Scoring sleep using respiration and movement-based features.

Rules derived from standard Rechtschaffen and Kales criteria were developed to accurately score rodent sleep into wake, rapid eye movement (REM) sleep, and non-REM sleep using movements detected by non-contact electric field (EF) sensors. • Using this method, rodent sleep can be scored using only respiratory and gross body movements as a validated, non-invasive alternative to electrode techniques. • The methodology and rules established for EF sensor-based sleep scoring were easily learned and implemented. • Examples of expert-scored files are included here to help novice scorers self-train to score sleep. Though validated in mice, sleep scoring using respiratory movements has the potential for application in other species and through other movement-based technologies beyond EF sensors.

Noninvasive three-state sleep-wake staging in mice using electric field sensors.

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.

Disc degeneration induces a mechano-sensitization of disc afferent nerve fibers that associates with low back pain.

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 naïve 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.

Phenotypic diversity and expression of GABAergic inhibitory interneurons during postnatal development in lumbar spinal cord of glutamic acid decarboxylase 67-green fluorescent protein mice.

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.

Expression and distribution of all dopamine receptor subtypes (D(1)-D(5)) in the mouse lumbar spinal cord: a real-time polymerase chain reaction and non-autoradiographic in situ hybridization study.

Dopamine is a catecholaminergic neuromodulatory transmitter that acts through five molecularly-distinct G protein-coupled receptor subtypes (D(1)-D(5)). 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 D(2) receptor expression, including in motoneurons and in the sensory encoding superficial dorsal horn (SDH). Laser capture microdissection (LCM) corroborated the predominance of D(2) 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 D(2) and D(5) receptors, while the incidence of D(1) and D(3) 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 D(2) receptors suggests a leading role for these receptors in dopaminergic actions on postsynaptic spinal neurons.

Reversal of the circadian expression of tyrosine-hydroxylase but not nitric oxide synthase levels in the spinal cord of dopamine D3 receptor knockout mice.

Circadian rhythms have been described for numerous transmitter synthesizing enzymes in the brain but rarely in spinal cord. We measured spinal tyrosine-hydroxylase (TH) and nitric oxide synthase (NOS) levels in the thoracic intermediolateral nucleus, the location of sympathetic preganglionic neurons, in male wild type (WT) and dopamine D(3) receptor knockout mice (D(3)KO). TH and NOS levels both displayed circadian patterns in WT and D(3)KO animals with overall reduced TH and increased NOS expression in the D(3)KO mice. The circadian pattern of NOS expression was similar in WT and D(3)KO mice. In contrast, TH expression was inverted in D(3)KO mice, with TH levels consistently lower than in WT throughout the day, but strongly increased temporarily 1 h prior to daylight. TH is the rate-limiting enzyme for the production of dopamine. Spinal dopamine dysfunction is implicated in a sleep disorder called restless legs syndrome (RLS). RLS follows a circadian rhythm and is relieved clinically by dopamine D(3) receptor agonists. Our observations of an altered circadian pattern in spinal dopamine synthesis in D(3)KO animals may provide insight into putative dopaminergic mechanisms contributing to RLS.

Human immunodeficiency virus type 1 Tat protein directly activates neuronal N-methyl-D-aspartate receptors at an allosteric zinc-sensitive site.

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.

Modulatory actions of serotonin, norepinephrine, dopamine, and acetylcholine in spinal cord deep dorsal horn neurons.

The deep dorsal horn represents a major site for the integration of spinal sensory information. The bulbospinal monoamine transmitters, released from serotonergic, noradrenergic, and dopaminergic systems, exert modulatory control over spinal sensory systems as does acetylcholine, an intrinsic spinal cord biogenic amine transmitter. Whole cell recordings of deep dorsal horn neurons in the rat spinal cord slice preparation were used to compare the cellular actions of serotonin, norepinephrine, dopamine, and acetylcholine on dorsal root stimulation-evoked afferent input and membrane cellular properties. In the majority of neurons, evoked excitatory postsynaptic potentials were depressed by the bulbospinal transmitters serotonin, norepinephrine, and dopamine. Although, the three descending transmitters could evoke common actions, in some neurons, individual transmitters evoked opposing actions. In comparison, acetylcholine generally facilitated the evoked responses, particularly the late, presumably N-methyl-D-aspartate receptor-mediated component. None of the transmitters modified neuronal passive membrane properties. In contrast, in response to depolarizing current steps, the biogenic amines significantly increased the number of spikes in 14/19 neurons that originally fired phasically (P < 0.01). Together, these results demonstrate that even though the deep dorsal horn contains many functionally distinct subpopulations of neurons, the bulbospinal monoamine transmitters can act at both synaptic and cellular sites to alter neuronal sensory integrative properties in a rather predictable manner, and clearly distinct from the actions of acetylcholine.

Serotonin 5-HT(2) receptor activation induces a long-lasting amplification of spinal reflex actions in the rat.

1. C-fibre activation induces a long-term potentiation (LTP) in the spinal flexion reflex in mammals, presumably to provide enhanced reflexive protection of damaged tissue from further injury. Descending monoaminergic pathways are thought to depress sensory input but may also amplify spinal reflexes; the mechanisms of this modulation within the spinal cord remain to be elucidated. 2. We used electrical stimulation of primary afferents and recordings of motor output, in the rat lumbar spinal cord maintained in vitro, to demonstrate that serotonin is capable of inducing a long-lasting increase in reflex strength at all ages examined (postnatal days 2-12). 3. Pharmacological analyses indicated an essential requirement for activation of 5-HT(2C) receptors while 5-HT(1A/1B), 5-HT(7) and 5-HT(2A) receptor activation was not required. In addition, primary afferent-evoked synaptic potentials recorded in a subpopulation of laminae III-VI spinal neurons were similarly facilitated by 5-HT. Thus, serotonin receptor-evoked facilitatory actions are complex, and may involve alterations in neuronal properties at both motoneuronal and pre-motoneuronal levels. 4. This study provides the first demonstration of a descending transmitter producing a long-lasting amplification in reflex strength, accomplished by activating a specific serotonin receptor subtype. It is suggested that brain modulatory systems regulate reflex pathways to function within an appropriate range of sensori-motor gain, facilitating reflexes in behavioural situations requiring increased sensory responsiveness.

Neurogenesis in postnatal mouse dorsal root ganglia.

Neurogenesis continues in various regions of the central nervous system (CNS) throughout life. As the mitogen basic fibroblast growth factor (bFGF) can proliferate neuronal precursors of CNS neurons in culture, and is also upregulated within adult dorsal root ganglia following axotomy, it is possible that the postnatal dorsal root ganglia contain bFGF-responsive neuronal precursors. We undertook cell culture of postnatal mouse dorsal root ganglia to demonstrate neurogenesis. Basic FGF induced a cellular proliferative response in dorsal root ganglia cell culture. After 2 weeks in serum-free medium containing bFGF, neurons were rarely observed. However, following removal of bFGF and addition of trophic factors, many cells were observed that morphologically resembled dorsal root ganglia neurons, stained for neuronal markers, and generated action potentials. Furthermore, bromodeoxyuridine, used as a marker of cytogenesis, was detected in neurofilament-160(+) and/or microtubule-associated protein-2(+) cells that morphologically resembled neurons. In addition to bFGF, epidermal growth factor, nerve growth factor, and sonic hedgehog were also capable of generating spherical cell clusters that contained cells that stained for neuronal markers following the addition of trophic factors. These results suggest that early postnatal dorsal root ganglia contain neural precursors that appear to proliferate in response to various factors and can then be induced to differentiate into neurons. In conclusion, the existence of neural precursors and the possibility of neurogenesis in postnatal dorsal root ganglia may provide a greater range of plasticity available to somatosensory systems during growth or following injury, perhaps to replace ineffectual or dying neurons.

Serotonin increases the incidence of primary afferent-evoked long-term depression in rat deep dorsal horn neurons.

5-hydroxytryptamine (5-HT) is released in spinal cord by descending systems that modulate somatosensory transmission and can potently depress primary afferent-evoked synaptic responses in dorsal horn neurons. Since primary afferent activity-induced long-term potentiation (LTP) may contribute to central sensitization of nociception, we studied the effects of 5-HT on the expression of sensory-evoked LTP and long-term depression (LTD) in deep dorsal horn (DDH) neurons. Whole cell, predominantly current clamp, recordings were obtained from DDH neurons in transverse slices of neonatal rat lumbar spinal cord. The effect of 5-HT on dorsal-root stimulation-evoked synaptic responses was tested before, during, or after high-frequency conditioning stimulation (CS). In most cells (80%), 5-HT caused a depression of the naïve synaptic response. Even though 5-HT depressed evoked responses, CS in the presence of 5-HT was not only still capable of inducing LTD but also increased its incidence from 54% in controls to 88% (P < 0.001). Activation of ligands selective for 5-HT(1A/1B) and 5-HT(1B), but not 5-HT(2A/2C) or 5-HT(3) receptors, best reproduced these actions. 5-HT also potently depressed postconditioning synaptic responses regardless of whether the induced plasticity was LTP or LTD. Our results demonstrate that in addition to depressing the amplitude of evoked sensory input, 5-HT can also control the direction of its long-term modifiability, favoring the expression of LTD. These findings demonstrate cellular mechanisms that may contribute to the descending serotonergic control of nociception.

Pharmacological characterization of serotonin receptor subtypes modulating primary afferent input to deep dorsal horn neurons in the neonatal rat.

Spinal cord slices and whole-cell patch clamp recordings were used to investigate the effects of serotonergic receptor ligands on dorsal root-evoked synaptic responses in deep dorsal horn (DDH) neurons of the neonatal rat at postnatal days (P) 3 - 6 and P10 - 14. Bath applied 5-hydroxytryptamine (5-HT) potently depressed synaptic responses in most neurons. Similarly, the 5-HT(1/7) receptor agonist, 5-carboxamidotryptamine (5-CT) depressed synaptic responses. This action was probably mediated by 5-HT(1A) receptor activation, since it occurred in the presence of the 5-HT(7) receptor antagonist clozapine and was not observed in the presence of NAN-190, a 5-HT(1A) receptor antagonist. In the absence of any agonist, 5-HT(1A) receptor antagonists often facilitated synaptic responses, suggesting that there is sufficient endogenous 5-HT to tonically activate 5-HT(1A) receptors. 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), the 5-HT(1A/7) receptor agonist, facilitated synaptic responses, an action probably mediated by 5-HT(7) receptors, since the facilitation could be reversed by subsequent application of the 5-HT(7) receptor antagonist clozapine. Agonists for the 5-HT(1B), 5-HT(2) and 5-HT(3) receptors exerted only modest modulatory actions. A pharmacological analysis of the depression evoked by 5-HT suggested an action partly mediated by 5-HT(1A) receptor activation, since antagonism of the 5-HT(1A) receptor with NAN-190 or WAY-100635 partly reversed 5-HT-evoked depression. In comparison, 5-HT(7) receptor activation could account for much of the 5-HT-evoked facilitation. We conclude that 5-HT is capable of modulating sensory input onto DDH neurons via several receptor subtypes, producing both facilitatory and depressant actions. Also, the actions of most receptor ligands on the evoked responses were similar within the first 2 postnatal weeks.

Diffuse distribution of sulforhodamine-labeled neurons during serotonin-evoked locomotion in the neonatal rat thoracolumbar spinal cord.

The fluorescent dye sulforhodamine-101 undergoes synaptic activity-dependent endocytotic uptake and consequent retrograde transport in presynaptic neurons. We used sulforhodamine to identify thoracolumbar spinal premotor neurons (T11-L6) activated during serotonin (5-HT) -induced hindlimb locomotor-like activity in the in vitro neonatal rat spinal cord preparation. Sulforhodamine labeling required locomotor-like activity because few neurons were labeled unless bath applied 5-HT recruited the locomotor rhythm. In contrast, N-methyl-D-aspartate (NMDA; 5 microM) profoundly increased spinal neuronal labeling irrespective of locomotor activity. The contribution of false-positive activity labeling during locomotion induced by application of NMDA with 5-HT (Kjaerulff et al. [1994] J Physiol (Lond). 478:265-273) necessitated the present re-mapping of sulforhodamine-labeled neurons. During 5-HT-evoked locomotion, the sulforhodamine-labeled neurons were diffusely scattered within the spinal cord with predominant labeling in lamina VII. Motor nuclei (lamina IX) and superficial laminae (I-II) were typically devoid of labeled cells in the isolated spinal cord. However, unilateral labeling of motoneurons was achieved when the ipsilateral hindlimb remained attached, suggesting that uptake in motoneurons requires an intact neuromuscular junction. The rostrocaudal incidence and distribution of labeled neurons was uniform in spinal segments L1-L5, with reduced numbers observed in thoracic and L6 spinal segments. Mean total cell labeling was less than 400 per spinal segment, suggesting recruitment from a very small fraction of the neurons contained within the spinal cord (calculated at < 0.1%). These results are consistent with the limited transfer of locomotor-related synaptic activity (Raastad et al. [1996] Neuron 17:729-738) and severe synaptic fatigue (Lev-Tov and Pinco [1992] J Physiol. 447:149-169; Pinco and Lev-Tov [1993] J Neurophysiol. 70:1151-1158; Fleoter and Lev-Tov [1993] J Neurophysiol. 70:2241-2250) observed in the neonatal rat spinal cord.

Thin slice CNS explants maintained on collagen-coated culture dishes.

We have developed a simple and inexpensive procedure for explant culture termed 'thin slice culture' that relies on the use of thin sections of CNS tissue ( < or = 150 microm) which adhere directly to the bottom of collagen-coated culture dishes (or glass coverslips within culture dishes). Microscopic visualization and tissue oxygenation are enhanced due to the reduced slice thickness, and the reduced volumes of incubation media required lessen the amount of expensive agents used (e.g. growth factors). We show that thin slice cultures of spinal cord, brainstem and hippocampus remain viable for at least several weeks and are suitable for many experimental approaches including time-dependent studies, immunocytochemistry and electrophysiology.

Whole cell recordings of lumbar motoneurons during locomotor-like activity in the in vitro neonatal rat spinal cord.

Whole cell current- and voltage-clamp recordings were obtained from lumbar motoneurons in the isolated neonatal rat spinal cord to characterize the behavior of motoneurons during neurochemically induced locomotor-like activity. Bath application of serotonin (10-100 muM) in combination with N-methyl-D-aspartate (1-12 muM) initially produced tonic membrane depolarization (mean = 26 mV), increased input resistance, decreased rheobase, and increased spike inactivation in response to depolarizing current pulse injections. After the initial tonic depolarization, rhythmic fluctuations of the motoneuron membrane potential (locomotor drive potentials; LDPs) developed that were modulated phasically in association with ventral root discharge. The peak and trough voltage levels of the LDP fluctuated above and below the membrane potential recorded immediately before the onset of rhythmic activity. Similarly, firing frequency was modulated above and below prelocomotion firing rates (in those motoneurons that displayed neurochemically induced tonic firing immediately before the onset of rhythmic activity). These observations are consistent with an alternation between phasic excitatory and inhibitory synaptic drives. The amplitude of LDPs and rhythmic excitatory drive current increased with membrane depolarization from -80 to -40 mV and then decreased with further depolarization, thus displaying nonlinear voltage-dependence. Faster frequency, small amplitude voltage fluctuations were observed superimposed on the depolarized phase of LDPs. In some motoneurons, the trajectory of these superimposed fluctuations was consistent with a synaptic origin, whereas in other cells, the regular sinusoidal appearance of the fluctuations and the occurrence of superimposed plateau potentials were more compatible with the activation of an intrinsic membrane property. One motoneuron displayed exclusively excitatory phasic drive, and another motoneuron was characterized by inhibitory phasic drive alone, during rhythmic activity. These findings are compatible with the concept of a central pattern generator that is capable of delivering both excitatory and inhibitory drive to motoneurons during locomotion. The data also suggest that the rhythmic excitatory and inhibitory outputs of the hypothetical half-center model can be dissociated and operate in isolation.

Neuronal excitatory properties of human immunodeficiency virus type 1 Tat protein.

Neuronal dysfunction and cell death in patients with human immunodeficiency virus type-1 (HIV-1) infection may be mediated by HIV-1 proteins and products released from infected cells. Two HIV-1 proteins, the envelope glycoprotein gp120 and nonstructural protein Tat, are neurotoxic. We have determined the neuroexcitatory properties of HIV-1 tat protein using patch-clamp recording techniques. When fmoles of Tat were applied extracellularly, it elicited dose-dependent depolarizations of human fetal neurons in culture and rat CA1 neurons in slices, both in the absence and presence of tetrodotoxin. These responses were voltage-dependent, reversed at approximately 0 mV, and were significantly increased by repetitive applications with no evidence of desensitization. That these responses to Tat were due to direct actions on neurons was supported by observations that Tat dose-dependently depolarized outside-out patches excised from cultured human neurons. Removal of extracellular Ca2+ decreased the responses both in neurons and membrane patches. This is the first demonstration that an HIV-1 protein can, in the absence of accessory cells, directly excite neurons and leads us to speculate that Tat may be a causative agent in HIV-1 neurotoxicity.

NMDA receptor-mediated oscillatory properties: potential role in rhythm generation in the mammalian spinal cord.

Previous studies have demonstrated that (1) NMDA receptor activation occurs during locomotor network operation in lower and higher vertebrates and (2) NMDA induces active membrane properties that can be expressed as intrinsic voltage fluctuations in cells located in the spinal cord of lower vertebrates, as well as in neurons located in supraspinal regions of the mammalian nervous system. This paper reviews recent data showing that NMDA can induce similar inherent membrane potential behavior in synaptically isolated motoneurons and interneurons in the mammalian (in vitro neonatal rat) spinal cord. These TTX-resistant voltage fluctuations include rhythmic oscillations and plateau potentials, as well as low-frequency long-lasting voltage shifts (LLVSs). 5-HT facilitates the transformation of LLVSs into oscillatory events, and 5-HT receptor antagonists have the reverse effect. In the absence of TTX, locomotor-related rhythmic drive potentials in spinal cord neurons can display nonlinear voltage behavior compatible with NMDA receptor activation, although other voltage-activated conductances are not excluded. Suppression of the nonlinear voltage response associated with NMDA receptor activation, via removal of Mg2+, disrupts locomotor patterns of network activity. The potential role of NMDA receptor activation in the operation of mammalian locomotor networks is discussed in the context of these recent observations.

Membrane properties of deep dorsal horn neurons from neonatal rat spinal cord in vitro.

Whole-cell patch-clamp recordings were undertaken to characterize and compare the membrane properties of deep dorsal horn neurons in transverse slices of rat lumbar spinal cord in two age groups, postnatal days (P) 3-6 and 9-16. In both age groups, significant correlations were observed between membrane time constant and cell resistance and between action potential height and its duration at half-maximal amplitude. Cell resistance and action potential half-width values were lower in the P9-16 age group. Neurons were divided into four categories based on their firing properties in response to intracellular current injection: single spike, phasic firing, repetitive firing, and delayed firing. The distribution of neurons within these categories was similar in both age groups which suggests that the firing properties of deep dorsal horn neurons are functionally differentiated at an early postnatal age.

Primary afferent-evoked synaptic plasticity in deep dorsal horn neurons from neonatal rat spinal cord in vitro.

Whole-cell patch clamp recordings of deep dorsal horn neurons were undertaken in 'thick' transverse slices to demonstrate plasticity of primary afferent-evoked synaptic responses following conditioning stimulation. Synaptic plasticity was observed in neurons throughout the age range examined (postnatal days 3-6 and 9-16) but only long-term depression (LTD) was evocable in older animals (P9-16). Both short- and long-latency synaptic responses could undergo long-term potentiation (LTP) and LTD suggesting that AMPA/kainate and NMDA receptor-evoked responses are modifiable.

A variation of the tissue print technique for studying isolated spinal cord cells in situ.

We present a novel variation of the tissue print technique which yields isolated spinal cells that retain their original topographical organization in situ. A light papain digestion combined with a low-melting-point agarose as the adhesive substrate is used to "print' a monolayer of cells from spinal cord slices. Printed cells are viable for studies using electrophysiological approaches. This technique provides a means to bridge the gap between properties of isolated cells and their topographical identity in spinal cord.