Synaptic drive in spinal motoneurons during scratch network activity.

Synaptic activity in motoneurons may provide unique insight in the relation between functional network activity and behavior. During scratch network activity in an ex vivo preparation from red-eared turtles ( Trachemys scripta elegans), excitatory and inhibitory synaptic current can be separated and quantified in voltage-clamp recordings. With this technique, we confirm the reciprocal synaptic excitation and inhibition in hip flexor motoneurons during ipsilateral scratching and show that out-of-phase inhibition and excitation also characterize hip extensor motoneurons during ipsi- and contralateral scratching. In contrast, inhibition precedes and partly overlaps excitation in hip flexor-like motoneurons and delays depolarization of membrane potential. We conclude that out-of-phase excitation and inhibition during rhythmic network activity is a common feature in spinal motoneurons. NEW & NOTEWORTHY During network activity, the firing pattern of individual neurons is shaped by their intrinsic conductances and synaptic input. Quantification of synaptic input is, therefore, essential to understand how the properties of individual neurons contribute to function and help to reveal the structure of the network. Here, we show how a combination of recording techniques can be used to quantify and compare the pattern of synaptic activity in different groups of motoneurons during rhythmic network activity.

Synaptic Excitation in Spinal Motoneurons Alternates with Synaptic Inhibition and Is Balanced by Outward Rectification during Rhythmic Motor Network Activity.

Regular firing in spinal motoneurons of red-eared turtles (Trachemys scripta elegans, either sex) evoked by steady depolarization at rest is replaced by irregular firing during functional network activity. The transition caused by increased input conductance and synaptic fluctuations in membrane potential was suggested to originate from intense concurrent inhibition and excitation. We show that the conductance increase in motoneurons during functional network activity is mainly caused by intrinsic outward rectification near threshold for action potentials by activation of voltage and Ca2+ gated K channels. Intrinsic outward rectification facilitates spiking by focusing synaptic depolarization near threshold for action potentials. By direct recording of synaptic currents, we also show that motoneurons are activated by out-of-phase peaks in excitation and inhibition during network activity, whereas continuous low-level concurrent inhibition and excitation may contribute to irregular firing.SIGNIFICANCE STATEMENT Neurons embedded in active neural networks can enter a high-conductance state. High-conductance states were observed in spinal motoneurons during rhythmic motor behavior. Assuming no change in intrinsic conductance, it was suggested that the high-conductance state in motoneurons originated from balanced inhibition and excitation. In this study, we demonstrate that intrinsic outward rectification significantly contributes to the high-conductance state. Outward rectification balances synaptic excitation and maintains membrane potential near spike threshold. In addition, direct synaptic current recordings show out-of-phase excitation and inhibition in motoneurons during rhythmic network activity.

Motor Neurons.

Motor neurons translate synaptic input from widely distributed premotor networks into patterns of action potentials that orchestrate motor unit force and motor behavior. Intercalated between the CNS and muscles, motor neurons add to and adjust the final motor command. The identity and functional properties of this facility in the path from synaptic sites to the motor axon is reviewed with emphasis on voltage sensitive ion channels and regulatory metabotropic transmitter pathways. The catalog of the intrinsic response properties, their underlying mechanisms, and regulation obtained from motoneurons in in vitro preparations is far from complete. Nevertheless, a foundation has been provided for pursuing functional significance of intrinsic response properties in motoneurons in vivo during motor behavior at levels from molecules to systems. © 2017 American Physiological Society. Compr Physiol 7:463-484, 2017.

Irregular Firing and High-Conductance States in Spinal Motoneurons during Scratching and Swimming.

UNLABELLED: Intense synaptic transmission during scratch network activity increases conductance and induces irregular firing in spinal motoneurons. It is not known whether this high-conductance state is a select feature for scratching or a property that goes with spinal motor network activity in general. Here we compare conductance and firing patterns in spinal motoneurons during network activity for scratching and swimming in an ex vivo carapace-spinal cord preparation from adult turtles (Trachemys scripta elegans). The pattern and relative engagement of motoneurons are distinctly different in scratching and swimming. Nevertheless, we found increased synaptic fluctuations in membrane potential, irregular firing, and increased conductance in spinal motoneurons during scratch and swim network activity. Our finding indicates that intense synaptic activation of motoneurons is a general feature of spinal motor network activity. SIGNIFICANCE STATEMENT: Neurons embedded in active neural networks can enter high-conductance states with irregular firing. This was previously shown for spinal motoneurons during scratching. Because scratching is highly specialized rhythmic behavior, it is not known whether high-conductance states and irregular firing are a peculiarity for motoneurons during scratching. Here, using intracellular recordings from motoneurons in an ex vivo carapace-spinal cord preparation from adult turtles, we demonstrate that irregular firing and high-conductance states are present not only during scratching but also during swimming. Our findings suggest that irregular firing and high-conductance states could be a general feature for motor behaviors.

Dense distributed processing in a hindlimb scratch motor network.

In reduced preparations, hindlimb movements can be generated by a minimal network of neurons in the limb innervating spinal segments. The network of neurons that generates real movements is less well delineated. In an ex vivo carapace-spinal cord preparation from adult turtles (Trachemys scripta elegans), we show that ventral horn interneurons in mid-thoracic spinal segments are functionally integrated in the hindlimb scratch network. First, mid-thoracic interneurons receive intense synaptic input during scratching and behave like neurons in the hindlimb enlargement. Second, some mid-thoracic interneurons activated during scratching project descending axons toward the hindlimb enlargement. Third, elimination of mid-thoracic segments leads to a weakening of scratch rhythmicity. We conclude that densely innervated interneurons in mid-thoracic segments contribute to hindlimb scratching and may be part of a distributed motor network that secures motor coherence.

Excitatory and inhibitory synaptic mechanisms at the first stage of integration in the electroreception system of the shark.

High impulse rate in afferent nerves is a common feature in many sensory systems that serve to accommodate a wide dynamic range. However, the first stage of integration should be endowed with specific properties that enable efficient handling of the incoming information. In elasmobranches, the afferent nerve originating from the ampullae of Lorenzini targets specific neurons located at the Dorsal Octavolateral Nucleus (DON), the first stage of integration in the electroreception system. Using intracellular recordings in an isolated brainstem preparation from the shark we analyze the properties of this afferent pathway. We found that stimulating the afferent nerve activates a mixture of excitatory and inhibitory synapses mediated by AMPA-like and GABAA receptors, respectively. The excitatory synapses that are extremely efficient in activating the postsynaptic neurons display unusual voltage dependence, enabling them to operate as a current source. The inhibitory input is powerful enough to completely eliminate the excitatory action of the afferent nerve but is ineffective regarding other excitatory inputs. These observations can be explained by the location and efficiency of the synapses. We conclude that the afferent nerve provides powerful and reliable excitatory input as well as a feed-forward inhibitory input, which is partially presynaptic in origin. These results question the cellular location within the DON where cancelation of expected incoming signals occurs.

Increased activity of pre-motor network does not change the excitability of motoneurons during protracted scratch initiation.

Intrinsic response properties of neurons change during network activity. These changes may reinforce the initiation of particular forms of network activity. If so, the involvement of neurons in particular behaviours in multifunctional networks could be determined by up- or down-regulation of their intrinsic excitability. Here we employed an experimental paradigm of protracted scratch initiation in the integrated carapace-spinal cord preparation of adult turtles (Chrysemys scripta elegans). The protracted initiation of scratch network activity allows us to investigate the excitability of motoneurons and pre-motor network activity in the time interval from the start of sensory stimulation until the onset of scratch activity. Our results suggest that increased activity in the pre-motor network facilitates the onset of scratch episodes but does not change the excitability of motoneurons at the onset of scratching.

Mathematical model of dopamine autoreceptors and uptake inhibitors and their influence on tonic and phasic dopamine signaling.

Dopamine (DA) D2-like autoreceptors are an important component of the DA system, but their influence on postsynaptic DA signaling is not well understood. They are, directly or indirectly, involved in drug abuse and in treatment of schizophrenia and attention deficit hyperactive disorder: DA autoreceptors influence the behavioral effect of cocaine and methylphenidate and may be the target of antipsychotic medications such as haloperidol. DA autoreceptors are active at two levels: Somatodendritic autoreceptors mainly influence firing rate of DA neurons, and presynaptic autoreceptors control release of neurotransmitter at axonal terminals. Here we develop a mathematical model that captures the dynamics of this dual autoregulation system. Our model predicts a biphasic autoreceptor response between DA terminals and somatodendritic regions that influences the postsynaptic integration of DAergic firing patterns. We applied our model to study how DA uptake inhibition affects the translation of DA cell firing into activation of postsynaptic DA receptors. While uptake inhibition increased tonic activation of low-affinity postsynaptic receptors, high-affinity state receptors saturated and thus became insensitive to phasic DA signaling. This effect had remarkable regional specificity: While high-affinity DA receptors saturated at low levels of uptake inhibition in nucleus accumbens, they only saturated at higher levels of uptake inhibition in dorsal striatum. Based on high-affinity receptor saturation, the model predicted that removal of autoreceptor control would lead to cocaine hypersensitivity.

Opposing Effects of Intrinsic Conductance and Correlated Synaptic Input on V-Fluctuations during Network Activity.

Neurons often receive massive concurrent bombardment of synaptic inhibition and excitation during functional network activity. This increases membrane conductance and causes fluctuations in membrane potential (V(m)) and spike timing. The conductance increase is commonly attributed to synaptic conductance, but also includes the intrinsic conductances recruited during network activity. These two sources of conductance have contrasting dynamic properties at sub-threshold membrane potentials. Synaptic transmitter gated conductance changes abruptly and briefly with each presynaptic action potential. If the spikes arrive at random times the changes in synaptic conductance are therefore stochastic and rapid during intense network activity. In comparison, sub-threshold intrinsic conductances vary smoothly in time. In the present study this discrepancy is investigated using two conductance-based models: a (1) compartment model and a (2) compartment with realistic slow intrinsic conductances. We examine the effects of varying the relative contributions of non-fluctuating intrinsic conductance with fluctuating concurrent inhibitory and excitatory synaptic conductance. For given levels of correlation in the synaptic input we find that the magnitude of the membrane fluctuations uniquely determines the relative contribution of synaptic and intrinsic conductance. We also quantify how V(m)-fluctuations vary with synaptic correlations for fixed ratios of synaptic and intrinsic conductance. Interestingly, the levels of V(m) -fluctuations and conductance observed experimentally during functional network activity leave little room for intrinsic conductance to contribute. Even without intrinsic conductances the variance in V(m) -fluctuations can only be explained by a high degree of correlated firing among presynaptic neurons.

Inhibition of motoneurons during the cutaneous silent period in the spinal cord of the turtle.

The transient suppression of motor activity in the spinal cord after a cutaneous stimulus is termed the cutaneous silent period (CSP). It is not known if CSP is due to suppression of the premotor network or direct inhibition of motoneurons. This issue was examined by intracellular recordings from motoneurons in the isolated carapace-spinal cord preparation from adult turtles during rhythmic scratch-like reflex. Electrical stimulation of cutaneous nerves induced CSP-like suppression of motor nerve firing during rhythmic network activity. The stimulus that generated the CSP-like suppression of motor activity evokes a polysynaptic compound synaptic potential in motoneurons and suppressed their firing. This compound synaptic potential was hyperpolarizing near threshold for action potentials and was associated with a substantial increase in conductance during the CSP in the motor pool. These results show that direct postsynaptic inhibition of motoneurons contributes to the CSP.

Voltage fluctuations in neurons: signal or noise?

Noise and variability are fundamental companions to ion channels and synapses and thus inescapable elements of brain function. The overriding unresolved issue is to what extent noise distorts and limits signaling on one hand and at the same time constitutes a crucial and fundamental enrichment that allows and facilitates complex adaptive behavior in an unpredictable world. Here we review the growing experimental evidence that functional network activity is associated with intense fluctuations in membrane potential and spike timing. We trace origins and consequences of noise and variability. Finally, we discuss noise-free neuronal signaling and detrimental and beneficial forms of noise in large-scale functional neural networks. Evidence that noise and variability in some cases go hand in hand with behavioral variability and increase behavioral choice, richness, and adaptability opens new avenues for future studies.

Motoneuron membrane potentials follow a time inhomogeneous jump diffusion process.

Stochastic leaky integrate-and-fire models are popular due to their simplicity and statistical tractability. They have been widely applied to gain understanding of the underlying mechanisms for spike timing in neurons, and have served as building blocks for more elaborate models. Especially the Ornstein-Uhlenbeck process is popular to describe the stochastic fluctuations in the membrane potential of a neuron, but also other models like the square-root model or models with a non-linear drift are sometimes applied. Data that can be described by such models have to be stationary and thus, the simple models can only be applied over short time windows. However, experimental data show varying time constants, state dependent noise, a graded firing threshold and time-inhomogeneous input. In the present study we build a jump diffusion model that incorporates these features, and introduce a firing mechanism with a state dependent intensity. In addition, we suggest statistical methods to estimate all unknown quantities and apply these to analyze turtle motoneuron membrane potentials. Finally, simulated and real data are compared and discussed. We find that a square-root diffusion describes the data much better than an Ornstein-Uhlenbeck process with constant diffusion coefficient. Further, the membrane time constant decreases with increasing depolarization, as expected from the increase in synaptic conductance. The network activity, which the neuron is exposed to, can be reasonably estimated to be a threshold version of the nerve output from the network. Moreover, the spiking characteristics are well described by a Poisson spike train with an intensity depending exponentially on the membrane potential.

Stereological estimate of the total number of neurons in spinal segment D9 of the red-eared turtle.

The red-eared turtle is an important animal model for investigating the neural activity in the spinal circuit that generates motor behavior. However, basic anatomical features, including the number of neurons in the spinal segments involved, are unknown. In the present study, we estimate the total number of neurons in segment D9 of the spinal cord in the red-eared turtle (Trachemys scripta elegans) using stereological cell counting methods. In transverse spinal cord sections stained with modified Giemsa, motoneurons (MNs), interneurons (INs), and non-neuronal cells were distinguished according to location and morphology. Each cell type was then counted separately using an optical disector with the cell nucleus as counting item. The number of cells in segment D9 was as follows (mean ± SE): MNs, 2049 ± 74; INs, 16,135 ± 316; non-neuronal cells, 47,504 ± 478 (n = 6). These results provide the first estimate of the total number of neurons in a spinal segment in a terrestrial vertebrate based on unbiased stereological methods and an upper bound on the number of neurons involved in segmental sensorimotor activity. These findings also form a crucial quantitative foundation for integrating electrophysiological data into mathematical circuit models.

Influence of phasic and tonic dopamine release on receptor activation.

Tonic and phasic dopamine release is implicated in learning, motivation, and motor functions. However, the relationship between spike patterns in dopaminergic neurons, the extracellular concentration of dopamine, and activation of dopamine receptors remains unresolved. In the present study, we develop a computational model of dopamine signaling that give insight into the relationship between the dynamics of release and occupancy of D(1) and D(2) receptors. The model is derived from first principles using experimental data. It has no free parameters and offers unbiased estimation of the boundaries of dopaminergic volume transmission. Bursts primarily increase occupancy of D(1) receptors, whereas pauses translate into low occupancy of D(1) and D(2) receptors. Phasic firing patterns, composed of bursts and pauses, reduce the average D(2) receptor occupancy and increase average D(1) receptor occupancy compared with equivalent tonic firing. Receptor occupancy is crucially dependent on synchrony and the balance between tonic and phasic firing modes. Our results provide quantitative insight in the dynamics of volume transmission and complement experimental data obtained with electrophysiology, positron emission tomography, microdialysis, amperometry, and voltammetry.

Signaling in large-scale neural networks.

We examine the recent finding that neurons in spinal motor circuits enter a high conductance state during functional network activity. The underlying concomitant increase in random inhibitory and excitatory synaptic activity leads to stochastic signal processing. The possible advantages of this metabolically costly organization are analyzed by comparing with synaptically less intense networks driven by the intrinsic response properties of the network neurons.

Intense synaptic activity enhances temporal resolution in spinal motoneurons.

In neurons, spike timing is determined by integration of synaptic potentials in delicate concert with intrinsic properties. Although the integration time is functionally crucial, it remains elusive during network activity. While mechanisms of rapid processing are well documented in sensory systems, agility in motor systems has received little attention. Here we analyze how intense synaptic activity affects integration time in spinal motoneurons during functional motor activity and report a 10-fold decrease. As a result, action potentials can only be predicted from the membrane potential within 10 ms of their occurrence and detected for less than 10 ms after their occurrence. Being shorter than the average inter-spike interval, the AHP has little effect on integration time and spike timing, which instead is entirely determined by fluctuations in membrane potential caused by the barrage of inhibitory and excitatory synaptic activity. By shortening the effective integration time, this intense synaptic input may serve to facilitate the generation of rapid changes in movements.

Organization of projection-specific interneurons in the spinal cord of the red-eared turtle.

Using differential retrograde axonal tracing, we identified motoneurons (MNs) and projection-specific interneuron (IN) classes in lumbar segment D9 of the adult red-eared turtle spinal cord. We characterized the distribution of these neurons in the transverse plane, and estimated their numbers and proportions. Different labeling paradigms allowed us to distinguish ipsilateral INs (IINs) from commissural INs (CINs), and to identify IINs and CINs with either ascending (a) axons, descending (d) axons, or axons that bifurcate to both ascend and descend (ad). Local interneurons with axons shorter than 1 segment in length were not studied. We show that most retrogradely labeled INs are located dorsal to the MNs, in the ventral horn, the intermediate zone and the dorsal horn. IINs predominate in the dorsal horn. CINs are located on average more medially than the IINs in the ventral horn and intermediate zone. Within the IIN and CIN populations, aINs and dINs overlap extensively. The adIINs and adCINs make up only a small fraction of the total number of INs and are scattered throughout much of the respective IIN and CIN domains. The proportions of IINs and CINs are about equal, as are the proportions of aIINs versus dIINs, of aCINs versus dCINs, and of adIINs versus adCINs. The findings are compared to the organization of lumbar spinal INs in other vertebrate species.

Balanced inhibition and excitation drive spike activity in spinal half-centers.

Many limb movements are composed of alternating flexions and extensions. However, the underlying spinal network mechanisms remain poorly defined. Here, we show that the intensity of synaptic excitation and inhibition in limb motoneurons varies in phase rather than out of phase during rhythmic scratchlike network activity in the turtle. Inhibition and excitation peak with the total neuron conductance during the depolarizing waves of scratch episodes. Furthermore, spike activity is driven by depolarizing synaptic transients rather than pacemaker properties. These findings show that balanced excitation and inhibition and irregular firing are fundamental motifs in certain spinal network functions.

Heterosynaptic modulation of the dorsal root potential in the turtle spinal cord in vitro.

In the somatosensory system, the flow of sensory information is regulated at early stages by presynaptic inhibition. Recent findings have shown that the mechanisms generating the primary afferent depolarization (PAD) associated with presynaptic inhibition are complex, with some components mediated by a non-spiking mechanism. How sensory inputs carried by neighbouring afferent fibres interact to regulate the generation of PAD, and thus presynaptic inhibition, is poorly known. Here, we investigated the interaction between neighbouring primary afferents for the generation of PAD in an in vitro preparation of the turtle spinal cord. To monitor PAD we recorded the dorsal root potential (DRP), while the simultaneous cord dorsum potential (CDP) was recorded to assess the population postsynaptic response. We found that the DRP and the CDP evoked by a primary afferent test stimulus was greatly reduced by a conditioning activation of neighbouring primary afferents. This depression had early and late components, mediated in part by GABAA and GABAB receptors, since they were reduced by bicuculline and SCH 50911 respectively. However, with the selective stimulation of C and Adelta fibres in the presence of TTX, the early and late depression of the DRP was replaced by facilitation of the GABAergic and glutamatergic components of the TTX-resistant DRP. Our findings suggest a subtle lateral excitatory interaction between primary afferents for the generation of PAD mediated by a non-spiking mechanism that may contribute to shaping of information transmitted by C and Adelta fibres in a spatially confined scale in analogy with the retina and olfactory bulb.