Development of multisensory convergence in the Xenopus optic tectum

Abstract

The adult Xenopus optic tectum receives and integrates visual and nonvisual sensory information. Nonvisual inputs include mechanosensory inputs from the lateral line, auditory, somatosensory, and vestibular systems. While much is known about the development of visual inputs in this species, almost nothing is known about the development of mechanosensory inputs to the tectum. In this study, we investigated mechanosensory inputs to the tectum during critical developmental stages (stages 42-49) in which the retinotectal map is being established. Tract-tracing studies using lipophilic dyes revealed a large projection between the hindbrain and the tectum as early as stage 42; this projection carries information from the Vth, VIIth, and VIIIth nerves. By directly stimulating hindbrain and visual inputs using an isolated whole-brain preparation, we found that all tectal cells studied received both visual and hindbrain input during these early developmental stages. Pharmacological data indicated that the hindbrain-tectal projection is glutamatergic and that there are no direct inhibitory hindbrain-tectal ascending projections. We found that unlike visual inputs, hindbrain inputs do not show a decrease in paired-pulse facilitation over this developmental period. Interestingly, over this developmental period, hindbrain inputs show a transient increase followed by a significant decrease in the alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA)/N-methyl-D-aspartate (NMDA) ratio and show no change in quantal size, both in contrast to visual inputs. Our data support a model by which fibers are added to the hindbrain-tectal projection across development. Nascent fibers form new synapses with tectal neurons and primarily activate NMDA receptors. At a time when retinal ganglion cells and their tectal synapses mature, hindbrain-tectal synapses are still undergoing a period of rapid synaptogenesis. This study supports the idea that immature tectal cells receive converging visual and mechanosensory information and indicates that the Xenopus tectum might be an ideal preparation to study the early development of potential multisensory interactions at the cellular level.

Fig. 1.

Mechanosensory inputs to the tectum. A: 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) injection into the trigeminal ganglion (Vth nerve). After entering the hindbrain the projection terminates rostrally and caudally in the ipsilateral hindbrain. B: DiI injection into the posterior nerve bundle carrying the facial (VIIth), acoustico-vestibular (VIIIth), and lateral-line nerves, reveals that projections also terminate in the rostral and caudal regions of the ipsilateral hindbrain. C: dye injection into the hindbrain shows a projection from the termini of primary sensory afferents to the contralateral tectum. D: schematic summarizing the input composing the hindbrain-tectal projection. The Vth, VIIth, and VIIIth nerves terminate on primary sensory nuclei in the ipsilateral hindbrain. The hindbrain-tectal projection arises from these primary sensory nuclei and travels to the contralateral tectum where it makes synapses with individual tectal neurons that also receive direct visual input from the retina. ON, optic nerve; Th, thalamus; Tec, tectum; HB, hindbrain; Cb, cerebellum; OC, otic capsule. *, injection site. All injections were done at least in triplicate. Images are maximal projections of confocal stacks. Orientation of images is the same as in D.

Fig. 2.

Development of convergence of hindbrain and visual inputs to the tectum. A: maximal projection of a confocal z-series for visual (green) and hindbrain (red) inputs to the tectum at different developmental stages. Each tectal lobe receives a projection from the eye and the hindbrain, which carry visual and mechanosensory information, respectively. These projections are present at all 3 developmental stages investigated here. —, the tectum outline. - - -, the tectal neuropil. Black box represents the region of interest used in B–D. Scale bars are 50 μm. B and C: normalized fluorescence values collected across the region of interest in the tectum indicated in A for the 2 different fluorescence channels representing visual and hindbrain projections. Fluorescence was averaged in the rostrocaudal axis. Each trace represents the fluorescence profile at a given depth (see methods). z-stacks were collected every 7.5 μm in the stage 42 and 44 tecta and at every 4 μm in the stage 48 tectum. Notice that the hindbrain projection terminates at a deeper layer than does the visual projection. D: averaged fluorescence for each channel at all depths across the rostrocaudal axis. This shows that even at stage 42, visual and hindbrain inputs overlap in the rostrocaudal direction in the tectum. Images are representative animals from each of the developmental stages. All injections at each stage were done at least in triplicate. T, tectum.

Fig. 3.

Tectal neurons receive both hindbrain and visual input. A: maximal projection of a confocal z-series of a stage 49 tectal neuron labeled with FITC-dextran. Projection is superimposed on a single optical plane of the transmitted light image to show the relationship of the neuron's dendritic arbor relative to the neuropil layer. - - -, border between cell body layer and the neuropil. Black box represents the region in which the fluorescence profiles in B were measured. B: normalized fluorescence values collected across the region of interest in the tectum indicated in A. Fluorescence was averaged in the rostrocaudal axis. Each trace represents the fluorescence profile at a given depth (see methods). z-stacks were collected every 1.6 μm. Notice that the dendritic arbor spans almost all entire neuropil region in the ventral to dorsal axis and overlaps with the termination depths of both hindbrain and visual inputs. C: schematic of stimulation and recording configuration for electrophysiological experiments. Mechanosensory pathways are activated by electrically stimulating the hindbrain, while visual inputs are activated by stimulating the optic nerve at the optic chiasm. Whole cell voltage-clamp recordings were performed from individual tectal neurons. D: monosynaptic responses could be evoked by either stimulating the hindbrain or visual inputs. All cells in which visual inputs could be evoked also had hindbrain-evoked responses and vice versa.

Fig. 4.

Hindbrain-tectal synapses are glutamatergic. A: excitatory postsynaptic currents (sEPSCs) in tectal neurons are blocked by co-application of the AMPA and N-methyl-d-aspartate (NMDA) receptor antagonists 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) and d-(−)-2-amino-5-phosphonopentanoic acid (d-AP5). B: in the presence of GABA receptor (GABAR) blocker PTX, the AMPAR antagonist NBQX blocks the evoked hindbrain-tectal currents at −60 mV. Depolarization of the tectal neurons to −20 mV reveals that hindbrain-tectal currents are also mediated by NMDA receptor (NMDAR). C: current (I)–voltage (V) relationship at hindbrain-tectal synapses in the presence of NBQX. I-V curve is voltage-dependent, consistent with the activation of NMDARs. D: there are no monosynaptic ascending inhibitory hindbrain-tectal connections. NBQX and d-AP5 eliminate all hindbrain-evoked synaptic transmission. Gray trace, control recording; black trace, in the presence of the drugs. The tectal neuron was depolarized to +5 mV, a potential at which inhibitory activity would be present. Stimulation of the hindbrain in the presence of these blockers reveals the absence of direct inhibitory hindbrain-tectal synapses.

Fig. 5.

There is no developmental change in paired-pulse ratio at hindbrain-tectal synapses. A: representative traces from tadpoles of each of the three stage epochs investigated, 42–43, 44–46, and 48–49. B: paired-pulse ratios expressed as the amplitude ratio between the peak values of excitatory postsynaptic currents 2 and 1 (EPSC2/EPSC1) for tadpoles from stages 42–43 (1.3 ± 0.07, n = 7); 44–46 (1.2 ± 0.3, n = 11); and 48–49 (1.0 ± 0.9, n = 11). P > 0.05, error bars are SE.

Fig. 6.

The AMPA/NMDA ratio at hindbrain-tectal synapses transiently increases and then decreases during development. A: representative tectal responses to hindbrain stimulation recorded at −60 mV and +60 mV. An average of 10 traces were averaged at −60 mV to represent the AMPA response and +60 mV to represent the NMDA response. Traces were from the same cell and superimposed. B: the average A/N ratio across development beginning with stage 42 (1.45 ± 0.3, n = 7) and showing a significant decrease between stage 44–46 (2.5 ± 0.6, n = 11) and 48–49 (1.0 ± 0.2, n = 15, P = 0.0281). Error bars are SE. A/N ratios were calculated as indicated in methods.

Fig. 7.

Input/output curves indicate degree of convergence of hindbrain-tectal inputs. Each line represents data points from a single cell; the line graphs summarize the data across cells and animals. A, left: representative traces showing the effects of a gradual increase in stimulation intensity on response amplitude in a representative stage 42 neuron. Right: summary of peak EPSC amplitudes from each cell plotted against stimulus intensity. All values were normalized according to methods. Individual input-output curves were grouped according to whether they showed one “jump” in amplitude of ≥65% of the maximal response size (type 1 responses, black lines, closed circles), or whether increases were more graded (type 2 responses, gray lines, open circles). We interpreted that cells in the 1st group would be innervated by one or few fibers, whereas those in the second group would be innervated by multiple fibers. In 55% (5 of 9) of stage 42/43 cells tested, the hindbrain-tectal projection was type 1. B and C: same as A but for older tadpoles. As the animals develop, the percentage of type 1 responses decreased to 33% (3 of 9) by stage 44–46 and to 14% (1 of 7) by stage 48/49. This indicates that over development the number of hindbrain fibers innervating a given tectal cell increases.

Fig. 8.

Quantal size of evoked hindbrain and visual inputs to tectal neurons across development. A: representative traces of tectal responses to direct stimulation of the hindbrain (HB) or visual (Vis) projections in the presence of Sr²⁺. B: asynchronous EPSC (aEPSC) amplitudes evoked by stimulation of different pathways that terminate on the same tectal cell are paired and connected by a line. The average evoked aEPSC amplitudes resulting from hindbrain and visual pathway stimulation are represented by black squares connected by a black line. We did not see consistent differences between aEPSC amplitudes of HB or Vis inputs to a given cell across development. C: average peak aEPSC amplitudes resulting from direct stimulation of the hindbrain across development (42–43: 9.06 ± 0.875 pA, n = 13; 44–46: 8.25 ± 0.97 pA, n = 10; 48–49: 7.36 ± 1.05 pA, n = 8). D: average aEPSC amplitudes resulting from direct stimulation of the visual inputs to tectal cells across development. The decrease in peak amplitude between stages 42–43 and 48–49 is significant (42–43: 11.03 ± 0.987 pA, n = 13; 44–46: 10.21 ± 1.207 pA, n = 10; 48–49: 6.71 ± 0.786 pA, n = 8; P = 0.006). E: aEPSC amplitude evoked by visual stimulation plotted against aEPSC amplitude evoked by HB stimulation for each tectal neuron investigated from all 3 developmental epochs. Circles = 42–43; bowties = 44–46, triangles 48–49.