized retinal projection. The GLv is also the target of substantial and topographically organized projections from the optic tectum and the visual wulst (hyperpallium). Tectal and retinal afferents terminate homotopically within the external GLv-neuropil. Efferents from the GLv follow a descending course through the tegmentum and can be traced into the medial pontine nucleus. At present, the cells of origin of the Tecto-GLv projection are only partially described. Here we characterized the laminar location, morphology, projection pattern, and neurochemical identity of these cells by means of neural tracer injections and intracellular fillings in slice preparations and extracellular tracer injections in vivo. The Tecto-GLv projection arises from a distinct subset of layer 10 bipolar neurons, whose apical dendrites show a complex transverse arborization at the level of layer 7. Axons of these bipolar cells arise from the apical dendrites and follow a course through the optic tract to finally form very fine and restricted terminal endings inside the GLv-neuropil. Double-label experiments showed that these bipolar cells were choline acetyltransferase (ChAT)-immunoreactive. Our results strongly suggest that Tecto-GLv neurons form a pathway by which integrated tectal activity rapidly feeds back to the GLv and exerts a focal cholinergic modulation of incoming retinal inputs.

Keywords: ChAT; GLv; birds; optic tectum; slice; vine-neuron.

Figure 1. Tectal and retinal terminals in the GLv

A) Transverse plane of a Giemsa staining showing the position of the GLv in a dorso-ventral orientation. B) Sagittal plane of the Giemsa staining showing the position of the GLv in a rostro-caudal orientation. White vertical line represent aproximatelly the location of the sections in C and D. C) Anterogradely labeled CTB-terminals in the GLv-ne after tectal injection into intermediate layers (injection site not shown). D) Labeled terminals in the Glv-ne after intraocular injection of CTB. Empty areas in the GLv-ne may probably due to uneven distribution of CTB into the vitreal chamber of the eye. Tectal and retinal afferents are topographic and coexist in close apposition into the GLv-ne.

Figure 2. Retrograde tracing of the TeO-GLv projection in vivo

A) Retrograde CTB-labeling of cells located mainly in layer 10 of the TeO after a GLv injection. B) Higher magnification of the labeled cells located in layer 10 of the TeO. Note the heavy labeled processes in the layer 7. C) Corresponding injection site in the lateral GLv. Orientation in A is the same as B. V = ventral; L = lateral.

Figure 3. Chicken slice containing the TeO-GLv connection

A) Macrophotography of a typical 500 μm slice used in this study. Circles and squares mark the location of extracellular injections of dextran amines into the TeO and GLv (performed in each slice) B) Giemsa staining of a 60 μm section showing tectum, pretectum and thalamus.. V = ventricle. Orientation: M = medial; V = ventral.

Figure 4. Anterograde tracing of the TeO-GLv projection

A) Two tectal injections of dextran-amine-alexa-546 (D-Alexa-546) in layer 9–10 of the TeO. B) Topographic terminals within the GLv-ne after the tectal injection. Orientation in B is the same as in A.

Figure 5. Retrograde tracing of the TeO-GLv projection

A) Composite image of five sections (separation between each section = 60μm) of retrogradely labeled cells with somata located in layer 10 of the TeO after three injections of BDA in the GLv-ne. B) Extracellular injections of BDA into the GLv-ne. Note how cells in GLv-li are also heavy labeled.

Figure 6. Retrograde labeling of cells in the TeO after BDA injections into GLv

A) Retrogradely labeled cells with nissl counterstain. Basal dendrites extend until layer 13, somata are located in layer 10, dendritic side-branches lie in layer 7 and apical dendrites extend until layer 2 of the TeO. B) Inset showing the basal dendrite ending in layer 13 (black arrowheads). C) Inset showing the soma and the basal dendrite (black arrowheads). D) Inset showing the apical dendrite, the axon and the dendritic side-branch (dsb) of a neuron (black arrowheads). E) Inset showing the apical dendrite ending and the axon of one vine-neuron. Note the 90° bending of the axons between layer 2 and SO.

Figure 7. Deconvolution and extended focus projection of a retrogradely labeled cell in the TeO after GLv injection of D-Alexa-546

Retrograde labeling shows that the axon splits from the apical denritic trunk at the level of layer 9. The apical dendrite side-branch massively in layer 7, continuing with a smaller branching in layer 4. Finally the apical dendrite ends in layer 2. Deconvolution used = Wiener. Orientation: L = lateral; V = ventral.

Figure 8. Intracellular filling of a vine-neuron with biocytin

The soma is located in layer 10 and the dendritic side-branch (dsb) in layer 7 of the TeO. Soma width = 5.0 μm; dendritic side-branch width = 66 μm.

Figure 9. Intracellular filling showing a tectal terminal in the GLv-ne

A) Composite image of two contiguous 60 μm sections of the terminal of a vine-neuron in the lateral part of the GLv-ne. B) and C) Inset showing the two sections where the terminal was observed.

Figure 10. Reconstruction of an intracellularly filled vine-neuron with terminals in the GLv

Figure shows a complete filled neuron with biocytin. Basal dendrite does not reach layer 13 as previously shown probably because it extend beyond the limits of the slice. The axon runs through the upper limit of the SO and the Ot before entering the GLv. Note the L-shape form of the axon entrance into the GLv and the butterfly-shape of the terminal in GLv-ne. 10, 9, 7 = layers of the TeO.

Figure 11. AntiChAT immunohistochemistry in the pigeon

A) Overview of antiChAT-DAB labeling. Left side shows the intact Ipc nucleus and the characteristic ChAT-li of the paintbrush endings radially oriented in the TeO. Arrowheads on the right side outline the neurochemically ablated Ipc. B) Inset of the ventral part of the optic tectum showing mainly anti-ChAT positive paintbrushes from the IPc neurons in layer 5. C) Anti-ChAT positive somata located in layer 10 of the TeO are observed after chemical ablation of the Ipc nucleus. Note that the heavy staining previously observed in layer 5 (panel B) is no longer observed.

Figure 12. AntiChAT immunohistochemistry in chicken slice

A) Overview of diencephalic and mesencephalic structures showing antiChAT-DAB labeling. B) Inset of the ventral part of the optic tectum showing anti-ChAT positive cells with the somata located in layer 10 of the TeO. Note that Layer 7 shows neurite immunoreactivity. V = ventricle.

Figure 13. Double labeling experiments showing cells located in layer 10 of the TeO

A) Retrograde labeling of a vine-neuron with D-Alexa-546 located in layer 10 of the TeO after a GLv-ne injection. B) ChAT Immunohistochemistry with alexa-488 of the same section showing labeled cells in layer 10 of the TeO. C) Merge of both images showing ChAT labeling of the vine-neuron. All retrograde cells labeled with A-546-Dextran were positive for ChAT (data not shown). Orientation: M = medial; D = dorsal.

Figure 14. Schematic drawing of proposed neural circuitry of the TeO-GLv projection

Cholinergic vine-neuron located in layer 10 of the TeO (red) projects topographically onto a presumable GABAergic GLv projection cell (brown). Glutamatergic retinal input from a retinal ganglion cell ends in GLv and TeO (blue). The gray shadings indicate the activation of a specific locus in the TeO and the concurrent activation of a homotopic locus in the GLv. V= ventricle