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Recovering appropriate motor behavior is a major problem following spinal cord injury because of the damage to neural processes descending from the brain
to the spinal cord. Increasing input efficiency from intact descending fibers will be critical for recovery. In order to recover effective motor behavior, connections
must be restored with the appropriate pattern and specificity that normally occurs during development. In this study we explore a novel solution; we examine the
possibility that Protocadherins (Pcdhs), a family of adhesion molecules in the CNS, play a role in establishing connection specificity. We have forced the expression
of Pcdhs in motoneurons of the chick embryo. Once Pcdh expression is established we isolate the spinal cord and utilize electrophysiological methods to test
whether changes in motoneuron connections have been generated by Pcdhs. We now have data from control embryos indicating that direct connections form
between motoneurons that innervate the same muscle but connections do not occur between motoneurons that innervate different muscles even though they
are in a position to make these connections. Further, we have preliminary results implying that connections between motoneurons innervating different muscles
do make connections following forced expression of Pcdhs. Therefore, observing the formation of these novel direct connections in transfected spinal cords would
support the role of Pcdhs in specifying synaptic contact and provides an initial step in re-establishing connections that have been damaged after spinal injury.
The pattern of Protocadherin expression at CNS synapses and the capability of Pcdhs in mediating intercellular adhesion makes them likely candidates for
establishing specific synaptic connections. Further, recent studies have demonstrated that knocking out large combinations of Pcdhs does reduce the number
of synaptic connections in the spinal cord. 1,2 Therefore, a better understanding of Pcdhs could lead to therapeutic strategies for treatment of spinal cord
injuries.
We take advantage of an accessible system in which femorotibialis and adductor motoneurons, make direct connections only to motoneurons innervating the same
muscle. Monosynaptic (direct) responses (with onset latency under 12ms) were observed in femorotibialis motoneurons after the stimulation of the femorotibialis
muscle nerve .
Similar motoneurons make direct connections with each other.
Motoneurons do not make direct connections to motoneurons innervating different muscles even though they are in an appropriate position to make such
connections. Therefore, the stimulation of a different type of muscle nerve (adductor) does not result a short latency response (under 12ms) in the other nerve
(i.e. femorotibialis).
Different motoneurons do not form direct connections.
In this study we have tested whether Protocadherins play a role in synaptic specificity by electroporating (transfecting) Pcdhs into the spinal cord, to see if this
produces novel connections. Then we isolate the cord for electrophysiological nerve recordings.
Possible direct connections between different type motoneurons?
In Ovo Electroporation
Transfection is carried out using in ovo electroporation which transfects many cells on one side of the cord. Spinal neurons are transfected with expression
plasmids (CMV-based expression vector, pCS2) carrying a combination of 2 gamma Pcdh and EGFP plasmids. Plasmids are mixed together and injected into
the spinal canal in the living embryo at embryonic day 3 (E3), before motoneuron connections form (see injected spinal canal image below). Using a BTX
electroporator an electrical field is applied across the cord to transfect the plasmids into many spinal neurons. EGFP marks the location of the Pcdh transfected
neurons, which can be seen under fluorescence microsocopy.
Spinal Cord Preparation
Embryos were allowed to develop until stage 36 (embryonic day 10), after motoneuron connections have formed. At E10 spinal cords are isolated from the
embryo in recirculating oxygenated saline bath with intact femorotibialis and adductor muscle nerves.
Extracellular Recordings
Cords were transferred to a recording chamber where femorotibialis and adductor nerves were drawn into suction electrodes that were used for stimulation and/or
recording (see image below.) At this point single pulses of 30μA were delivered to either muscle nerve while recording the other. Direct or monosynaptic
connections were indicated by onset latencies under 12ms while indirect connections polysynaptic) showed responses 12ms or more after the stimulus.
1. In non-transfected spinal cords there are normally no direct connections between motoneurons innervating different muscles.
Femorotibialis muscle nerve recoding from normal embryo in response to a single pulse stimulus to the adductor muscle nerve does not exhibit a direct
monosynaptic response (<12ms), but rather shows an indirect or polysynaptic response (>12ms). This indicates that there are no direct connections between
femorotibialis and adductor motoneurons, but a strong indirect connection through a spinal interneuron.
2. Protocadherin-expressing spinal cords show the formation of novel connections between montoneurons innervating different muscles.
The image shows EGFP expression, a marker for the Pcdh expression, in neurons and the axons of femorotibialis and adductor motoneurons. Pcdhs are
expressed on the right side of the cord (lower) but not on the left side (upper).
A: Recording obtained from the non-transfected side of a spinal cord, adductor stimulation recording from femorotibialis motoneurons. No direct response is
observed (< 12ms, within the boxed region), indicating that there are no direct connections between femorotibialis and adductor motoneurons. B: Same, but on
the side transfected with Pcdhs. Here, femorotibialis motoneurons respond to stimulation of the adductor muscle nerve with a burst of discharge at monosynaptic
latencies (< 12ms, within the boxed region) suggesting the presence of novel direct connections between femorotibialis and adductor motoneurons on the
Pcdh-expressing side.
Under normal conditions, direct synaptic connections occur between motoneurons innervating the same muscle but not between motoneurons innervating
different muscles.
Preliminary studies find changes in synaptic connectivity after transfection of motoneurons with Pcdhs, which suggests that Protocadherins may cause the
formation of novel direct connections between motoneurons that innervate different muscles.
These preliminary findings support the idea that Protocadherins play a role in synaptic specificity and this could be important in helping to develop treatments
for the recovery of motor behavior after spinal cord injury.
This material is based upon work supported by the Howard Hughes Medical Institute under Grant No.52005873 and by the NSF under Grant No. 0616097.
1.Weiner, J.A., Xiaozhong, W., Tapia, J.C. & Sanes, J.R. (2005) Gamma protocadherins are required for synaptic development in the spinal cord. Proceedings
of the Natikonal Academy of Science of the United States of America 102(1), 8-14.
2.Xiaozhong, W., Weiner, J.A., Levi, S., Craig, A.M., Bradley, A., Sanes, J.R. (2002) Gamma protocadherins are required for survival of spinal
interneurons. Neuron 36(5), 843-854.
3.Xu et al. (2007) Developmental reorganization of the output of a GABAergic interneural circuit. Neurophysiology 97, 2769-2779.
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