A multitarget basal ganglia dopaminergic and GABAergic transplantation strategy enhances behavioural recovery in parkinsonian rats.

The current transplantation paradigm for Parkinson's disease that places foetal dopaminergic cells in the striatum neither normalizes neuronal activity in basal ganglia structures such as the substantia nigra (SN) and subthalamic nucleus (STN) nor leads to complete functional recovery. It was hypothesized that restoration of parkinsonian deficits requires inhibition of the pathological overactivity of the STN and SN in addition to restoration of dopaminergic activity in the striatum. To achieve inhibition, a multitargeted basal ganglia transplantation strategy using GABAergic cells derived from either foetal striatal primordia (FSP) cells or human neural precursor cells (hNPCs) expanded in suspension bioreactors was investigated. In hemiparkinsonian rats, transplantation of foetal rat dopaminergic cells in the striatum in conjunction with GABAergic grafts in the STN and/or SN promoted significant improvement in forelimb akinesia and motor function compared to transplantation of intrastriatal dopaminergic grafts alone or in conjunction with undifferentiated hNPCs. In culture, FSP cells exhibited neuronal electrophysiological properties. However, recordings from GABAergic hNPCs revealed limited ionic conductances and an inability to fire action potentials. Despite this, they were almost as efficacious as FSP cells in inducing functional recovery following transplantation, suggesting that such recovery may have been mediated by secretion of GABA rather than by functional integration into the host. Thus, restoration of dopaminergic activity to the striatum in concert with inhibition of the STN and SN by GABAergic grafts may be beneficial for improving clinical outcomes in patients with Parkinson's disease and potential clinical application of this strategy may be enhanced by the use of differentiated hNPCs.

Heterogeneity of V2-derived interneurons in the adult mouse spinal cord.

Spinal neurons and networks that generate rhythmic locomotor activity remain incompletely defined, prompting the use of molecular biological strategies to label populations of neurons in the postnatal mouse. During spinal cord development, expression of Lhx3 in the absence of Isl1 specifies a V2 interneuronal fate. In this study, postnatal V2-derived interneurons were identified by yellow fluorescent protein (YFP) expression in the double-transgenic offspring of Lhx3Cre/+ x thy1-loxP-stop-loxP-YFP mice. While some motoneurons were labelled, several populations of interneurons predominantly located in lamina VII could also be distinguished. Small interneurons were located throughout the spinal cord whereas larger interneurons were concentrated in the lumbar enlargement. Some V2-derived interneurons were propriospinal, with axons that bifurcated in the lateral funiculus. V2-derived interneurons gave rise to populations of both excitatory and inhibitory interneurons in approximately equal proportions, as demonstrated by in situ hybridization with VGLUT2 mRNA. Immunohistochemical studies revealed YFP+ boutons throughout the spinal cord. Both glutamatergic and glycinergic YFP+ boutons were observed in lamina IX where many apposed motoneuron somata. GABAergic YFP+ boutons were also observed in lamina IX, and they did not form P-boutons. At P0, more than half of the YFP+ interneurons expressed Chx10 and thus were derived from the V2a subclass. In adult mice, there was an increase in Fos expression in V2-derived interneurons following locomotion, indicating that these neurons are active during this behaviour. The heterogeneity of V2-derived interneurons in adult mice indicates that physiologically distinct subpopulations, including last-order interneurons, arise from these embryonically defined neurons.

Heterogeneous electrotonic coupling and synchronization of rhythmic bursting activity in mouse Hb9 interneurons.

The neurons and mechanisms involved in mammalian spinal cord networks that produce rhythmic locomotor activity remain largely undefined. Hb9 interneurons, a small population of discretely localized interneurons in the mouse spinal cord, are conditionally bursting neurons. Here we applied potassium channel blockers with the aim of increasing neuronal excitability and observed that under these conditions, postnatal Hb9 interneurons exhibited bursts of action potentials with underlying voltage-independent spikelets. The bursts were insensitive to antagonists to fast chemical synaptic transmission, and the bursting and spikelets were blocked by tetrodotoxin. Calcium imaging studies using 2-photon excitation in spinal cord slices revealed that clustered Hb9 interneurons exhibited synchronous and occasional asynchronous, calcium transients that were also insensitive to fast synaptic transmission blockade. All transients were blocked by the gap junction blocker carbenoxolone. Paired whole cell patch-clamp recordings of Hb9 interneurons in the late postnatal mouse revealed common chemical synaptic inputs but no evidence of current transfer (i.e., electrotonic coupling) between the neurons. However, Hb9 and a previously defined population of non-Hb9 interneurons were electrotonically coupled. In the absence of fast chemical transmission in the whole spinal cord preparation, 2-photon excitation calcium imaging revealed bursting activity of Hb9 interneurons synchronous with rhythmic ventral root output. Thus Hb9 interneurons are both endogenous bursters and rhythmically active within a heterogeneous electrotonically coupled network. A network with these properties could produce the wide range of stable rhythms necessary for locomotor activity.

Rapid pH and PO2 changes in the tissue recording chamber during stoppage of a gas-equilibrated perfusate: effects on calcium currents in ventral horn neurons.

In vitro studies often use bicarbonate-buffered saline solutions to mimic the normal extracellular environment of tissues. These solutions are typically equilibrated with gaseous O2 and CO2, the latter interacting with bicarbonate ions to maintain a physiological pH. In vitro tissue chambers, like those used for electrophysiology, are usually continually perfused with the gassed buffer, but stopping the perfusion to add expensive chemicals or acquire imaging data is a common practice. The present study demonstrates that this procedure leads to rapid (< 30 s) increases in pH and decreases in PO2 of the detained solution in the tissue chamber. During the first 200 s, pH increased by 0.4 units and resulted in a 25% PO2 reduction of the detained solution. The rates of these changes were dependent on the volume of solution in the chamber. In experiments using acute transverse slices from the lumbar spinal cord of neonatal (postnatal day 0-10) mice, perfusion stoppage of the same duration was accompanied by a 34.7% enhancement of the peak voltage-gated calcium current recorded from ventral horn neurons. In these cells both low voltage-activated and high voltage-activated currents were affected. These currents were unaffected by decreasing PO2 when a CO2-independent buffer was used, suggesting that changes in pH were responsible for the observed effects. It is concluded that the procedure of stopping a bicarbonate/CO2-buffered perfusate results in rapid changes in pH and PO2 of the solution detained in the tissue chamber, and that these changes have the potential to covertly influence experimental results.

Mechanisms underlying the early phase of spike frequency adaptation in mouse spinal motoneurones.

Spike frequency adaptation (SFA) is a fundamental property of repetitive firing in motoneurones (MNs). Early SFA (occurring over several hundred milliseconds) is thought to be important in the initiation of muscular contraction. To date the mechanisms underlying SFA in spinal MNs remain unclear. In the present study, we used both whole-cell patch-clamp recordings of MNs in lumbar spinal cord slices prepared from motor functionally mature mice and computer modelling of spinal MNs to investigate the mechanisms underlying SFA. Pharmacological blocking agents applied during whole-cell recordings in current-clamp mode demonstrated that the medium AHP conductance (apamin), BK-type Ca2+ -dependent K+ channels (iberiotoxin), voltage-activated Ca2+ channels (CdCl2), M-current (linopirdine) and persistent Na+ currents (riluzole) are all unnecessary for SFA. Measurements of Na+ channel availability including action potential amplitude, action potential threshold and maximum depolarization rate of the action potential were found to correlate with instantaneous firing frequency suggesting that the availability of fast, inactivating Na+ channels is involved in SFA. Characterization of this Na+ conductance in voltage-clamp mode demonstrated that it undergoes slow inactivation with a time course similar to that of SFA. When experimentally measured parameters for the fast, inactivating Na+ conductance (including slow inactivation) were incorporated into a MN model, SFA could be faithfully reproduced. The removal of slow inactivation from this model was sufficient to remove SFA. These data indicate that slow inactivation of the fast, inactivating Na+ conductance is likely to be the key mechanism underlying early SFA in spinal MNs.

Characterization of calcium currents in functionally mature mouse spinal motoneurons.

Motoneurons integrate synaptic input and produce output in the form of trains of action potentials such that appropriate muscle contraction occurs. Motoneuronal calcium currents play an important role in the production of this repetitive firing. Because these currents change in the postnatal period, it is necessary to study them in animals in which the motor system is 'functionally mature', that is, animals that are able to weight-bear and walk. In this study, calcium currents were recorded using whole-cell patch-clamp techniques from large (> 20 microm) ventral horn cells in lumbar spinal cord slices prepared from mature mice. Ninety percent (nine out of 10) of the recorded cells processed for choline acetyltransferase were found to be cholinergic, confirming their identity as motoneurons. A small number of motoneurons were found to have currents with low-voltage-activated (T-type) characteristics. Pharmacological dissection of the high-voltage-activated current demonstrated omega-agatoxin-TK- (P/Q-type), omega-conotoxin GVIA- (N-type), and dihydropyridine- and FPL-64176-sensitive (L-type) components. A cadmium-sensitive component of the current that was insensitive to these chemicals (R-type) was also seen in these cells. These results indicate that the calcium current in lumbar spinal motoneurons from functionally mature mice is mediated by a number of different channel subtypes. The characterization of these calcium channels in mature mammalian motoneurons will allow for the future study of their modulation and their roles during behaviours such as locomotion.

Dendritic L-type calcium currents in mouse spinal motoneurons: implications for bistability.

The intrinsic properties of mammalian spinal motoneurons provide them with the capability to produce high rates of sustained firing in response to transient inputs (bistability). Even though it has been suggested that a persistent dendritic calcium current is responsible for the depolarizing drive underlying this firing property, such a current has not been demonstrated in these cells. In this study, calcium currents are recorded from functionally mature mouse spinal motoneurons using somatic whole-cell patch-clamp techniques. Under these conditions a component of the current demonstrated kinetics consistent with a current originating at a site spatially segregated from the soma. In response to step commands this component was seen as a late-onset, low amplitude persistent current whilst in response to depolarizing-repolarizing ramp commands a low voltage clockwise current hysteresis was recorded. Simulations using a neuromorphic motoneuron model could reproduce these currents only if a noninactivating calcium conductance was placed in the dendritic compartments. Pharmacological studies demonstrated that both the late-onset and hysteretic currents demonstrated sensitivity to both dihydropyridines and the L-channel activator FPL-64176. Furthermore, the alpha1D subunits of L-type calcium channels were immunohistochemically demonstrated on motoneuronal dendrites. It is concluded that there are dendritically located L-type channels in mammalian motoneurons capable of mediating a persistent depolarizing drive to the soma and which probably mediate the bistable behaviour of these cells.

Development of L-type calcium channels and a nifedipine-sensitive motor activity in the postnatal mouse spinal cord.

Intrinsic membrane properties are important in the regulation of motoneuronal output during such behaviours as locomotion. A conductance through L-type calcium channels has been implicated as an essential component in the transduction of motoneuronal input to output during locomotion. Given the developmental changes in calcium currents occurring postnatally in some neurons, and the increasing interest in the study of spinal locomotor output in neonatal preparations, experiments were conducted to investigate the postnatal development of L-type calcium channels in mouse motoneurons. This was assessed both physiologically, using a chemically induced rhythmic motor output, and anatomically, using immunohistochemical methods. The electrophysiological data were obtained during rhythmic bursting produced by application of N-methyl-D-aspartate (NMDA) and strychnine to the isolated spinal cord at various postnatal ages. The L-type calcium channel blocker nifedipine has no effect on this ventral root bursting in postnatal day (P) P2-P5 animals, but reversibly reduced the amplitude and/or burst duration of this activity in animals greater than P7. The immunohistochemical evidence demonstrates a dramatic change in the cellular profile of both the alpha1C and alpha1D subunits of L-type calcium channels during postnatal development; the labelling of both subunits increases with age, approximating the adult pattern by P18. These results demonstrate that in the spinal cord, the L-type calcium channel profile develops both physiologically and anatomically in the early postnatal period. This development parallels the development of the mature functional behaviours of weight bearing and walking, and may be necessary for the production of complex motor behaviour in the mature mammal.

An in vitro functionally mature mouse spinal cord preparation for the study of spinal motor networks.

An in vitro isolated whole spinal cord preparation has been developed in 'motor functionally mature' mice; that is mice of developmental maturity sufficient to weight-bear and walk. In balb/c mice this stage occurs at around postnatal day 10 (P10). Administration of strychnine elicited synchronous activity bilaterally in lumbar ventral roots. Rhythmic alternating locomotor-like activity could be produced by application of a combination of serotonin (5-HT), N-methyl-d-aspartate (NMDA), and dopamine in animals up to P12. Using a live cell-dead cell assay, it is demonstrated that there are primarily viable cells throughout the lumbar spinal cord. The viability of descending pathways was demonstrated with stimulation of the mid-thoracic white matter tracts. In addition, polysynaptic segmental reflexes could be elicited. Although usually absent in whole cord preparations, monosynaptic reflexes could invariably be elicited following longitudinal midline hemisection, leading to the possible explanation that there might be an active crossed pathway producing presynaptic inhibition of primary afferent terminals. The data demonstrate that this functionally mature spinal cord preparation can be used for the study of spinal cord physiology including locomotion.

Human immunodeficiency virus type 1 tat activates non-N-methyl-D-aspartate excitatory amino acid receptors and causes neurotoxicity.

The human immunodeficiency virus type 1 (HIV-1) protein Tat is known to be released from HIV-1-infected cells. We show that micromolar concentrations of Tat depolarized young rat and adult human neurons. In addition, Tat, at similar concentrations, was toxic to human fetal neurons in culture. Tat-induced responses were insensitive to the Na+ channel blocker tetrodotoxin, suggesting a direct effect of Tat on neurons. Tat-induced depolarizations and cytotoxicity were blocked by the excitatory amino acid antagonist kynurenate. The N-methyl-D-aspartate receptor antagonist D-2-amino-5-phosphonovalerate had little effect on Tat-induced depolarizations but did provide protection from Tat neurotoxicity. These results suggest that Tat, released from HIV-1-infected cells, may be an important mediator of neurotoxicity observed in HIV-1 encephalopathy.

Mechanical entrainment of fictive locomotion in the decerebrate cat.

1. We examined the ability of muscular and joint afferents from the hip region to entrain fictive locomotion evoked by stimulation of the mesencephalic locomotor region in the decerebrate cat by mechanically imposed, sinusoidal hip flexion and extension movements. 2. A method is presented for qualitative and quantitative analysis of entrainment. 3. Hip joint capsular afferents were shown by denervation experiments to be unnecessary for mediating locomotor entrainment. 4. As the population of muscular afferents was progressively decreased by selective denervation, the strength of entrainment concomitantly decreased, even though a few as two small intrinsic hip muscles were still effective in producing entrainment. The ability to entrain locomotion was abolished with complete ipsilateral denervation. 5. Entrainment was observed with low amplitude hip angular displacement of 5-20 degrees, which would be expected to activate low-threshold, stretch-sensitive muscle afferents. 6. The extensor burst activity occurred during the period of imposed hip flexion, which corresponded to passive stretching and loading of the extensor muscles, while the flexor burst activity occurred during the latter portion of the imposed hip extension, which corresponded to passive stretching of the flexor muscles (when attached) and release of the extensors. During harmonic entrainment, the match of hip cycle duration and step cycle duration was accomplished by a variation in extensor electroneurogram (ENG) burst duration. These results are consistent with a positive feedback mechanism where low-threshold afferent activity from the extensor musculature is used by the rhythm generator to prolong the extension phase of locomotion. 7. A hip cycle frequency-dependent phase shift of ENG activity was observed. This may indicate that the locomotor rhythm generator is dependent on more than just static positional or threshold load information for modulation of the step cycle frequency and switching between flexion and extension phases. 8. Subharmonic forms of entrainment were observed when the number of innervated muscles was markedly reduced. The occurrence of subharmonic entrainment characterizes the locomotor rhythm generator as a nonlinear oscillator. 9. To modulate the stepping frequency, the afferent pathways responsible for entrainment must be directly connected to the neural circuitry responsible for rhythm generation. The rhythm generating interneurons must receive a high degree of convergence from afferents arising from a variety of muscles spanning the hip joint.

Transmission in a locomotor-related group Ib pathway from hindlimb extensor muscles in the cat.

It has been previously shown that phasic stimulation of group I afferents from ankle and knee extensor muscles may entrain and/or reset the intrinsic locomotor rhythm; these afferents are thus acting on motoneurones through the spinal rhythm generators. It was also concluded that the major part of these effects originates from Golgi tendon organ Ib afferents. Transmission in this pathway to lumbar motoneurones has now been investigated during fictive locomotion in spinal cats injected with nialamide and L-DOPA, and in decerebrate cats with stimulation of the mesencephalic locomotor region. In spinal cats injected with nialamide and L-DOPA, it was possible to evoke long-latency, long-lasting reflexes upon stimulation of high threshold afferents before spontaneous fictive locomotion commenced. During that period, stimulation of ankle and knee extensor group I afferents evoked oligosynaptic excitation of extensor motoneurones, rather than the "classical" Ib inhibition. Furthermore, a premotoneuronal convergence (spatial facilitation) between this group I excitation and the crossed extensor reflex was established. During fictive locomotion, in both preparations, the transmissions in these groups I pathway was phasically modulated within the step cycle. During the flexor phase, the group I input cut the depolarised (active) phase in flexor motoneurones and evoked EPSPs in extensor motoneurones; during the extensor phase the group I input evoked smaller EPSPs in extensor motoneurones and had virtually no effect on flexor motoneurones. The above results suggest that the group I input from extensor muscles is transmitted through the spinal rhythm generator and more particularly, through the extensor "half-centre". The locomotor-related group I excitation had a central latency of 3.5-4.0 ms. The excitation from ankle extensors to ankle extensors remained after a spinal transection at the caudal part of L6 segment; the interneurones must therefore be located in the L7 and S1 spinal segments. Candidate interneurones for mediating these actions were recorded extracellularly in lamina VII of the 7th lumbar segment. Responses to different peripheral nerve stimulation (high threshold afferents and group I afferents bilaterally) were in concordance with the convergence studies in motoneurones. The interneurones were rhythmically active in the appropriate phases of the fictive locomotor cycle, as predicted by their response patterns. The synaptic input to, and the projection of these candidate interneurones must be fully identified before their possible role as components of the spinal locomotor network can be evaluated.

Voltage-dependent excitation of motoneurones from spinal locomotor centres in the cat.

Lumbar motoneurones were recorded intracellularly during fictive locomotion induced by stimulation of the mesencephalic locomotor region in decerebrate cats. After blocking the action potentials using intracellular QX-314, and by using a discontinuous current clamp, it is shown that the excitatory component of the locomotor drive potentials behaves in a voltage-dependent manner, such that its amplitude increases with depolarisation. As the input to motoneurones during locomotion is comprised of alternating excitation and inhibition, it was desirable to examine the excitatory input in relative isolation. This was accomplished in spinalised decerebrate cats treated with nialamide and L-dihydroxy-phenylalanine (L-DOPA) by studying the excitatory post-synaptic potentials (EPSPs) evoked from the "flexor reflex afferents" (FRA) and extensor Ib afferents, both of which are likely to be mediated via the locomotor network. As expected, these EPSPs also demonstrate a voltage-dependent increase in amplitude. In addition, the input to motoneurones from the network for scratching, which is thought to share interneurones with the locomotor network, also results in voltage-dependent excitation. The possible underlying mechanisms of NMDA-mediated excitation and plateau potentials are discussed: both may contribute to the observed effect. It is suggested that this nonlinear increase in excitation contributes to the mechanisms involved in the production of the high rates of repetitive firing of motoneurones typically seen during locomotion, thus ensuring appropriate muscle contraction.

Control of functional systems in the brainstem and spinal cord.

Progress has been made in the identification of cells, circuits, and networks involved in certain important subcortical functional systems, including swallowing, chewing, posture and locomotion, and in the shared mechanisms for selecting the network for specific motor tasks, including a role for excitatory amino acids for network activation, the shaping of the network by inhibitory control, and the selection of inputs and modulation of outputs by monoamines and other agents.

On the regulation of repetitive firing in lumbar motoneurones during fictive locomotion in the cat.

Repetitive firing of motoneurones was examined in decerebrate, unanaesthetised, paralysed cats in which fictive locomotion was induced by stimulation of the mesencephalic locomotor region. Repetitive firing produced by sustained intracellular current injection was compared with repetitive firing observed during fictive locomotion in 17 motoneurones. During similar interspike intervals, the afterhyperpolarisations (AHPs) during fictive locomotion were decreased in amplitude compared to the AHPs following action potentials produced by sustained depolarising current injections. Action potentials were evoked in 10 motoneurones by the injection of short duration pulses of depolarising current throughout the step cycles. When compared to the AHPs evoked at rest, the AHPs during fictive locomotion were reduced in amplitude at similar membrane potentials. The post-spike trajectories were also compared in different phases of the step cycle. The AHPs following these spikes were reduced in amplitude particularly in the depolarised phases of the step cycles. The frequency-current (f-I) relations of 7 motoneurones were examined in the presence and absence of fictive locomotion. Primary ranges of firing were observed in all cells in the absence of fictive locomotion. In most cells (6/7), however, there was no relation between the amount of current injected and the frequency of repetitive firing during fictive locomotion. In one cell, there was a large increase in the slope of the f-I relation. It is suggested that this increase in slope resulted from a reduction in the AHP conductance; furthermore, the usual elimination of the relation is consistent with the suggestions that the repetitive firing in motoneurones during fictive locomotion is not produced by somatic depolarisation alone, and that motoneurones do not behave as simple input-output devices during this behaviour. The correlation of firing level with increasing firing frequency which has previously been demonstrated during repetitive firing produced by afferent stimulation or by somatic current injection is not present during fictive locomotion. This lends further support to the suggestion that motoneurone repetitive firing during fictive locomotion is not produced or regulated by somatic depolarisation. It is suggested that although motoneurones possess the intrinsic ability to fire repetitively in response to somatic depolarisation, the nervous system need not rely on this ability in order to produce repetitive firing during motor acts. This capability to modify or bypass specific motoneuronal properties may lend the nervous system a high degree of control over its motor output.

The effects of caffeine on ischemic neuronal injury as determined by magnetic resonance imaging and histopathology.

The effects of caffeine on ischemic neuronal injury were determined in rats subjected to forebrain ischemia induced by bilateral carotid occlusion and controlled hypotension (50 mmHg for 10 min). High resolution (100 microns) multi-slice, multi-echo magnetic resonance images were obtained daily for three consecutive days post-operatively in sham-operated rats and in rats that received either saline vehicle (controls), a single i.v. injection of 10 mg/kg caffeine 30 min prior to an ischemic insult (acute caffeine group), or up to 90 mg/kg per day of caffeine for three consecutive weeks prior to an ischemic insult (chronic caffeine group). Rats in the control group exhibited enhanced magnetic resonance image intensity in the striatum 24 h after ischemia which increased in the striatum and also appeared in the hippocampus after 48 h, and which began to resolve in both regions by 72 h post-ischemia. Histopathological analysis of each rat following the final magnetic resonance examination showed that ischemic neuronal injury was strictly confined to the brain regions showing magnetic resonance image changes. Acute caffeine rats showed accelerated changes in the magnetic resonance images, with increased hippocampal intensity appearing at 24 h post-ischemia. Although there was magnetic resonance evidence of accelerated injury, quantitative analysis of the histopathological data at 72 h showed no significant difference in the extent of neuronal injury in any brain region between control-ischemic and acute caffeine rats. Nine out of 11 rats in the chronic caffeine group showed no magnetic resonance image changes over the three study days. Chronic caffeine rats had significantly less neuronal damage in all vulnerable brain regions than either of the other groups of ischemic rats. The accelerated ischemic injury in rats treated with an acute dose of caffeine may occur secondary to antagonism of adenosine receptors, whereas protection from ischemic injury following chronic administration of caffeine may be mediated by up-regulation of adenosine receptors.