Shawn Hochman

Restless legs syndrome: Revisiting the dopamine hypothesis from the spinal cord perspective

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 D2-like (D2, D3, and D4) 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 D3 receptor knockout (D3KO) mice. Excessive locomotor behavior is evident in both sets of animals, and D3KO 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 D3KO mouse might serve as a relevant animal model to study the underlying mechanisms of RLS.

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.

Conversion of the Modulatory Actions of Dopamine on Spinal Reflexes from Depression to Facilitation in D3 Receptor Knock-Out Mice

Descending monoaminergic systems modulate spinal cord function, yet spinal dopaminergic actions are poorly understood. Using the in vitro lumbar 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 D3 receptor agonists pergolide and PD 128907 reduced MSR amplitude in WT but not D3KO mice. Conversely, D3 receptor 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 μm dopamine 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 D3 receptors to limit reflex excitability. Moreover, in D3 ligand-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 D3 agonists, and likely involve increased reflex excitability, the D3KO mouse putatively explains how impaired D3 activity could contribute to this sleep disorder.

NMDA Receptor-mediated Oscillatory Properties: Potential Role in Rhythm Generation in the Mammalian Spinal Cord

Abstract: 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 similiar 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.

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 μM Zn2+ abolished Tat-evoked responses in all neurons tested. Thus, Tat appears to excite neurons via direct activation of the NMDA receptor at an allosteric Zn2+-sensitive site.

NMDA Receptor Activation Triggers Voltage Oscillations, Plateau Potentials and Burstinq in Neonatal Rat Lumbar Motoneurons ln Vitro

Whole‐cell recordings of lumbar motoneurons in the intact neonatal rat spinal cord in vitro were undertaken to examine the effects of Kmethyl‐D‐aspartate (NMDA) receptor activation on membrane behaviour. Bath application of NMDA induced rhythmic voltage oscillations of 5.9 ± 2.1 mV (SD) at a frequency of 4.4 ± 1.5 Hz. Amplitude, but not frequency, of the voltage oscillations was membrane potential‐dependent. Voltage oscillations could recruit action potentials and/or plateau potentials with or without superimposed bursting. Blockade of synaptic transmission with tetrodotoxin (TTX) sometimes resulted in a loss of oscillatory activity which could then be restored by increasing the NMDA concentration. After application of TTX, the trajectory of NMDA‐induced oscillations was similar to the trajectory induced in the presence of intact synaptic networks, although the mean oscillation duration was longer and the oscillation frequency was slower (1.8 ± 1.1 Hz). Current ramps delivered after bath application of NMDA demonstrated bistable membrane properties which may underlie the plateau potentials. Injection of intracellular current pulses could initiate, entrain and terminate individual plateau potentials. The results suggest that membrane depolarization produced by oscillations may activate other intrinsic conductances which generate plateau potentials, thereby providing the neuron with enhanced voltage sensitivity, compared to that produced by NMDA receptor activation alone. These oscillatory events may have a role in the regulation of motor output in a variety of rhythmic behaviours including locomotion.

Expression and distribution of all dopamine receptor subtypes (D1 – D5) in the mouse lumbar spinal cord: A real-time PCR and non-autoradiographic in situ hybridization study

Abstract 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.

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 μM) 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 (Kjærulff 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. J. Comp. Neurol. 423:590–602, 2000.

Lamina VII neurons are rhythmically active during locomotor-like activity in the neonatal rat spinal cord

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.

Characterization of Fetal and Postnatal Enteric Neuronal Cell Lines With Improvement in Intestinal Neural Function

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 gamma-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 degrees C, and interferon-gamma for 24 or 48 hours, and then transferred to 39 degrees 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 degrees C and 39 degrees C. Nestin, S-100beta, and alpha-smooth muscle actin were expressed minimally at 39 degrees 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 Hirschsprungs disease.