Michael J. O'Donovan

Early Functional Impairment of Sensory-Motor Connectivity in a Mouse Model of Spinal Muscular Atrophy

To define alterations of neuronal connectivity that occur during motor neuron degeneration, we characterized the function and structure of spinal circuitry in spinal muscular atrophy (SMA) model mice. SMA motor neurons show reduced proprioceptive reflexes that correlate with decreased number and function of synapses on motor neuron somata and proximal dendrites. These abnormalities occur at an early stage of disease in motor neurons innervating proximal hindlimb muscles and medial motor neurons innervating axial muscles, but only at end-stage disease in motor neurons innervating distal hindlimb muscles. Motor neuron loss follows afferent synapse loss with the same temporal and topographical pattern. Trichostatin A, which improves motor behavior and survival of SMA mice, partially restores spinal reflexes, illustrating the reversibility of these synaptic defects. Deafferentation of motor neurons is an early event in SMA and may be a primary cause of motor dysfunction that is amenable to therapeutic intervention.

Real-time imaging of neurons retrogradely and anterogradely labelled with calcium-sensitive dyes

Membrane-impermeant calcium indicator dyes were used to retrogradely label dorsal root ganglia, spinal motoneurons and interneurons in the spinal cord of the chick embryo. The dyes were also used to label anterogradely primary afferent axons in the spinal cord and synaptic endings in the ciliary ganglion. Labelled neurons were imaged using digital videomicroscopy. Motoneurons and dorsal root ganglion cells exhibited a frequency-dependent change in fluorescence during antidromic stimulation. Single antidromic stimuli resulted in fluorescence transients that could be resolved in individual cells in real time. In addition, fluorescence changes could be recorded in motoneurons during episodes of bursting generated by rhythmic synaptic inputs from premotor networks. Stimulus-induced fluorescence signals were also detected in axons and synaptic endings labelled anterogradely. Optical signals were largely abolished in the absence of extracellular calcium. The results show that calcium changes can now be measured in identified populations of neurons and presynaptic terminals. The strong dependence of these signals on impulse activity suggests that the technique will be useful for monitoring the activity of identified neuronal populations. The calcium-dependent fluorescence signal probably results from cytosolic dye derived from diffusion which may limit the technique to situations in which the dye can be applied close (< 1 cm) to cell bodies.

Calcium imaging of rhythmic network activity in the developing spinal cord of the chick embryo

Video-rate imaging of spinal neurons loaded with calcium-sensitive dyes was used to investigate the calcium dynamics and cellular organization of spontaneously active rhythm-generating networks in the spinal cord of E9-E12 chick embryos. Spinal neurons were loaded with bath-applied fura-2am. Motoneurons were also loaded by retrograde labeling with dextran-conjugated, calcium-sensitive dyes. Dye-filled motoneurons exhibited large fluorescent changes during antidromic stimulation of motor nerves, and an increase in the 340/380 fura fluorescence ratio that is indicative of increased intracellular free calcium. Rhythmic fluorescence changes in phase with motoneuron electrical activity were recorded from motoneurons and interneurons during episodes of evoked or spontaneous rhythmic motor activity. Fluorescent responses were present in the cytosol and in the perinuclear region, during antidromic stimulation and network-driven rhythmic activity. Optically active cells were mapped during rhythmic activity, revealing a widespread distribution in the transverse and horizontal planes of the spinal cord with the highest proportion in the ventrolateral part of the cord. Fluorescent signals were synchronized in different regions of the cord and were similar in time course in the lateral motor column and in the intermediate region. In the dorsal region the rhythm was less pronounced and the signal decayed after a large initial transient. Video-rate fluorescent measurements from individual cells confirmed that fluorescent signals were synchronized in interneurons and in motoneurons although the time course of the signal could vary between cells. Some of the interneurons exhibited tonic elevations of fluorescence for the duration of the episode whereas others were rhythmically active in phase with motoneurons. At the onset of each cycle of rhythmic activity the earliest fluorescent change occurred ventrolaterally, in and around the lateral motor column, from which it spread to the rest of the cord. The results suggest that neurons in the ventrolateral part of the spinal cord are important for rhythmogenesis and that axons traveling in the ventrolateral white matter may be involved in the rhythmic excitation of motoneurons and interneurons. The widespread synchrony of the rhythmic calcium transients may reflect the existence of extensive excitatory interconnections between spinal neurons. The network-driven calcium elevations in the cytosol and the perinuclear region may be important in mediating activity-dependent effects on the development of spinal neurons and networks.

Synaptic depression: A dynamic regulator of synaptic communication with varied functional roles

It appears that a bewildering array of processes can contribute to the mechanism of synaptic depression. Markram and Tsodyks argued that the depression they observed in cortical cells was probably dependent on the amount of transmitter released, as indicated in the model (Fig. 1Fig. 1), although changes in the affinity of postsynaptic glutamate receptors could not be excluded[9xMarkram, H. and Tsodyks, M. Nature. 1996; 882: 807–810Crossref | Scopus (510)See all References[9]. However, in studies of the visual cortex several postsynaptic factors have been eliminated because synaptic depression at excitatory synapses is unaffected by postsynaptic receptor blockade with the non-NMDA receptor antagonist CNQX or by reduction of receptor desensitization by cyclohexemide (J.A. Varela et al., unpublished observations). In developing muscle afferent–motoneuron contacts a presynaptic mechanism for depression has also been implicated because depression is abolished when transmitter release is decreased by low extracellular Ca2+ concentrations or bath-application of the GABAB agonist, baclofen[5xLev-Tov, A. and Pinco, M. J. Physiol. 1992; 447: 149–169Crossref | PubMedSee all References[5]. In cultured spinal cord neurons transmitter depletion is probably also responsible for synaptic depression at some synapses. However, at other synapses in such cultures, the depression might be due to failure of the presynaptic action potential to invade all of the terminals[6xStreit, J., Luscher, C., and Luscher, H-R. J. Neurophysiol. 1992; 68: 1793–1803PubMedSee all References[6]. It has also been proposed that synaptic depression might be caused by a form of negative feedback. This can occur if excess transmitter leaks from synaptic sites to engage presynaptic autoreceptors which decrease transmitter release[8xDeisz, R.A. and Prince, D.A. J. Physiol. 1989; 412: 513–541PubMedSee all References, 21xScanziani, M. et al. Nature. 1997; 385: 630–634Crossref | PubMed | Scopus (357)See all References], or by an activity-dependent reduction in the presynaptic calcium currents responsible for transmitter release[22xJia, M. and Nelson, P.G. J. Neurophysiol. 1986; 56: 1257–1267PubMedSee all References[22]. Finally, alterations in the sensitivity of postsynaptic receptors have also been implicated in synaptic depression[23xNumann, R.E. and Wong, R.K. Neurosci. Lett. 1984; 47: 289–294Crossref | PubMed | Scopus (58)See all References[23].It should be clear from this review that synaptic depression has the potential to be an important contributor to the properties and function of networks. But are these interesting properties actually engaged in active networks? This will be a difficult question to answer because it will require modifying the degree of synaptic depression within a network and documenting the effects on the network output. This may be easier to accomplish in rhythmically active networks whose output can be easily measured. It will be considerably more difficult to establish whether or not synaptic depression actually enhances the dynamic sensitivity of networks, both for experimental reasons and because we do not know how such information is actually used in functioning networks.Finally, it will be important in future studies to establish whether synaptic depression is actively regulated by the nervous system and whether or not its effects can be modified by events in the postsynaptic cell. For example, it is possible that active conductances in dendrites might be capable of compensating or modifying the effects of depression at some synapses.Perhaps many different classes of synapse can express both depression and facilitation. Certainly, there is evidence that both processes can be co-expressed at the same synapse. Can the nervous system control the relative expression of either process at individual synapses? Is synaptic depression a property that is regulated retrogradely by the postsynaptic cell, as has been proposed for developing synapses in the spinal cord[24xSeebach, B.S. and Mendell, L.M. J. Neurophysiol. 1996; 76: 3875–3885PubMedSee all References[24]?

Dual personality of GABA/glycine-mediated depolarizations in immature spinal cord

The inhibitory action of glycine and GABA in adult neurons consists of both shunting incoming excitations and moving the membrane potential away from the action potential (AP) threshold. By contrast, in immature neurons, inhibitory postsynaptic potentials (IPSPs) are depolarizing; it is generally accepted that, despite their depolarizing action, these IPSPs are inhibitory because of the shunting action of the Cl− conductance increase. Here we investigated the integration of depolarizing IPSPs (dIPSPs) with excitatory inputs in the neonatal rodent spinal cord by means of both intracellular recordings from lumbar motoneurons and a simulation using the compartment model program “Neuron.” We show that the ability of IPSPs to suppress suprathreshold excitatory events depends on ECl and the location of inhibitory synapses. The depolarization outlasts the conductance changes and spreads electrotonically in the somatodendritic tree, whereas the shunting effect is restricted and local. As a consequence, dIPSPs facilitated AP generation by subthreshold excitatory events in the late phase of the response. The window of facilitation became wider as ECl was more depolarized and started earlier as inhibitory synapses were moved away from the excitatory input. GAD65/67 immunohistochemistry demonstrated the existence of distal inhibitory synapses on motoneurons in the neonatal rodent spinal cord. This study demonstrates that small dIPSPs can either inhibit or facilitate excitatory inputs depending on timing and location. Our results raise the possibility that inhibitory synapses exert a facilitatory action on distant excitatory inputs and slight changes of ECl may have important consequences for network processing.

Reduced gap junctional coupling leads to uncorrelated motor neuron firing and precocious neuromuscular synapse elimination

During late embryonic and early postnatal life, neuromuscular junctions undergo synapse elimination that is modulated by patterns of motor neuron activity. Here, we test the hypothesis that reduced spinal neuron gap junctional coupling decreases temporally correlated motor neuron activity that, in turn, modulates neuromuscular synapse elimination, by using mutant mice lacking connexin 40 (Cx40), a developmentally regulated gap junction protein expressed in motor and other spinal neurons. In Cx40−/− mice, electrical coupling among lumbar motor neurons, measured by whole-cell recordings, was reduced, and single motor unit recordings in awake, behaving neonates showed that temporally correlated motor neuron activity was also reduced. Immunostaining and intracellular recording showed that the neuromuscular synapse elimination was accelerated in muscles from Cx40−/− mice compared with WT littermates. Our work shows that gap junctional coupling modulates neuronal activity patterns that, in turn, mediate synaptic competition, a process that shapes synaptic circuitry in the developing brain.

Primary Afferent Synapses on Developing and Adult Renshaw Cells

The mechanisms that diversify adult interneurons from a few pools of embryonic neurons are unknown. Renshaw cells, Ia inhibitory interneurons (IaINs), and possibly other types of mammalian spinal interneurons have common embryonic origins within the V1 group. However, in contrast to IaINs and other V1-derived interneurons, adult Renshaw cells receive motor axon synapses and lack proprioceptive inputs. Here, we investigated how this specific pattern of connectivity emerges during the development of Renshaw cells. Tract tracing and immunocytochemical markers [parvalbumin and vesicular glutamate transporter 1 (VGLUT1)] showed that most embryonic (embryonic day 18) Renshaw cells lack dorsal root inputs, but more than half received dorsal root synapses by postnatal day 0 (P0) and this input spread to all Renshaw cells by P10–P15. Electrophysiological recordings in neonates indicated that this input is functional and evokes Renshaw cell firing. VGLUT1-IR bouton density on Renshaw cells increased until P15 but thereafter decreased because of limited synapse proliferation coupled with the enlargement of Renshaw cell dendrites. In parallel, Renshaw cell postsynaptic densities apposed to VGLUT1-IR synapses became smaller in adult compared with P15. In contrast, vesicular acetylcholine transporter-IR motor axon synapses contact embryonic Renshaw cells and proliferate postnatally matching Renshaw cell growth. Like other V1 neurons, Renshaw cells are thus competent to receive sensory synapses. However, after P15, these sensory inputs appear deselected through arrested proliferation and synapse weakening. Thus, Renshaw cells shift from integrating sensory and motor inputs in neonates to predominantly motor inputs in adult. Similar synaptic weight shifts on interneurons may be involved in the maturation of motor reflexes and locomotor circuitry.

Motoneurons Dedicated to Either Forward or Backward Locomotion in the Nematode Caenorhabditis elegans

Multifunctional motoneurons and muscles, which are active during forward and backward locomotion are ubiquitous in animal models. However, studies in the nematode Caenorhabditis elegans suggest that some locomotor motoneurons are necessary only for forward locomotion (dorsal B-motoneurons, DB), while others (dorsal A-motoneurons, DA) are necessary only for backward locomotion. We tested this hypothesis directly by recording the activity of these motoneurons during semirestrained locomotion. For this purpose, we used epifluorescence imaging of the genetically encoded calcium sensor cameleon, expressed in specific motoneurons, while monitoring locomotor behavior through the microscope condenser using a second camera. We found that ventral and dorsal B-motoneurons (DB and VB) were coactive during forward locomotion while ventral A-motoneurons (VA) were only active during backward locomotion. The signals we recorded correlated with the direction of locomotion but not with the faster undulatory cycles. To our knowledge, these are the first recordings of motoneuron activity in C. elegans and the only direction-dedicated motoneurons described to date.

Supraspinal facilitation of cutaneous polysynaptic EPSPs in cat medial gastrocnemius motoneurons

SummaryWe examined the characteristics of postsynaptic potentials (PSPs) produced in antidromically-identified medial gastrocnemius (MG) α-motoneurons by electrical stimulation of low threshold (< 3×T) distal limb cutaneous afferents in the sural (SUR) nerve in adult cats anesthetized with α-chloralose, together with the effects on SUR PSPs of supraspinal conditioning stimulation of the contralateral red nucleus (RN) and pyramidal tract (PT). In the majority of MG motoneurons, SUR afferents with electrical thresholds < 1.5×T produced early excitatory synaptic potentials (EPSPs) with minimum central latency of about 2.0 ms, suggesting activation of a trisynaptic segmental pathway with two interposed interneurons. Such early EPSPs were often detectable with stimuli < 1.2×T, as determined by recording the compound action potential in the sciatic nerve and from the first appearance of the N1 wave of the cord dorsum potential. Inhibitory synaptic potentials (IPSPs) were regularly produced by SUR volleys of only slightly greater strength (often as low as 1.3×T) and these had minimum central latencies of about 3.0 ms (about 1.0 ms longer than the earliest EPSPs), suggesting a three interneuron central pathway.Repetitive stimulation of RN and PT regularly produced facilitation of both EPSP and IPSP components in the SUR response, suggesting that these supraspinal systems directly or indirectly excite some of the same interneurons that convey the SUR effects to MG motoneurons. When using very low strength SUR stimuli, PT conditioning produced relatively pure facilitation of the SUR EPSPs but with larger SUR volleys, PT clearly facilitated both EPSPs and IPSPs. RN conditioning produced more parallel facilitation of SUR EPSPs and IPSPs. Supraspinal control of the polysynaptic pathway producing SUR EPSPs is of particular interest because of earlier evidence that this pathway is differentially distributed to motoneurons of fast twitch versus slow twitch MG motor units.

Modeling Spontaneous Activity in the Developing Spinal Cord Using Activity-Dependent Variations of Intracellular Chloride

We investigated how spontaneous activity is generated in developing, hyperexcitable networks. We focused our study on the embryonic chick spinal cord, a preparation that exhibits rhythmic discharge on multiple timescales: slow episodes (lasting minutes) and faster intraepisode cycling (∼1 Hz frequency). For this purpose, we developed a mean field model of a recurrent network with slow chloride dynamics and a fast depression variable. We showed that the model, in addition to providing a biophysical mechanism for the slow dynamics, was able to account for the experimentally observed activity. The model made predictions on how interval and duration of episodes are affected when changing chloride-mediated synaptic transmission or chloride flux across cell membrane. These predictions guided experiments, and the model results were compared with experimental data obtained with electrophysiological recordings. We found agreement when transmission was affected through changes in synaptic conductance and good qualitative agreement when chloride flux was varied through changes in external chloride concentration or in the rate of the Na+-K+-2Cl- cotransporter. Furthermore, the model made predictions about the time course of intracellular chloride concentration and chloride reversal potential and how these are affected by changes in synaptic conductance. Based on the comparison between modeling and experimental results, we propose that chloride dynamics could be an important mechanism in rhythm generation in the developing chick spinal cord.