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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.

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.

The trace amines (TAs) are a family of endogenous amines with structural, metabolic, physiological and pharmacological similarities to classical monoamine neurotransmitters. The TA family includes tyramine, octopamine, β-phenylethylamine (PEA), and tryptamine (Figure 1). Their presence has been well documented in all vertebrate and invertebrate species studied. Like the classical monoamines, the TAs are synthesized via enzymatic decarboxylation of the precursor aromatic amino acids phenylalanine, tyrosine, and tryptophan with aromatic L-amino acid decarboxylase (AADC; also called DOPA decarboxylase). For decades, researchers have known that these amines are present in the brain. A large earlier literature asserted their role as endogenous neuromodulators of monoaminergic excitability and neurotransmission, challenging the more conventional view of the TAs as metabolic byproducts that interfere with classical monoamine modulatory functions (Berry, 2004).

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.

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.

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.

Each spinal neuron has a receptive field that corresponds to stimulation of a specific area of skin or subcutaneous tissue. Receptive fields are plastic and can be altered during development and injury but the actions of neuromodulators, such as serotonin (5-hydroxytryptamine, 5-HT) on receptive field properties are not well known. We used stimulation of multiple adjacent dorsal root spinal segments as a measure of ‘receptive field size’ to determine the effects of 5-HT on multi-segmental convergent input onto neurons in laminae IV–VII. Whole-cell patch-clamp recordings were undertaken in the in vitro hemisected thoracolumbar spinal cord of rats aged 8–10 days old. Based on synaptic responses, neurons could be divided into two predominant groups and 5-HT exerted different effects on these groups. The first group received excitatory post-synaptic potentials (EPSPs) from the homonymous dorsal root but inhibitory post-synaptic potentials (IPSPs) with increasing amplitude from more distant dorsal roots. In this group, 5-HT preferentially depressed the IPSPs from adjacent nerve roots while leaving the EPSP intact. The second group received short-latency EPSPs from all segments stimulated and 5-HT potently depressed all synaptic input. In both populations the depressant actions of 5-HT increased with dose (0.1–10.0 μM). Bicuculline and strychnine did not affect the 5-HT induced short-latency synaptic depression. These results suggest that descending serotonergic systems depress spinal sensory convergence in a graded and differentiated manner. The findings are discussed in relation to the modulation of nociceptive signaling.

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional afferent fibers monitor other characteristics of the muscle environment, including metabolite buildup, temperature, and nociceptive stimuli. Overall, abnormal activation of sensory neurons can lead to movement disorders or chronic pain syndromes. We describe the isolation of the extensor digitorum longus (EDL) muscle and nerve for in vitro study of stretch-evoked afferent responses in the adult mouse. Sensory activity is recorded from the nerve with a suction electrode and individual afferents can be analyzed using spike sorting software. In vitro preparations allow for well controlled studies on sensory afferents without the potential confounds of anesthesia or altered muscle perfusion. Here we describe a protocol to identify and test the response of muscle spindle afferents to stretch. Importantly, this preparation also supports the study of other subtypes of muscle afferents, response properties following drug application and the incorporation of powerful genetic approaches and disease models in mice.

Gain control of primary afferent neurotransmission at their intraspinal terminals occurs by several mechanisms including primary afferent depolarization (PAD). PAD produces presynaptic inhibition via a reduction in transmitter release. While it is known that descending monoaminergic pathways complexly regulate sensory processing, the extent these actions include modulation of afferent-evoked PAD remains uncertain. We investigated the effects of serotonin (5HT), dopamine (DA) and noradrenaline (NA) on afferent transmission and PAD. Responses were evoked by stimulation of myelinated hindlimb cutaneous and muscle afferents in the isolated neonatal mouse spinal cord. Monosynaptic responses were examined in the deep dorsal horn either as population excitatory synaptic responses (recorded as extracellular field potentials; EFPs) or intracellular excitatory postsynaptic currents (EPSCs). The magnitude of PAD generated intraspinally was estimated from electrotonically back-propagating dorsal root potentials (DRPs) recorded on lumbar dorsal roots. 5HT depressed the DRP by 76%. Monosynaptic actions were similarly depressed by 5HT (EFPs 54%; EPSCs 75%) but with a slower time course. This suggests that depression of monosynaptic EFPs and DRPs occurs by independent mechanisms. DA and NA had similar depressant actions on DRPs but weaker effects on EFPs. IC50 values for DRP depression were 0.6, 0.8 and 1.0 µM for 5HT, DA and NA, respectively. Depression of DRPs by monoamines was nearly-identical in both muscle and cutaneous afferent-evoked responses, supporting a global modulation of the multimodal afferents stimulated. 5HT, DA and NA produced no change in the compound antidromic potentials evoked by intraspinal microstimulation indicating that depression of the DRP is unrelated to direct changes in the excitability of intraspinal afferent fibers, but due to metabotropic receptor activation. In summary, both myelinated afferent-evoked DRPs and monosynaptic transmission in the dorsal horn are broadly reduced by descending monoamine transmitters. These actions likely integrate with modulatory actions elsewhere to reconfigure spinal circuits during motor behaviors.

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.

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.

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.

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.

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.

The midsagittally-sectioned lumbar spinal cord with thoracic segments intact retains the capacity for locomotor-like activity. Intracellular recordings were used to characterize the activity and concurrently label lumbar neurons in lamina VII, an area previously implicated in the generation of locomotion. Sharp electrodes were shown to preferentially impale larger neurons. These neurons undergo rhythmic voltage oscillations, presumably synaptically driven, during locomotor-like activity induced by bath application of N-methyl-D-aspartate and 5-hydroxytryptamine. This supports the hypothesis that synaptic activity recruits neurons in lamina VII that are associated with locomotor behavior.

The neonatal rodent spinal cord maintained in vitro is a powerful model system to understand the central properties of spinal circuits generating mammalian locomotion. We describe three enabling approaches that incorporate afferent input and attached hindlimbs. (i) Sacral dorsal column stimulation recruits and strengthens ongoing locomotor-like activity, and implementation of a closed positive-feedback paradigm is shown to support its stimulation as an untapped therapeutic site for locomotor modulation. (ii) The spinal cord hindlimbs-restrained preparation allows suction electrode electromyographic recordings from many muscles. Inducible complex motor patterns resemble natural locomotion, and insights into circuit organization are demonstrated during spontaneous motor burst 'deletions', or following sensory stimuli such as tail and paw pinch. (iii) The spinal cord hindlimbs-pendant preparation produces unrestrained hindlimb stepping. It incorporates mechanical limb perturbations, kinematic analyses, ground reaction force monitoring, and the use of treadmills to study spinal circuit operation with movement-related patterns of sensory feedback while providing for stable whole-cell recordings from spinal neurons. Such techniques promise to provide important additional insights into locomotor circuit organization.

We utilized an in vitro adult mouse extensor digitorum longus (EDL) nerve-attached preparation to characterize the responses of muscle spindle afferents to ramp-and-hold stretch and sinusoidal vibratory stimuli. Responses were measured at both room (24°C) and muscle body temperature (34°C). Muscle spindle afferent static firing frequencies increased linearly in response to increasing stretch lengths to accurately encode the magnitude of muscle stretch (tested at 2.5%, 5% and 7.5% of resting length [Lo]). Peak firing frequency increased with ramp speeds (20% Lo/sec, 40% Lo/sec, and 60% Lo/sec). As a population, muscle spindle afferents could entrain 1:1 to sinusoidal vibrations throughout the frequency (10–100 Hz) and amplitude ranges tested (5–100 µm). Most units preferentially entrained to vibration frequencies close to their baseline steady-state firing frequencies. Cooling the muscle to 24°C decreased baseline firing frequency and units correspondingly entrained to slower frequency vibrations. The ramp component of stretch generated dynamic firing responses. These responses and related measures of dynamic sensitivity were not able to categorize units as primary (group Ia) or secondary (group II) even when tested with more extreme length changes (10% Lo). We conclude that the population of spindle afferents combines to encode stretch in a smoothly graded manner over the physiological range of lengths and speeds tested. Overall, spindle afferent response properties were comparable to those seen in other species, supporting subsequent use of the mouse genetic model system for studies on spindle function and dysfunction in an isolated muscle-nerve preparation.

Spinal cord sympathetic preganglionic neurons (SPNs) integrate activity from descending and sensory systems to determine the final central output of the sympathetic nervous system. The intermediolateral column (IML) has the highest number and density of SPNs and, within this region, SPN somas are found in distinct clusters within thoracic and upper lumbar spinal segments. Whereas SPNs exhibit a rostrocaudal gradient of end-target projections, individual clusters contain SPNs with diverse functional roles. Here we explored diversity in the electrophysiological properties observed in Hb9-eGFP–identified SPNs in the IML of neonatal mice. Overall, mouse SPN intrinsic membrane properties were comparable with those seen in other species. A wide range of values was obtained for all measured properties (up to a 10-fold difference), suggesting that IML neurons are highly differentiated. Using linear regression we found strong correlations between many cellular properties, including input resistance, rheobase, time constant, action potential shape, and degree of spike accommodation. The best predictor of cell function was rheobase, which correlated well with firing frequency–injected current ( f– I) slopes as well as other passive and active membrane properties. The range in rheobase suggests that IML neurons have a recruitment order with stronger synaptic drives required for maximal recruitment. Using cluster analysis, we identified at least four subpopulations of SPNs, including one with a long time constant, low rheobase, and high f– I gain. We thus propose that the IML contains populations of neurons that are differentiable by their membrane properties and hypothesize they represent diverse functional classes.

Dorsal root-evoked stimulation of sensory afferents in the hemisected in vitro rat spinal cord produces reflex output, recorded on the ventral roots. Transient spinal 5-HT2Creceptor activation induces a long-lasting facilitation of these reflexes (LLFR) by largely unknown mechanisms. Two Sprague–Dawley substrains were used to characterize network properties involved in this serotonin (5-HT) receptor–mediated reflex plasticity. Serotonin more easily produced LLFR in one substrain and a long-lasting depression of reflexes (LLDR) in the other. Interestingly, LLFR and LLDR were bidirectionally interconvertible using 5-HT2A/2Cand 5-HT1Areceptor agonists, respectively, regardless of substrain. LLFR was predominantly Aβ afferent fiber mediated, consistent with prominent 5-HT2Creceptor expression in the Aβ fiber projection territories (deeper spinal laminae). Reflex facilitation involved an unmasking of polysynaptic pathways and an increased receptive field size. LLFR emerged even when reflexes were evoked three to five times/h, indicating an activity independent induction. Both the NMDA and AMPA/kainate receptor–mediated components of the reflex could be facilitated, and facilitation was dependent on 5-HT receptor activation alone, not on coincident reflex activation in the presence of 5-HT. Selective blockade of GABAAand/or glycine receptors also did not prevent reflex amplification and so are not required for LLFR. Indeed, a more robust response was seen after blockade of spinal inhibition, indicating that inhibitory processes serve to limit reflex amplification. Overall we demonstrate that the serotonergic system has the capacity to induce long-lasting bidirectional changes in reflex strength in a manner that is nonassociative and independent of evoked activity or activation of ionotropic excitatory and inhibitory receptors.

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.

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.

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.

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.

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.

Descending serotonergic, noradrenergic, and dopaminergic systems project diffusely to sensory, motor and autonomic spinal cord regions. Using neonatal mice, this study examined monoaminergic modulation of visceral sensory input and sympathetic preganglionic output. Whole-cell recordings from sympathetic preganglionic neurons (SPNs) in spinal cord slice demonstrated that serotonin, noradrenaline, and dopamine modulated SPN excitability. Serotonin depolarized all, while noradrenaline and dopamine depolarized most SPNs. Serotonin and noradrenaline also increased SPN current-evoked firing frequency, while both increases and decreases were seen with dopamine. In an in vitro thoracolumbar spinal cord/sympathetic chain preparation, stimulation of splanchnic nerve visceral afferents evoked reflexes and subthreshold population synaptic potentials in thoracic ventral roots that were dose-dependently depressed by the monoamines. Visceral afferent stimulation also evoked bicuculline-sensitive dorsal root potentials thought to reflect presynaptic inhibition via primary afferent depolarization. These dorsal root potentials were likewise dose-dependently depressed by the monoamines. Concomitant monoaminergic depression of population afferent synaptic transmission recorded as dorsal horn field potentials was also seen. Collectively, serotonin, norepinephrine and dopamine were shown to exert broad and comparable modulatory regulation of viscero-sympathetic function. The general facilitation of SPN efferent excitability with simultaneous depression of visceral afferent-evoked motor output suggests that descending monoaminergic systems reconfigure spinal cord autonomic function away from visceral sensory influence. Coincident monoaminergic reductions in dorsal horn responses support a multifaceted modulatory shift in the encoding of spinal visceral afferent activity. Similar monoamine-induced changes have been observed for somatic sensorimotor function, suggesting an integrative modulatory response on spinal autonomic and somatic function.

The external urethral sphincter (EUS) and external anal sphincter (EAS) are striated muscles that function to maintain urinary and fecal continence respectively. This study examines the short-latency synaptic input from a variety of cutaneous perineal and muscle/cutaneous hindlimb afferents to the motoneurons innervating these muscles. Intracellular recordings from anti dromically identified EUS and EAS motoneurons provided records of the postsynaptic potentials (PSPs) produced by electrical stimulation of peripheral afferents in decerebrate or chloralose anesthetized cats. Excitatory postsynaptic potentials (EPSPs) were produced in most EUS and EAS motoneurons by stimulation of ipsilateral and contralateral sensory pudendal (SPud) and superficial perineal (SPeri) cutaneous nerves. The shortest cen tral latencies in the study (1.5 ms) suggest that there are disynaptic excitatory, in addition to tri-and oligosynap tic, connections within these reflex pathways. EPSPs mixed with longer latency inhibitory potentials (E/I PSPs) were observed in both motoneuron populations but were found more frequently in EAS motoneurons. These E/I PSPs were evoked more often from contralat eral afferents than from ipsilateral afferents. Cutaneous nerves innervating the hindlimb had weaker if any synaptic effects on sphincter motoneurons. Stimulation of ipsilateral hindlimb muscle nerves rarely produced PSPs in EUS motoneurons and had weak synaptic actions on EAS motoneurons. In 2 of 22 animals (both decerebrate), large inhibitory potentials predominated over early small EPSPs suggesting that inhibitory pathways from these afferents to sphincter motoneurons can be released under certain circumstances. The relation between the segmental afferents to EUS and EAS motoneurons and the neural circuitry influencing them during micturition and defecation are discussed.

Membrane electrical properties of motoneurons innervating the striated muscle of the external urethral and anal sphincters were examined in the decerebrate cat. Both populations of motoneurons had similar electrical properties (mean pooled values; conduction velocity 48 m/s, membrane time constant 3.3 ms, afterhyperpolarization (AHP) duration 97 ms, membrane input resistance 2.2 MΩ, rheobase 3.3 nA, and threshold voltage 8.1 mV). Although a portion of cells in both subpopulations of sphincter motoneurons displayed subthreshold conductances (i.e. sag, anomalous rectification), the incidence was higher in the external urethral sphincter motoneurons. The sphincter motoneuron membrane properties were likened to ‘slow’ hindlimb motoneurons and the low rheobase values suggest that the sphincter motoneurons are easily recruited.