Abstract
Synapse elimination and strengthening are central mechanisms for the developmental organization of excitatory neuronal networks. Little is known, however, about whether these processes are also involved in establishing precise inhibitory circuits. We examined the development of functional connectivity before hearing onset in rats in the tonotopically organized, glycinergic pathway from the medial nucleus of the trapezoid body (MNTB) to the lateral superior olive (LSO), which is part of the mammalian sound localization system. We found that LSO neurons became functionally disconnected from ∼75% of their initial inputs, resulting in a two-fold sharpening of functional topography. This was accompanied by a 12-fold increase in the synaptic conductance generated by maintained individual inputs. Functional elimination of MNTB–LSO synapses was restricted to the period when these glycinergic/GABAergic synapses are excitatory. These results provide new insights into the mechanisms by which precisely organized inhibitory circuits are established during development.
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Acknowledgements
We thank N.K. Baba, E. Frank, D. Gillespie, E. Rubel and D. Simons for discussions and comments on the manuscript, I. Ehrlich for determining MNTB boundaries and N. Allman for technical support. We are also grateful to R. Givens for php-glutamate and to B. Schmidt for BC204 GABA. This work was supported by the National Institute on Deafness and Other Communicative Disorders (DC04199), the Alfred P. Sloan foundation, a Presidential Early Career Award (K.K.) and the Center for Neural Basis of Cognition (G.K.).
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Supplementary Fig 1.
MNTB-elicited calcium responses in the immature superior olivary complex. Areas of calcium responses are outlined (different colors represent different stimulation trials) and overlaid on corresponding bright-field images. Calcium responses occur consistently in the LSO and only on rare occasions also in a few other nuclei such as the medial superior olive (MSO) and superior paraolivary nucleus (SPN). The absence of consistent responses in areas outside the LSO indicates that calcium responses in the immature LSO are elicited by monosynaptic MNTB-LSO connections rather than by polysynaptic pathways between the MNTB and LSO. Slices were prepared from P2 rats and bulk-labeled with Fura-2 AM. Changes in intracellular calcium concentrations were measured using 340/380 nm ratio-imaging on an inverted microscope (Nikon Eclipse TE200) with a 10x objective (NA: 0.5). For details of staining and imaging procedures, see ref. 24. The MNTB was stimulated electrically with bipolar stimulation electrodes (two pulses at 30 μA in A and 40 μA in B, 0.1 ms duration, separated by 20 ms). To increase spike activity and increase the detection of calcium responses, 10 mM TEA was included in the perfusion medium. Ionotropic glutamate receptors were blocked by CNQX (20 μM) and D,L- APV (100 μM) to isolate glycinergic/GABAergic calcium responses. Stimulus-induced calcium responses were detected by subtracting the ratio-image immediately taken before electrical stimulation from the first ratio-image taken after MNTB-stimulation. The resulting δratio-image was smoothed by a 3 x 3 Gaussian filter (Adobe Photoshop), thresholded, and overlaid onto the corresponding bright-field image. To map the entire superior olivary complex, 4-6 areas in the superior olivary complex were sequentially imaged and assembled using the corresponding bright-field images for orientation. At each position, responses to two stimulus trials were measured and all responses were overlaid over the composite image. (JPG 72 kb)
Supplementary Fig 2.
Absence of intra-nuclear synaptic connections between LSO neurons in neonatal rats as revealed by focal uncaging of glutamate. Locations of uncaging sites are indicated by circles and are overlaid on a brightfield video picture taken during the mapping experiments. LSO boundaries are outlined in black. Uncaging sites, which failed to elicit currents in the recorded LSO neuron, are marked in yellow; uncaging sites, which elicited currents, are marked in green. In all 6 LSO neurons tested, responses could only be elicited from 4-5 stimulation sites located in close vicinity to LSO cell bodies (location of cell bodies indicated by the tip of the recording electrode). Successful stimulation sites form elongated response areas (4.3 ± 0.2 locations, n = 6). The long axis of response areas matched the orientation of the bipolar dendrites of the recorded neurons as observed at 40x magnification during seal formation. Notably, responses were never elicited from medio-lateral ("tonotopically") distant stimulation sites. In 3 neurons (right columns), response areas were remapped after application of 1 μM TTX to block spike-elicited synaptic transmission. In two neurons, input patterns were completely unaffected by TTX, and in one neuron only one single input site could not be reproduced in TTX (lower row). This TTX-insensitivity indicates that responses resulted from direct stimulation of LSO dendrites rather then the activation of local intra-nuclear connections. Slices were prepared from P3-P4 animals and recordings were performed in voltage-clamp using a K-gluconate-based pipette solution (see Methods). (JPG 104 kb)
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Kim, G., Kandler, K. Elimination and strengthening of glycinergic/GABAergic connections during tonotopic map formation. Nat Neurosci 6, 282–290 (2003). https://doi.org/10.1038/nn1015
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DOI: https://doi.org/10.1038/nn1015
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