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Summary Objective We aimed to investigate mechano-sensitivity at the afferent nerve fibers projecting to degenerated intervertebral disc (IVD) and nociceptive behaviour in a rat model of low back pain (LBP). Design Animal model with LBP was established by lumbar 4/5 IVD puncture and nucleus pulposus aspiration. In vivo single nerve recordings (n = 121) were introduced to measure discharge frequency at the afferent nerve fiber innervating the IVD during mechanical stimulations (von Frey filament or intradiscal pressure). Nerve growth factor (NGF) expression levels in the IVD (n = 20) were assessed by Western blot. LBP-related behaviour (n = 22) was assessed by measuring changes in rearing, mechanical paw-withdrawal threshold, and dynamic weight bearing in a freely walking rat. Inhibitory effect of morphine on the neuronal excitability (n = 19) and painful behaviour (n = 28) was also assessed. Results Compared to those with sham or naive IVD, animal group with degenerated IVD displayed the sensitized neuronal responses and painful behaviour, with hyperexcitability of the afferent nerve fibers in any range of mechanical stimulations (von Frey filament stimulation; 1, 2, and 26 g; intradiscal pressure, 1,500–3,000 mm Hg), strong upregulation of NGF (200–250 % increase), and LBP-like behaviour such as failure of rearing, front limbs-dependent walking pattern, and hypersensitivity in hind-paws. However, the neuronal hyperexcitability and pain behaviour were attenuated after local (30 μM) or systemic (3 mg kg−1) morphine administration. Conclusions Our study suggests that enhanced mechano-sensitivity at the afferent nerve fiber innervating degenerated IVD is deeply correlated with LBP development, which supports the hypothesis that hyperexcited responses at the nerve fibers represent a decisive source of LBP.

The thoracic paravertebral sympathetic chain postganglionic neurons (tSPNs) represent the predominant sympathetic control of vascular function in the trunk and upper extremities. tSPNs cluster to form ganglia linked by an interganglionic nerve and receive multisegmental convergent and divergent synaptic input from cholinergic sympathetic preganglionic neurons of the spinal cord (Blackman and Purves, 1969; Lichtman et al., 1980 ). Studies in the past have focused on cervical and lumbar chain ganglia in multiple species, but few have examined the thoracic chain ganglia, whose location and diminutive size make them less conducive to experimentation. Seminal studies on the integrative properties of preganglionic axonal projections onto tSPNs were performed in guinea pig (Blackman and Purves, 1969; Lichtman et al., 1980 ), but as mice have become the accepted mammalian genetic model organism, there is need to reproduce and expand on these studies in this smaller model. We describe an ex vivo approach that enables electrophysiological, calcium imaging, and optogenetic assessment of convergence, divergence, and studies on pre- to postganglionic synaptic transmission, as well as whole-cell recordings from individual tSPNs. Preganglionic axonal connections from intact ventral roots and interganglionic nerves across multiple segments can be stimulated to evoke compound action potential responses in individual thoracic ganglia as recorded with suction electrodes. Chemical block of synaptic transmission differentiates spiking of preganglionic axons from synaptically-recruited tSPNs. Further dissection, including removal of the sympathetic chain, enables whole-cell patch clamp recordings from individual tSPNs for characterization of cellular and synaptic properties.

Somatosensory input strength can be modulated by primary afferent depolarization (PAD) generated predominantly via presynaptic GABAA receptors on afferent terminals. We investigated whether ionotropic nicotinic acetylcholine receptors (nAChRs) also provide modulatory actions, focusing on myelinated afferent excitability in in vitro murine spinal cord nerve-attached models. Primary afferent stimulation-evoked synaptic transmission was recorded in the deep dorsal horn as extracellular field potentials (EFPs), whereas concurrently recorded dorsal root potentials (DRPs) were used as an indirect measure of PAD. Changes in afferent membrane excitability were simultaneously measured as direct current (DC)-shifts in membrane polarization recorded in dorsal roots or peripheral nerves. The broad nAChR antagonist d-tubocurarine (d-TC) selectively and strongly depressed Aδ-evoked synaptic EFPs (36% of control) coincident with similarly depressed A-fiber DRP (43% of control), whereas afferent electrical excitability remained unchanged. In comparison, acetylcholine (ACh) and the nAChR agonists, epibatidine and nicotine, reduced afferent excitability by generating coincident depolarizing DC-shifts in peripheral axons and intraspinally. Progressive depolarization corresponded temporally with the emergence of spontaneous axonal spiking and reductions in the DRP and all afferent-evoked synaptic actions (31%–37% of control). Loss of evoked response was long-lasting, independent of DC repolarization, and likely due to mechanisms initiated by spontaneous C-fiber activity. DC-shifts were blocked with d-TC but not GABAA receptor blockers and retained after tetrodotoxin block of voltage-gated Na+ channels. Notably, actions tested were comparable between three mouse strains, in rat, and when performed in different labs. Thus, nAChRs can regulate afferent excitability via two distinct mechanisms: by central Aδ-afferent actions, and by transient extrasynaptic axonal activation of high-threshold primary afferents. NEW & NOTEWORTHY Primary afferents express many nicotinic ACh receptor (nAChR) subtypes but whether activation is linked to presynaptic inhibition, facilitation, or more complex and selective activity modulation is unknown. Recordings of afferent-evoked responses in the lumbar spinal cord identified two nAChR-mediated modulatory actions: 1) selective control of Aδ afferent transmission and 2) robust changes in axonal excitability initiated via extrasynaptic shifts in DC polarization. This work broadens the diversity of presynaptic modulation of primary afferents by nAChRs.

Somatosensory information can be modulated at the spinal cord level by primary afferent depolarization (PAD), known to produce presynaptic inhibition (PSI) by decreasing neurotransmitter release through the activation of presynaptic ionotropic receptors. Descending monoaminergic systems also modulate somatosensory processing. We investigated the role of D(1)-like and D(2)-like receptors on pathways mediating PAD in the hemisected spinal cord of neonatal mice. We recorded low-threshold evoked dorsal root potentials (DRPs) and population monosynaptic responses as extracellular field potentials (EFPs). We used a paired-pulse conditioning-test protocol to assess homosynaptic and heterosynaptic depression of evoked EFPs to discriminate between dopaminergic effects on afferent synaptic efficacy and/or on pathways mediating PAD, respectively. DA (10 μM) depressed low-threshold evoked DRPs by 43%, with no effect on EFPs. These depressant effects on DRPs were mimicked by the D(2)-like receptor agonist quinpirole (35%). Moreover, by using selective antagonists at D(2)-like receptors (encompassing the D(2), D(3), and D(4) subtypes), we found that the D(2) and D(3) receptor subtypes participate in the quinpirole depressant inhibitory effects of pathways mediating PAD.

Study Objective Validate a novel method for sleep-wake staging in mice using noninvasive electric field (EF) sensors. Methods Mice were implanted with electroencephalogram (EEG) and electromyogram (EMG) electrodes and housed individually. Noninvasive EF sensors were attached to the exterior of each chamber to record respiration and other movement simultaneously with EEG, EMG, and video. A sleep-wake scoring method based on EF sensor data was developed with reference to EEG/EMG and then validated by three expert scorers. Additionally, novice scorers without sleep-wake scoring experience were self-trained to score sleep using only the EF sensor data, and results were compared to those from expert scorers. Lastly, ability to capture three-state sleep-wake staging with EF sensors attached to traditional mouse home-cages was tested. Results EF sensors quantified wake, rapid eye movement (REM) sleep, and non-REM sleep with high agreement (>93%) and comparable inter- and intra-scorer error as EEG/EMG. Novice scorers successfully learned sleep-wake scoring using only EF sensor data and scoring criteria, and achieved high agreement with expert scorers (>91%). When applied to traditional home-cages, EF sensors enabled classification of three-state (wake, NREM and REM) sleep-wake independent of EEG/EMG. Conclusions EF sensors score three-state sleep-wake architecture with high agreement to conventional EEG/EMG sleep-wake scoring 1) without invasive surgery, 2) from outside the home-cage, and 3) and without requiring specialized training or equipment. EF sensors provide an alternative method to assess rodent sleep for animal models and research laboratories in which EEG/EMG is not possible or where noninvasive approaches are preferred.

Mapping the expression of transcription factors in the mouse spinal cord has identified ten progenitor domains, four of which are cardinal classes of molecularly defined, ventrally located interneurons that are integrated in the locomotor circuitry. This review focuses on the properties of these interneuronal populations and their contribution to hindlimb locomotor central pattern generation. Interneuronal populations are categorized based on their excitatory or inhibitory functions and their axonal projections as predictors of their role in locomotor rhythm generation and coordination. The synaptic connectivity and functions of these interneurons in the locomotor central pattern generators (CPGs) have been assessed by correlating their activity patterns with motor output responses to rhythmogenic neurochemicals and sensory and descending fibers stimulations as well as analyzing kinematic gait patterns in adult mice. The observed complex organization of interneurons in the locomotor CPG circuitry, some with seemingly similar physiological functions, reflects the intricate repertoire associated with mammalian motor control and is consistent with high transcriptional heterogeneity arising from cardinal interneuronal classes. This review discusses insights derived from recent studies to describe innovative approaches and limitations in experimental model systems and to identify missing links in current investigational enterprise.

Somatosensory afferent transmission strength is controlled by several presynaptic mechanisms that reduce transmitter release at the spinal cord level. We focused this investigation on the role of α-adrenoceptors in modulating sensory transmission in low-threshold myelinated afferents and in pathways mediating primary afferent depolarization (PAD) of neonatal mouse spinal cord. We hypothesized that the activation of α-adrenoceptors depresses low threshold-evoked synaptic transmission and inhibits pathways mediating PAD. Extracellular field potentials (EFPs) recorded in the deep dorsal horn assessed adrenergic modulation of population monosynaptic transmission, while dorsal root potentials (DRPs) recorded at root entry zone assessed adrenergic modulation of PAD. We found that noradrenaline (NA) and the α1-adrenoceptor agonists phenylephrine and cirazoline depressed synaptic transmission (by 15, 14 and 22%, respectively). DRPs were also depressed by NA, phenylephrine and cirazoline (by 62, 30, and 64%, respectively), and by the α2-adrenoceptor agonist clonidine, although to a lower extent (20%). We conclude that NA depresses monosynaptic transmission of myelinated afferents onto deep dorsal horn neurons via α1-adrenoceptors and inhibits interneuronal pathways mediating PAD through the activation of α1- and α2-adrenoceptors. The functional significance of these modulatory actions in shaping cutaneous and muscle sensory information during motor behaviors requires further study.

BackgroundRodent sleep scoring in principally reliant on electroencephalogram (EEG) and electromyogram (EMG), but this approach is invasive, can be expensive, and requires expertise and specialized equipment. Affordable, simple to use, and noninvasive ways to accurately quantify rodent sleep are needed.New methodWe developed and validated a new method for sleep-wake staging in mice using cost-effective, noninvasive electric field (EF) sensors that detect respiration and other movements. We validated recordings from EF sensors attached to the exterior of specialty chambers used to continuously capture sleep with EEG/EMG, then compared this to EF sensors attached to vivarium home-cages.ResultsEF sensors quantified 3-state sleep architecture (wake, rapid eye movement – REM – sleep, and non-REM sleep) with high agreement (>93%) and comparable inter- and intra-scorer error as expert EEG/EMG scoring. Novices given an instruction document with examples were able to score sleep comparable to expert scorers (>91% agreement). Additionally, EF sensors were able to quantify 3-state sleep scoring in traditional mouse home cages.Comparison with existing methodMost noninvasive sleep assessment technology requires animal contact, altered cage environments, and/or can only discern 2 states of arousal (wake or asleep). The EF sensors are able to discriminate REM from non-REM sleep accurately and from outside the animal’s home cage.ConclusionsEF sensors provide a simple and reliable method to accurately score 3-state sleep architecture; (i) from outside the typical home cage, (ii) where noninvasive approaches are preferred, or (iii) which EEG/EMG is not possible.Graphical Abstract

Spinal cord stimulation (SCS) is used clinically to limit chronic pain, but fundamental questions remain on the identity of axonal populations recruited. We developed an ex vivo adult mouse spinal cord preparation to assess recruitment following delivery of clinically analogous stimuli determined by downscaling a finite element model of clinical SCS. Analogous electric field distributions were generated with 300-µm × 300-µm electrodes positioned 200 µm above the dorsal column (DC) with stimulation between 50 and 200 µA. We compared axonal recruitment using electrodes of comparable size and stimulus amplitudes when contacting the caudal thoracic DC and at 200 or 600 μm above. Antidromic responses recorded distally from the DC, the adjacent Lissauer tract (LT), and in dorsal roots (DRs) were found to be amplitude and site dependent. Responses in the DC included a unique component not seen in DRs, having the lowest SCS recruitment amplitude and fastest conduction velocity. At 200 μm above, mean cathodic SCS recruitment threshold for axons in DRs and LT were 2.6 and 4.4 times higher, respectively, than DC threshold. SCS recruited primary afferents in all (up to 8) caudal segments sampled. Whereas A and C fibers could be recruited at nearby segments, only A fiber recruitment and synaptically mediated dorsal root reflexes were observed in more distant (lumbar) segments. In sum, clinically analogous SCS led to multisegmental recruitment of several somatosensory-encoding axonal populations. Most striking is the possibility that the lowest threshold recruitment of a nonprimary afferent population in the DC are postsynaptic dorsal column tract cells (PSDCs) projecting to gracile nuclei.NEW & NOTEWORTHY Spinal cord stimulation (SCS) is used clinically to control pain. To identify axonal populations recruited, finite element modeling identified scaling parameters to deliver clinically analogous SCS in an ex vivo adult mouse spinal cord preparation. Results showed that SCS first recruited an axonal population in the dorsal column at a threshold severalfold lower than primary afferents. These putative postsynaptic dorsal column tract cells may represent a previously unconsidered population responsible for SCS-induced paresthesias necessary for analgesia.

Unobtrusive monitoring of physio-behavioral variables from animals can minimize variability in preclinical research and thereby maximize the potential for clinical translation. In this paper, we present the design, implementation, and validation of an instrumented nest providing continuous recordings of seismocardiogram (SCG) signals and skin temperature. SCG represents the chest-wall vibrations associated with the heartbeat, and can potentially provide a measure by which individual heartbeats can be detected without the need for electrodes or implantable devices. A non-contact electric field sensor placed in proximity to the animal in the nest was also used to detect respiratory dynamics. The setup was tested with a total of six anesthetized mice. To understand the effects of mouse positioning within the nest on signal quality, the error in heartbeat detection at different positions of the sensor on the body was quantified, with a simultaneously-obtained electrocardiogram (ECG) as the reference standard. At the optimal placement determined with this approach, multiple perturbations were performed such as pinching, changing ambient temperature, and norepinephrine injection to modulate physiology and assess measurement capability. Heartbeat intervals obtained from the ECG and SCG during the perturbations were correlated (R2=0.82) and were in agreement according to Bland-Altman methods (bias: 0.006ms, 95% confidence interval: [-3.79, 3.78]ms) suggesting that SCG can be reliably used for unobtrusive heartbeat detection. Accordingly, the setup can provide a means by which individual heartbeats – and thereby heart rate and heart rate variability indices – can be quantified without the need for any sensors to be attached to the body of the animal.

In humans, exercises involving slowed respiratory rate (SRR) counter autonomic sympathetic bias and reduce responses to stressors, including in individuals with various degrees of autonomic dysfunction. In the rat, we examined whether operant conditioning could lead to reductions in respiratory rate (RR) and performed preliminary studies to assess whether conditioned SRR was sufficient to decrease physiological and behavioral responsiveness to stressors. RR was continuously monitored during 20 2-h sessions using whole body plethysmography. SRR conditioned, but not yoked control rats, were able to turn off aversive visual stimulation (intermittent bright light) by slowing their breathing below a preset target of 80 breaths/min. SRR conditioned rats greatly increased the incidence of breaths below the target RR over training, with average resting RR decreasing from 92 to 81 breaths/min. These effects were significant as a group and vs. yoked controls. Preliminary studies in a subset of conditioned rats revealed behavioral changes suggestive of reduced reactivity to stressful and nociceptive stimuli. In these same rats, intermittent sessions without visual reinforcement and a post-training priming stressor (acute restraint) demonstrated that conditioned rats retained reduced RR vs. controls in the absence of conditioning. In conclusion, we present the first successful attempt to operantly condition reduced RR in an animal model. Although further studies are needed to clarify the physio-behavioral concomitants of slowed breathing, the developed model may aid subsequent neurophysiological inquiries on the role of slow breathing in stress reduction.

Thoracic paravertebral sympathetic postganglionic neurons (tSPNs) comprise the final integrative output of the distributed sympathetic nervous system controlling vascular and thermoregulatory systems. Considered a non-integrating relay, what little is known of tSPN intrinsic excitability has been determined by sharp microelectrodes with presumed impalement injury. We thus undertook the first electrophysiological characterization of tSPN cellular properties using whole-cell recordings and coupled results with a conductance-based model to explore the principles governing their excitability in adult mice of both sexes. Recorded membrane resistance and time constant values were an order of magnitude greater than values previously obtained, leading to a demonstrable capacity for synaptic integration in driving recruitment. Variation in membrane resistivity was the primary determinant controlling cell excitability with vastly lower currents required for tSPN recruitment. Unlike previous microelectrode recordings in mouse which observed inability to sustain firing, all tSPNs were capable of repetitive firing. Computational modeling demonstrated that observed differences are explained by introduction of a microelectrode impalement injury conductance. Overall, tSPNs largely linearly encoded injected current magnitudes over a broad frequency range with distinct subpopulations differentiable based on repetitive firing signatures. Thus, whole-cell recordings reveal tSPNs have more dramatically amplified excitability than previously thought, with greater intrinsic capacity for synaptic integration and with the ability for maintained firing to support sustained actions on vasomotor tone and thermoregulatory function. Rather than acting as a relay, these studies support a more responsive role and possible intrinsic capacity for tSPNs to drive sympathetic autonomic function.

Afferent feedback alters muscle activity during locomotion and must be tightly controlled. As primary afferent depolarization-induced presynaptic inhibition (PAD-PSI) regulates afferent signaling, we investigated hindlimb PAD-PSI during locomotion in an in vitro rat spinal cord-hindlimb preparation. We compared the relation of PAD-PSI, measured as dorsal root potentials (DRPs), to observed ipsilateral and contralateral limb endpoint forces. Afferents activated during stance-phase force strongly and proportionately influenced DRP magnitude in the swinging limb. Responses increased with locomotor frequency. Electrical stimulation of contralateral afferents also preferentially evoked DRPs in the opposite limb during swing (flexion). Nerve lesioning, in conjunction with kinematic results, support a prominent contribution from toe Golgi tendon organ afferents. Thus, force-dependent afferent feedback during stance binds interlimb sensorimotor state to a proportional PAD-PSI in the swinging limb, presumably to optimize interlimb coordination. These results complement known actions of ipsilateral afferents on PAD-PSI during locomotion.

Background Numerous environmental and genetic factors can contribute significantly to behavioral and cardiorespiratory variability observed experimentally. Affordable technologies that allow for noninvasive home cage capture of physio-behavioral variables should enhance understanding of inter-animal variability including after experimental interventions. New method We assessed whether EPIC electric field sensors (Plessey Semiconductors) embedded within or attached externally to a rodent’s home cage could accurately record respiration, heart rate, and motor behaviors. Comparison with existing methods Current systems for quantification of behavioral variables require expensive specialty equipment, while measures of respiratory and heart rate are often provided by surgically implanted or chronically affixed devices. Results Sensors accurately encoded imposed sinusoidal changes in electric field tested at frequencies ranging from 0.5–100 Hz. Mini-metronome arm movements were easily detected, but response magnitude was highly distance dependent. Sensors accurately reported respiration during whole-body plethysmography. In anesthetized rodents, PVC tube-embedded sensors provided accurate mechanical detection of both respiratory and heart rate. Comparable success was seen in naturally behaving animals at rest or sleeping when sensors were attached externally. Video-verified motor behaviors (sniffing, grooming, chewing, and rearing) were detectable and largely separable by their characteristic voltage fluctuations. Larger movement-related events had comparably larger voltage dynamics that easily allowed for a broad approximation of overall motor activity. Spectrograms were used to quickly depict characteristic frequencies in long-lasting recordings, while filtering and thresholding software allowed for detection and quantification of movement-related physio-behavioral events. Conclusions EPIC electric field sensors provide a means for affordable non-contact home cage detection of physio-behavioral variables.

Presynaptic inhibition is a powerful mechanism for selectively and dynamically gating sensory inputs entering the spinal cord. We investigated how hindlimb mechanics influence presynaptic inhibition during locomotion using pioneering approaches in an in vitro spinal cord–hindlimb preparation. We recorded lumbar dorsal root potentials to measure primary afferent depolarization-mediated presynaptic inhibition and compared their dependence on hindlimb endpoint forces, motor output, and joint kinematics. We found that stance-phase force on the opposite limb, particularly at toe contact, strongly influenced the magnitude and timing of afferent presynaptic inhibition in the swinging limb. Presynaptic inhibition increased in proportion to opposite limb force, as well as locomotor frequency. This form of presynaptic inhibition binds the sensorimotor states of the two limbs, adjusting sensory inflow to the swing limb based on forces generated by the stance limb. Functionally, it may serve to adjust swing-phase sensory transmission based on locomotor task, speed, and step-to-step environmental perturbations.

Understanding how sensory feedback can be used to facilitate locomotion is vital for designing effective locomotor rehabilitation strategies for spinal cord injury. Here we describe an in vitro spinal cord-hindlimb preparation (SCHP) that can be used to answer clinically relevant questions about the interaction of sensory feedback and spinal locomotor circuitry. The SCHP, composed of the exposed rodent spinal cord with hindlimbs intact, combines the neural accessibility and manipulability of classic in vitro isolated cord preparations with intact sensory feedback and many of the behavioral analysis techniques used in the clinic, such as kinematics, treadmill walking, and electromyography. Using the SCHP, we demonstrate the importance of task-appropriate limb loading and posture for establishing intact rat-like kinematic trajectories and muscle activation patterns. We also show that applying stepping-like movements to the limbs, similar to those applied during physical therapist-assisted or robot-guided bo...

Restless legs syndrome (RLS) is a common sensorimotor disorder characterized by abnormal sensations that occur primarily at rest or during sleep, which are alleviated by movement of the affected limb. The pathophysiology of RLS remains unclear, although roles for dopamine dysfunction and brain iron deficiency have been proposed. The hypothalamic A11 dopaminergic circuit is used to explain the dopamine dysfunction in RLS and the potential therapeutic actions of dopamine D2 agonists. Modulation of central and peripher- al neuronal circuits may also explain the potential therapeutic sites of action of opioids, adenosine receptor ligands, and voltage-gated calcium channel α2δ ligands in RLS. The known and possible therapeutic benefits of these agents and their relationship to dopaminergic dysfunction in RLS are discussed in this review.

Primary afferent neurotransmission is the fundamental first step in the central processing of sensory stimuli and is controlled by pre- and postsynaptic inhibitory mechanisms. Presynaptic inhibition (PSI) is probably the more powerful form of inhibitory control in all primary afferent fibers. A major mechanism producing afferent PSI is via a channel-mediated depolarization of their intraspinal terminals, which can be recorded extracellularly as a dorsal root potential (DRP). Based on measures of DRP latency it has been inferred that this primary afferent depolarization (PAD) of low-threshold afferents is mediated by minimally trisynaptic pathways with pharmacologically identified GABAergic interneurons forming last-order axo-axonic synapses onto afferent terminals. There is still no "squeaky clean" evidence of this organization. This paper describes recent and historical work that supports the existence of PAD occurring by more direct pathways and with a complex pharmacology that questions the proprietary role of GABA and GABA(A) receptors in this process. Cholinergic transmission in particular may contribute significantly to PAD, including via direct release from primary afferents.

Primary afferent neurotransmission is the fundamental first step in the central processing of sensory stimuli. A major mechanism producing afferent presynaptic inhibition is via a channel-mediated depolarization of their intraspinal terminals which can be recorded extracellularly as a dorsal root potential (DRP). Based on measures of DRP latency it has been inferred that this primary afferent depolarization (PAD) of low-threshold afferents is mediated by minimally trisynaptic pathways with GABAergic interneurons forming last-order axoaxonic synapses onto afferent terminals. We used anin vitrorat spinal cord preparation under conditions that restrict synaptic transmission to test whether more direct low-threshold pathways can produce PAD. Mephenesin or high divalent cation solutions were used to limit oligosynaptic transmission. Recordings of synaptic currents in dorsal horn neurons and population synaptic potentials in ventral roots provided evidence that conventional transmission was chiefly restricted to monosynaptic actions. Under these conditions, DRP amplitude was largely unchanged but with faster time to peak and reduced duration. Similar results were obtained following stimulation of peripheral nerves. Even following near complete block of transmission with high Mg2+/low Ca2+-containing solution, the evoked DRP was reduced but not blocked. In comparison, in nominally Ca2+-free or EGTA-containing solution, the DRP was completely blocked confirming that Ca2+entry mediated synaptic transmission is required for DRP genesis. Overall these results demonstrate that PAD of low-threshold primary afferents can occur by more direct synaptic mechanisms, including the possibility of direct negative-feedback or nonspiking dendroaxonic pathways.

The synthesis enzyme glutamic acid decarboxylase (GAD65 or GAD67) identifies neurons as GABAergic. Recent studies have characterized the physiological properties of spinal cord GABAergic interneurons using lines of GAD67-green fluorescent protein (GFP) transgenic mice. A more complete characterization of their phenotype is required to better understand the role of this population of inhibitory neurons in spinal cord function. Here, we characterize the distribution of lumbar spinal cord GAD67-GFP neurons at postnatal days (P) 0, 7, and 14, and adult based on their co-expression with GABA and determine the molecular phenotype of GAD67-GFP neurons at P14 based on the expression of various neuropeptides, calcium binding proteins, and other markers. At all ages >67% of GFP(+) neurons were also GABA(+). With increasing age; (i) GFP(+) and GABA(+) cell numbers declined, (ii) ventral horn GFP(+) and GABA(+) neurons vanished, and (iii) somatic labeling was reduced while terminal labeling increased. At P14, vasoactive intestinal peptide and bombesin were expressed in approximately 63% and approximately 35% of GFP(+) cells, respectively. Somatostatin was found in a small number of neurons, whereas calcitonin gene-related peptide never co-localized with GFP. Moderate co-expression was found for all the Ca(2+) binding proteins examined. Notably, most laminae I-II parvalbumin(+) neurons were also GFP(+). Neurogranin, a protein kinase C substrate, was found in approximately 1/2 of GFP(+) cells. Lastly, while only 7% of GFP(+) cells contain nitric oxide synthase (NOS), these cells represent a large fraction of all NOS(+) cells. We conclude that GAD67-GFP neurons represent the majority of spinal GABAergic neurons and that mouse dorsal horn GAD67-GFP(+) neurons comprise a phenotypically diverse population.

Dopamine (DA) acts through five receptor subtypes (D1-D5). We compared expression levels and distribution patterns of all DA mRNA receptors in the spinal cord of wild-type (WT) and loss of function D3 receptor knockout (D3KO) animals. D3 mRNA expression was increased in D3KO, but no D3 receptor protein was associated with cell membranes, supporting the previously reported lack of function. In contrast, mRNA expression levels and distribution patterns of D1, D2, D4, and D5 receptors were similar between WT and D3KO animals. We conclude that D3KO spinal neurons do not compensate for the loss of function of the D3 receptor with changes in the other DA receptor subtypes. This supports use of D3KO animals as a model to provide insight into D3 receptor dysfunction in the spinal cord.

Background & Aims: The isolation and culture of primary enteric neurons is a difficult process and yields a small number of neurons. We developed fetal and postnatal enteric neuronal cell lines using H-2K b -tsA58 transgenic mice (immortomice) that have a temperature-sensitive mutation of the SV40 large tumor antigen gene under the control of an interferon γ–inducible H-2K b promoter element. Methods: Enteric neuronal precursors were isolated from the intestines of E13-mouse fetuses and second day postnatal mice using magnetic immunoselection with a p75NTR antibody. The cells were maintained at the permissive temperature, 33°C, and interferon-γ for 24 or 48 hours, and then transferred to 39°C in the presence of glial cell line–derived neurotrophic factor for 7 days for further differentiation. Neuronal markers were assessed by reverse-transcription polymerase chain reaction, Western blot, and immunocytochemistry. Neuronal function was assessed by transplanting these cells into the colons of Piebald or nNOS −/− mice. Results: Expression analysis of cells showed the presence of neuronal markers peripherin, PGP9.5, HuD, tau, synaptic marker synaptophysin, characteristic receptors of enteric neurons, Ret, and 5-hydroxytryptamine–receptor subtypes at 33°C and 39°C. Nestin, S-100β, and α-smooth muscle actin were expressed minimally at 39°C. Glial cell line–derived neurotrophic factor resulted in increased phosphorylation of Akt in these cells, similar to primary enteric neurons. Transplantation of cells into the piebald or nNOS −/− mice colon improved colonic motility. Conclusions: We have developed novel enteric neuronal cell lines that have neuronal characteristics similar to primary enteric neurons. These cells can help us in understanding newer therapeutic options for Hirschsprung's disease.

Dopamine is a catecholaminergic neuromodulatory transmitter that acts through five molecularly-distinct G protein–coupled receptor subtypes (D1–D5). In the mammalian spinal cord, dopaminergic axon collaterals arise predominantly from the A11 region of the dorsoposterior hypothalamus and project diffusely throughout the spinal neuraxis. Dopaminergic modulatory actions are implicated in sensory, motor and autonomic functions in the spinal cord but the expression properties of the different dopamine receptors in the spinal cord remain incomplete. Here we determined the presence and the regional distribution of all dopamine receptor subtypes in mouse spinal cord cells by means of quantitative real time polymerase chain reaction (PCR) and digoxigenin-label in situ hybridization. Real-time PCR demonstrated that all dopamine receptors are expressed in the spinal cord with strongly dominant D2 receptor expression, including in motoneurons and in the sensory encoding superficial dorsal horn (SDH). Laser capture microdissection (LCM) corroborated the predominance of D2 receptor expression in SDH and motoneurons. In situ hybridization of lumbar cord revealed that expression for all dopamine receptors was largely in the gray matter, including motoneurons, and distributed diffusely in labeled cell subpopulations in most or all laminae. The highest incidence of cellular labeling was observed for D2 and D5 receptors, while the incidence of D1 and D3 receptor expression was least. We conclude that the expression and extensive postsynaptic distribution of all known dopamine receptors in spinal cord correspond well with the broad descending dopaminergic projection territory supporting a widespread dopaminergic control over spinal neuronal systems. The dominant expression of D2 receptors suggests a leading role for these receptors in dopaminergic actions on postsynaptic spinal neurons.

A new, scalable process for microfabrication of a silicone-based, elastic multi-electrode array (MEA) is presented. The device is constructed by spinning poly(dimethylsiloxane) (PDMS) silicone elastomer onto a glass slide, depositing and patterning gold to construct wires and electrodes, spinning on a second PDMS layer, and then micropatterning the second PDMS layer to expose electrode contacts. The micropatterning of PDMS involves a custom reactive ion etch (RIE) process that preserves the underlying gold thin film. Once completed, the device can be removed from the glass slide for conformal interfacing with neural tissue. Prototype MEAs feature electrodes smaller than those known to be reported on silicone substrate (60 microm diameter exposed electrode area) and were capable of selectively stimulating the surface of the in vitro isolated spinal cord of the juvenile rat. Stretchable serpentine traces were also incorporated into the functional PDMS-based MEA, and their implementation and testing is described.

Restless legs syndrome (RLS) involves abnormal limb sensations that diminish with motor activity, worsen at rest, have a circadian peak in expression in the evening and at night, and can severely disrupt sleep. Primary treatment is directed at CNS dopaminergic systems, particularly activation of D(2)-like (D(2), D(3), and D(4)) receptors. Although RLS affects 2% to 15% of the general population, the neural circuitry contributing to RLS remains speculative, and there is currently no accepted animal model to enable detailed mechanistic analyses. Traditional views suggest that RLS arises from supraspinal sources which favor facilitation of the flexor reflex and emergence of the RLS phenotype. The authors forward the hypothesis that RLS reflects a dysfunction of the little-studied dorsoposterior hypothalamic dopaminergic A11 cell group. They assert that, as the sole source of spinal dopamine, reduced drive in this system can lead to spinal network changes wholly consistent with RLS. The authors summarize their recent investigations on spinal cord dopamine dysfunction that rely on lesions centered on A11, and on studies in D(3) receptor knockout (D(3)KO) mice. Excessive locomotor behavior is evident in both sets of animals, and D(3)KO mice exhibit facilitation rather than the expected depression of spinal reflexes in the presence of dopamine as well as a reversal in their circadian expression of the rate-limiting enzyme for dopamine synthesis, tyrosine hydroxylase. Taken together, these findings are consistent with an involvement of spinal dopamine dysfunction in the etiology of RLS, and they argue that the D(3)KO mouse might serve as a relevant animal model to study the underlying mechanisms of RLS.

Spiking activity in motor axons represents the final central coding for muscle contraction. Recurrent collaterals in spinal cord from these same axons are known to offer a negative feedback control of motor output via a class of interposed inhibitory interneurons. Here we demonstrate that, during noradrenergic drive, a previously unknown recurrent excitatory pathway is unmasked and expressed. These excitatory projections are shown to have broad bilateral actions within and between hindlimb spinal segments and can alter ongoing pattern-generating motor behaviors. Thus, motor output strength is controlled via central positive and negative feedback loops, undoubtedly to provide a greater flexibility and dynamic range of control. That this novel function is regulated by a descending neuromodulatory transmitter indicates a conditional recruitment during certain behavioral states as part of the central noradrenergic arousal apparatus.

The trace amines (TAs), tryptamine, tyramine, and β-phenylethylamine, are synthesized from precursor amino acids via aromatic-L-amino acid decarboxylase (AADC). We explored their role in the neuromodulation of neonatal rat spinal cord motor circuits. We first showed that the spinal cord contains the substrates for TA biosynthesis (AADC) and for receptor-mediated actions via trace amine-associated receptors (TAARs) 1 and 4. We next examined the actions of the TAs on motor activity using the in vitro isolated neonatal rat spinal cord. Tyramine and tryptamine most consistently increased motor activity with prominent direct actions on motoneurons. In the presence of N-methyl-D-aspartate, all applied TAs supported expression of a locomotor-like activity (LLA) that was indistinguishable from that ordinarily observed with serotonin, suggesting that the TAs act on common central pattern generating neurons. The TAs also generated distinctive complex rhythms characterized by episodic bouts of LLA. TA actions on locomotor circuits did not require interaction with descending monoaminergic projections since evoked LLA was maintained following block of all Na(+)-dependent monoamine transporters or the vesicular monoamine transporter. Instead, TA (tryptamine and tyramine) actions depended on intracellular uptake via pentamidine-sensitive Na(+)-independent membrane transporters. Requirement for intracellular transport is consistent with the TAs having much slower LLA onset than serotonin and for activation of intracellular TAARs. To test for endogenous actions following biosynthesis, we increased intracellular amino acid levels with cycloheximide. LLA emerged and included distinctive TA-like episodic bouts. In summary, we provided anatomical and functional evidence of the TAs as an intrinsic spinal monoaminergic modulatory system capable of promoting recruitment of locomotor circuits independent of the descending monoamines. These actions support their known sympathomimetic function.

Lamina I is a sensory relay region containing projection cells and local interneurons involved in thermal and nociceptive signaling. These neurons differ in morphology, sensory response modality, and firing characteristics. We examined intrinsic properties of mouse lamina I GABAergic neurons expressing enhanced green fluorescent protein (EGFP). GABAergic neuron identity was confirmed by a high correspondence between GABA immunolabeling and EGFP fluorescence. Morphologies of these EGFP+/GABA+ cells were multipolar (65%), fusiform (31%), and pyramidal (4%). In whole cell recordings, cells fired a single spike (44%), tonically (35%), or an initial burst (21%) in response to current steps, representing a subset of reported lamina I firing properties. Membrane properties of tonic and initial burst cells were indistinguishable and these neurons may represent one functional population because, in individual neurons, their firing patterns could interconvert. Single spike cells were less excitable with lower membrane resistivity and higher rheobase. Most fusiform cells (64%) fired tonically while most multipolar cells (56%) fired single spikes. In summary, lamina I inhibitory interneurons are functionally divisible into at least two major groups both of which presumably function to limit excitatory transmission.

Descending monoaminergic systems modulate spinal cord function, yet spinal dopaminergic actions are poorly understood. Using thein vitrolumbar cord, we studied the effects of dopamine and D2-like receptor ligands on spinal reflexes in wild-type (WT) and D3-receptor knock-out mice (D3KO).Low dopamine levels (1 μm) decreased the monosynaptic “stretch” reflex (MSR) amplitude in WT animals and increased it in D3KO animals. Higher dopamine concentrations (10-100 μm) decreased MSR amplitudes in both groups, but always more strongly in WT. Like low dopamine, the D3receptor agonists pergolide and PD 128907 reduced MSR amplitude in WT but not D3KO mice. Conversely, D3receptor antagonists (GR 103691 and nafadotride) increased the MSR in WT but not in D3KO mice. In comparison, D2-preferring agonists bromocriptine and quinpirole depressed the MSR in both groups. Low dopamine (1-5 μm) also depressed longer-latency (presumably polysynaptic) reflexes in WT but facilitated responses in D3KO mice. Additionally, in some experiments (e.g., during 10 μmdopamine or pergolide in WT), polysynaptic reflexes were facilitated in parallel to MSR depression, demonstrating differential modulatory control of these reflex circuits. Thus, low dopamine activates D3receptors to limit reflex excitability. Moreover, in D3ligand-insensitive mice, excitatory actions are unmasked, functionally converting the modulatory action of dopamine from depression to facilitation.Restless legs syndrome (RLS) is a CNS disorder involving abnormal limb sensations. Because RLS symptoms peak at night when dopamine levels are lowest, are relieved by D3agonists, and likely involve increased reflex excitability, the D3KO mouse putatively explains how impaired D3activity could contribute to this sleep disorder.

Object. One of the current challenges in neurobiology is to ensure that neural precursor cells differentiate into specific neuron types, so that they can be used for transplantation purposes in patients with neuron loss. The goal of this study was to determine if spinal cord precursor cells could differentiate into motor neurons both in culture and following transplantation into a transected sciatic nerve. Methods. In cultures with trophic factors, neurons differentiate from embryonic precursor cells and express motor neuronal markers such as choline acetyltransferase (ChAT), Islet-1, and REG2. Reverse transcription—polymerase chain reaction analysis has also demonstrated the expression of Islet-1 in differentiated cultures. A coculture preparation of neurospheres and skeletal myocytes was used to show the formation of neuromuscular connections between precursor cell—derived neurons and myocytes both immunohistochemically and electrophysiologically. Following various survival intervals, precursor cells transplanted distal to a transection of the sciatic nerve differentiated into neurons expressing the motor neuron markers ChAT and the α11.2 (class C, L-type) voltage-sensitive Ca++ channel subunit. These cells extended axons into the muscle, where they formed cholinergic terminals. Conclusions. These results demonstrate that motor neurons can differentiate from spinal cord neural precursor cells grown in culture as well as following transplantation into a transected peripheral nerve.

The human immunodeficiency virus type 1 (HIV-1) regulatory protein Tat is neurotoxic and may be involved in the neuropathogenesis of HIV-1 dementia, in part via N-methyl-D-aspartate (NMDA) receptor activation. Here, in acutely isolated rat hippocampal neurons, Tat evoked inward currents reversing near 0 mV, with a negative slope conductance region characteristic of NMDA receptor activation. Although the NMDA receptor antagonist ketamine blocked Tat's actions, competitive glutamate- and glycine-binding site antagonists were ineffective (AP-5 and 5,7-dichlorokynurenate, respectively). Evidence for Tat acting at a distinct modulatory site on the NR1 subunit of NMDA receptors was provided by findings that 1 microM Zn(2+) abolished Tat-evoked responses in all neurons tested. Thus, Tat appears to excite neurons via direct activation of the NMDA receptor at an allosteric Zn(2+)-sensitive site.

Because oligodendrocytes and their precursors possess receptors for classical transmitters, and neurotransmitters such as glutamate and noradrenaline can mediate oligodendroglial proliferation and differentiation, it is possible that other neurotransmitters can also exert regulatory roles in oligodendrocyte function. We used mitogen-proliferated multipotent neuroepithelial precursors (neurospheres) and identified oligodendroglia that expressed markers traditionally found in cholinergic neurons. Regardless of culture conditions, there existed a large population of cells that resembled oligodendrocytes morphologically and coexpressed the oligodendrocyte-specific marker galactocerebroside (GalC) and the acetylcholine (ACh)-synthesizing enzyme choline acetyltransferase (ChAT). These cells did not express neuronal markers, and whole-cell recordings from cells with similar morphology displayed only outward currents in response to depolarizing voltage steps, further supporting their oligodendroglial identity. Another cholinergic marker, the vesicular ACh transporter, was also detected in GalC(+) oligodendrocytes. Furthermore, neurospheres cultured in the presence of the cholinergic receptor antagonist atropine showed a decrease in the number of GalC(+) spheres, implicating the muscarinic ACh receptor in oligodendrocyte development. The actions of neurotrophins and ciliary neurotrophic factor (CNTF) on these ChAT(+) oligodendrocytes were examined. Among these, CNTF treatment significantly increased oligodendrocytic process outgrowth. These results demonstrate classical cholinergic neuronal markers in oligodendrocytes as well as an effect of muscarinic receptor blockade on oligodendrocyte differentiation.