Ia afferents also make specific connections with Renshaw cells, Hb9 interneurons29
and other interneurons but little is known about the development of these connections. Most of our knowledge on the organization of proprioceptive inputs to interneurons comes from experiments in the adult cat spinal cord30,31
. For example, cat Ia inhibitory interneurons (IaINs), which mediate reciprocal inhibition between antagonistic pools, receive inputs from Ia muscle afferents that appear to be stronger and more specific than on other interneurons (i.e., Ia/Ib interneurons, group II interneurons). This conclusion is based on the strong coupling between IaINs and Ia afferent inputs, their ability to follow relatively high frequencies of Ia afferent discharge in response to muscle stretch, and the fact that they do not receive convergent inputs from other proprioceptors31,32,33
. In contrast, cat Renshaw cells, which mediate recurrent inhibition of homonymous and synergistic motor neurons, do not appear to receive direct connections from Ia fibers or any other low-threshold afferent fibers in the adult spinal cord34,35
. The mechanisms by which Ia afferents select specific interneuronal targets within the ventral horn are unknown.
Although muscle afferent inputs to Renshaw cells cannot be detected functionally in the adult cat, work in the chick embryo demonstrated that muscle afferents make monosynaptic connections with R-interneurons, a class of avian embryonic inhibitory interneuron thought to be similar to the mammalian Renshaw cell36
. However, because afferent inputs on mammalian Renshaw cells had not previously been studied in neonatal animals, it was not clear if these connections are also formed in mammalian development, or if they were unique to avians. To address this issue, we analyzed muscle afferent inputs to Renshaw cells in late embryonic, postnatal and adult rats and mice37
Using a variety of anatomical methods to define proprioceptive primary afferents (VGluT1-IR, parvalbumin-IR, anterograde tracing from dorsal roots), we found that murine Renshaw cells receive few primary afferent inputs during late embryonic development when Ia afferent connections to motor neurons became functional (~E16-E17)15,38
. However, the fraction of Renshaw cells that receive spindle afferent inputs gradually increases to ~50% at P0, 100% by P10, and these inputs are maintained in all adult Renshaw cells (). This result was surprising since previous in vivo
studies from adult cat had reported that Renshaw cells cannot be monosynapticaly activated by dorsal root stimulation.
Developmental changes in the number of synaptic inputs onto Renshaw cells derived from dorsal root and motor axon afferents
To investigate whether the anatomically identified inputs were functional in newborn animals, we obtained whole cell recordings from identified Renshaw cells in the isolated spinal cord of the neonatal mouse. Most (11 of 12) Renshaw interneurons displayed a short-latency response following dorsal root stimulation (). This response was determined to be monosynaptic because the onset of the evoked EPSP exhibited very little jitter during high frequency stimulation and the average latency of the response (~6ms) was similar to the latency of monosynaptic afferent-evoked EPSPs recorded from motor neurons. In conclusion, our morphological and anatomical evidence demonstrates the presence of functional sensory afferent synaptic inputs onto neonatal Renshaw cells.
Because Renshaw cells are located in the ventral most regions of laminae VII and IX, we assumed these projections must come from Ia afferents since no other sensory afferent class is known to project this ventrally in the spinal cord39
. This raises the question of why Renshaw cells do not seem to have functional monosynaptic Ia afferent inputs in the adult cat. One possible explanation is that Ia afferent inputs do not make synaptic contacts on Renshaw cells in all species. To date most physiological studies of adult Renshaw cells have been conducted in the cat, while our findings were obtained in neonatal mice. However, one study conducted in fetal cats40
provided evidence compatible with the presence of monosynaptic dorsal root inputs on embryonic Renshaw cells. Moreover, our morphological data revealed VGLUT1 sensory synapses on adult Renshaw cells in cats, similar to those found in rodents. Thus the available data seem to rule out the possibility of species differences. An alternative explanation is that Renshaw cells lose this functional input during postnatal development by weakening its synaptic effectiveness, despite maintaining a significant number of synaptic contacts.