Intrapallidal injection of 6-hydroxydopamine induced changes in dopamine innervation and neuronal activity of globus pallidus.

The globus pallidus (GP) plays an important role in basal ganglia circuitry. In contrast to the well-characterized actions of dopamine on striatal neurons, the functional role of the dopamine innervation of GP is still not clearly determined. The present study aimed to investigate the effects of intrapallidal injection of 6-hydroxydopamine (6-OHDA) on rotational behavior induced by apomorphine, on the loss of dopamine cell bodies in the substantia nigra pars compacta (SNc) and fibers in the GP and striatum and on in vivo extracellularly-recorded GP neurons in the rat. Injection of 6-OHDA into GP induced severe loss of tyrosine hydroxylase-immunoreactive (TH-IR) fibers in GP (-85%) with a reduction in the number of TH-IR cell bodies in the SNc (-52%) and fibers in the striatum (-50%). S.c. injection of apomorphine in these rats induced a moderate number of turns (26+/-6 turns/5 min). Electrophysiological recordings show that 6-OHDA injection in GP induced a significant decrease of the firing rate of GP neurons (16.02+/-1.11 versus 24.14+/-1.58 spikes/sec in control animals and 22.83+/-1.28 in sham animals, one-way ANOVA, P<0.0001) without any change in the firing pattern (chi(2)=1.03, df=4, P=0.90). Our results support the premise of the existence of collaterals of SNc dopaminergic axons projecting to the striatum and GP and that dopamine plays a role in the modulation of the firing rate but not the firing pattern of GP neurons. Our data provide important insights into the functional role of the SNc-GP dopaminergic pathway suggesting that dopamine depletion in GP may participate in the development of motor disabilities.

Effects of 6-hydroxydopamine-induced severe or partial lesion of the nigrostriatal pathway on the neuronal activity of pallido-subthalamic network in the rat.

The origin of changes in the neuronal activity of the globus pallidus (GP) and the subthalamic nucleus (STN) in animal models of Parkinson's disease (PD) is still controversial. The aim of the study was to investigate the neuronal activity of STN and GP neurons under urethane anesthesia in an early and in an advanced stage PD rat model. 6-Hydroxydopamine (6-OHDA) injection into the striatum induced a partial lesion of dopamine cells in the substantia nigra pars compacta (SNc) and fibers in the striatum. The GP firing rate decreased significantly with no significant change of the pattern. 6-OHDA injection into the SNc induced a total or subtotal lesion without any change in the firing rate and patterns of GP neurons. Concerning the STN, after partial lesion, the firing rate remained unchanged but the firing pattern significantly changed towards a more irregular and bursty pattern. In rats with total or subtotal lesion of the SNc the firing rate increased significantly and the relative amount of tonic neurons significantly decreased. Our results demonstrate that neuronal reactivity in the basal ganglia network considerably differs in the early versus late stage model of PD. We showed that the pathological activity of STN neurons after severe lesion is not mediated by the GP. Moreover, the unchanged activity of GP neurons is likely to be a consequence of the STN hyperactivity. These data suggest that in the GP-STN-GP network, the excitatory influence of the STN-GP pathway overrides that of the GABAergic GP-STN pathway, questioning the classical model of basal ganglia organization.

Distribution and regulation of L-type calcium channels in deep dorsal horn neurons after sciatic nerve injury in rats.

Deep dorsal horn neurons are involved in the processing of nociceptive information in the spinal cord. Previous studies revealed a role of the intrinsic bioelectrical properties (plateau potentials) of deep dorsal horn neuron in neuronal hyperexcitability, indicating their function in pain sensitization. These properties were considered to rely on L-type calcium currents. Two different isotypes of L-type calcium channel alpha 1 subunit have been cloned (Ca(V)1.2 and Ca(V)1.3). Both are known to be expressed in the spinal cord. However, no data were available on their subcellular localization. Moreover, possible changes in Ca(V)1.2 and Ca(V)1.3 expression had never been investigated in nerve injury models. Our study provides evidence for a differential expression of Ca(V)1.2 and Ca(V)1.3 subunits in the somato-dendritic compartment of deep dorsal horn neurons. Ca(V)1.2 immunoreactivity is restricted to the soma and proximal dendrites whereas Ca(V)1.3 immunoreactivity is found in the whole somato-dendritic compartment, up to distal dendritic segments. Moreover, these specific immunoreactive patterns are also found in electrophysiologically identified deep dorsal horn neurons expressing plateau potentials. After nerve injury, namely total axotomy or partial nerve ligation, Ca(V)1.2 and Ca(V)1.3 expression undergo differential changes, showing up- and down-regulation, respectively, both at the protein and at the mRNA levels. Taken together, our data support the role of L-type calcium channels in the control of intrinsic biolectrical regenerative properties. Furthermore, Ca(V)1.2 and Ca(V)1.3 subunits may have distinct and specific roles in sensory processing in the dorsal horn of the spinal cord, the former being most likely involved in long-term changes after nerve injury.

Expression of vesicular glutamate transporters, VGLUT1 and VGLUT2, in cholinergic spinal motoneurons.

Mammalian spinal motoneurons are cholinergic neurons that have long been suspected to use also glutamate as a neurotransmitter. We report that VGLUT1 and VGLUT2, two subtypes of vesicular glutamate transporters, are expressed in rat spinal motoneurons. Both proteins are present in somato-dendritic compartments as well as in axon terminals in primary cultures of immunopurified motoneurons and sections of spinal cord from adult rat. However, VGLUT1 and VGLUT2 are not found at neuromuscular junctions of skeletal muscles. After intracellular injection of biocytin in motoneurons, VGLUT2 is observed in anterogradely labelled terminals contacting Renshaw inhibitory interneurons. These VGLUT2- and VGLUT1-positive terminals do not express VAChT, the vesicular acetylcholine transporter. Overall, our study establishes for the first time that (i) mammalian spinal motoneurons express vesicular glutamate transporters, (ii) these motoneurons have the potential to release glutamate (in addition to acetylcholine) at terminals contacting Renshaw cells, and finally (iii) the VGLUTs are not present at neuromuscular synapses of skeletal muscles.

Effect of microiontophoretic application of dopamine on subthalamic nucleus neuronal activity in normal rats and in rats with unilateral lesion of the nigrostriatal pathway.

The subthalamic nucleus (STN) receives dopamine inputs from the substantia nigra but their implication in the pathophysiology of parkinsonism is still debated. Extracellular microrecordings were used to study the effect of microiontophoretic injection of dopamine and the D1 receptor agonist SKF 38393 on the activity of STN neurons in normal and 6-hydroxydopamine-lesioned rats under urethane anaesthesia. Dopamine and SKF induced an increase in the firing rate of the majority of STN neurons in both normal and 6-OHDA rats. In rats with 6-OHDA lesions, the percentage of firing rate increase did not differ from that of controls. When GABA, glutamate and dopamine were all applied to the same individual STN neurons, GABA induced an inhibitory effect and glutamate and dopamine caused an excitatory effect in both groups. This excitatory response was suppressed by the application of GABA. Systemic administration of apomorphine provoked a decrease in the firing rate of STN neurons in rats with 6-OHDA lesions. These results show that dopamine exerts an excitatory influence on STN neurons, suggesting that the inhibitory effect induced by the systemic injection of apomorphine is due to the GABAergic inputs from the globus pallidus as predicted by the current model of basal ganglia organization. In addition, we show that dopamine, GABA and glutamate can act on the same STN neuron and that GABA can reverse the excitatory effect of dopamine and glutamate, suggesting the predominant influence of GABAergic inputs to the subthalamic nucleus.

Time-course of changes in firing rates and firing patterns of subthalamic nucleus neuronal activity after 6-OHDA-induced dopamine depletion in rats.

The subthalamic nucleus (STN) plays a key role in motor control. Disorganization of its neuronal activity is implicated in the manifestation of parkinsonian motor symptoms. The aim of the present work was to study the time-course of changes in the firing activity of STN neurons in a rat model of parkinsonism. Electrophysiological recordings were done in normal rats and four groups of rats at different time points after 6-hydroxydopamine (6-OHDA) microinjection into the pars compacta of substantia nigra (SNc). Results showed a significant decrease in firing rate during the first and second weeks post lesion (5.53+/-0.56 and 7.66+/-0.73 spikes/s, respectively) compared to normal rats (11.13+/-0.59 spikes/s). From the 3rd week after 6-OHDA injection the firing rates returned toward baseline, with an average of 9.71+/-0.51 spikes/s during the 3rd week and 11.13+/-0.71 spikes/s during the 4th week. With regard to firing pattern, the majority of STN cells (90%) discharged regularly or slightly irregularly in normal animals. Only 4% exhibited burst activity and 6% had mixed firing patterns. After SNc-lesion, the percentage of cells exhibiting burst and mixed patterns increased progressively from 35% during the first week to 56% at week 4 post-lesion. In sum, these experiments revealed that the firing rate of STN neurons was altered only transiently following nigral lesions, whereas a progressive and stable change in the firing pattern was observed up to 4 weeks post lesion, suggesting that the persistence of bursts firing more closely relates to the motor pathologies of this rat model of parkinsonism.

Intrasubthalamic injection of 6-hydroxydopamine induces changes in the firing rate and pattern of subthalamic nucleus neurons in the rat.

The subthalamic nucleus (STN) receives dopaminergic projections from the substantia nigra pars compacta (SNc). To investigate the role of direct and indirect dopaminergic influences on STN neurons, the spontaneous activity was studied in four groups of animals: normal rats, rats with intrasubthalamic or intranigral injection of 6-hydroxydopamine (6-OHDA), and sham STN injection rats by using extracellular recordings 4 weeks postsurgery. After intrasubthalamic injection of 6-OHDA, the mean firing rate significantly decreased (7.29 +/- 0.39 spikes/sec, P < 0.01 vs. 11.13 +/- 0.59 spikes/sec in normal or 11.26 +/- 0.57 spikes/sec in sham group), and the percentage of STN neurons discharging regularly decreased significantly (81%, P < 0.05 vs. 90% in normal group or P < 0.01 vs. 92% in sham group) and that of bursty cells increased (19%, P < 0.05 vs. 10%; in normal group or P < 0.01 vs. 8% in sham group). In the group of rats with SNc lesion, the firing rate of subthalamic neurons did not show a significant difference (11.61 +/- 0.81 spikes/sec) compared with normal group. However, the firing pattern was dramatically changed: 74% of cells exhibited bursty pattern and only 26% of cells discharged regularly or slightly irregularly. Immunohistochemical results showed that intrasubthalamic injection of 6-OHDA induced a marked degeneration of dopaminergic cells in the lateral part of the ipsilateral SNc, whereas 6-OHDA injection into the SNc induced a total in situ lesion of dopamine cells. These results suggest that the SNc exerts an excitatory influence on STN neurons and that the loss of this dopaminergic projection could, at least partially, account for the changes in the firing pattern of STN neurons in the 6-OHDA rat model of parkinsonism.

Unilateral lesion of the nigrostriatal pathway induces an increase of neuronal activity of the pedunculopontine nucleus, which is reversed by the lesion of the subthalamic nucleus in the rat.

The role of the pedunculopontine nucleus (PPN) in the pathophysiology of Parkinson's disease is still unclear. Using microrecordings, we investigated the changes occurring in PPN neurons after lesions of the substantia nigra compacta (SNc) and the role of the subthalamic nucleus (STN) in these changes. In normal rats the firing rate of PPN neurons was 10.6 +/- 1.4 spikes/s, the majority of neurons (91%) having a regular firing pattern, 6% irregular and 3% in bursts. In rats with 6-hydroxydopamine lesions of the SNc, the firing rate increased significantly to 18.3 +/- 3.0 spikes/s compared with normal rats. In addition, the firing pattern changed significantly: 70% of the neurons discharged regularly, 27% irregularly and 3% in bursts. In rats with ibotenic acid lesions of the STN, the firing rate decreased significantly to 7.2 +/- 0.9 spikes/s and the firing pattern changed significantly: 50% of the neurons discharged regularly, 43% irregularly and 7% in bursts. The rats with combined SNc and STN lesions showed no change in the firing rate (8.5 +/- 1.0 spikes/s) compared to normal rats. The firing pattern changed significantly: 69% of the cells discharged regularly, 26% irregularly and 5% in bursts. These findings demonstrate that PPN neurons are overactive and more irregular in the 6-hydroxydopamine-lesioned rats, suggesting the implication of this nucleus in the pathophysiology of parkinsonism. Moreover, the fact that STN lesions induced a reduction in the firing rate of the PPN in normal rats and a normalization of the firing rate in rats with 6-hydroxydopamine lesions suggests that this nucleus is under major control of the STN.

Changes in the firing pattern of globus pallidus neurons after the degeneration of nigrostriatal pathway are mediated by the subthalamic nucleus in the rat.

Changes in the neuronal activity of globus pallidus (GP) have been shown in animal models of parkinsonism. In order to study the implication of the subthalamic nucleus (STN) in these changes, the effects of STN lesions alone or in combination with 6-hydroxydopamine (6-OHDA) -induced damage to the substantia nigra compacta (SNc) were examined in rats using electrophysiological recordings of GP cells. In normal rats, the firing rate was 22.1+/-1.4 spikes/s. The pattern was regular in 45%, irregular in 49% and bursty in 6% of the cases. In rats with STN lesions, the firing rate of GP units (20.15+/-1.25 spikes/s) did not differ from that of normal rats and only regular (46%) and irregular (54%) cells were found; a bursty pattern was not observed. 6-OHDA lesions of the SNc induced no change in the firing rate of GP neurons (21.5+/-1.4 spikes/s, P>0.05) but a significant decrease in the percentage of regular cells (27%, P<0.001), a significant increase in burst cells (21%, P<0.001) with no change in the percentage of irregular units (52%) were observed. In rats with combined SNc and STN lesions, the firing pattern did not change from that of normal rats. The present results show that STN lesions induced the disappearance of bursts in normal rats and normalization of firing pattern in the GP units of rats with 6-OHDA lesions suggesting that the STN plays an important role in the modulation of the pattern of activity of GP neurons which may account for the therapeutic effect of STN lesions in Parkinson's disease.

Effect of high-frequency stimulation of the subthalamic nucleus on the neuronal activities of the substantia nigra pars reticulata and ventrolateral nucleus of the thalamus in the rat.

Electrophysiological recordings were made in anaesthetized rats to investigate the mode of function of high-frequency stimulation of the subthalamic nucleus used as a therapeutic approach for Parkinson's disease. High-frequency electrical stimulation of the subthalamic nucleus (130 Hz) induced a net decrease in activity of all cells recorded around the site of stimulation in the subthalamic nucleus. It also caused an inhibition of the majority of neurons recorded in the substantia nigra pars reticulata in normal rats (94%) and in rats with 6-hydroxydopamine lesions of the substantia nigra pars compacta (90%) or with ibotenic acid lesions of the globus pallidus (79.5%). The majority of cells recorded in the ventrolateral nucleus of the thalamus responded with an increase in their activity (84%). These results show that high-frequency stimulation of the subthalamic nucleus induces a reduction of the excitatory glutamatergic output from the subthalamic nucleus which results in deactivation of substantia nigra pars reticulata neurons. The reduction in tonic inhibitory drive of nigral neurons induces a disinhibition of activity in the ventrolateral motor thalamic nucleus, which should result in activation of the motor cortical system.

Muscle transfection by electroporation with high-voltage and short-pulse currents provides high-level and long-lasting gene expression.

Gene transfer into muscle by electroporation with low-voltage and long-pulse (LV/LP, 100 V/50 msec) currents was shown to be more efficient than simple intramuscular DNA injection. Nevertheless, transgene expression declined from day 7 and only reached 10% of the maximum 3 weeks after electroporation. We have optimized electroporation conditions including voltage, pulse number, and the amount of injected luciferase-encoding plasmid DNA in the tibialis anterior muscle. Using high-voltage and short-pulse (HV/SP, 900 V/100 microsec) currents, we observed an average 500-fold increase in luciferase expression, in comparison with nonelectroporated muscle. Moreover, sustained and long-lasting gene expression was observed for at least 6 months. When we compared HV/SP currents with LV/LP currents, luciferase expression was similar 24 hr after electroporation. One month later, whereas luciferase expression was stable in muscle electroporated with HV/SP currents, it decreased 600-fold in muscle electroporated with LV/LP currents. In conclusion, electroporation with high-voltage and short-pulse currents provides high-level and long-lasting gene expression in muscle.

Therapeutic efficacy of the thymidine kinase/ganciclovir system on large experimental gliomas: a nuclear magnetic resonance imaging study.

Contradictory experimental results and human trials have questioned the clinical relevance of the HSVtk/ganciclovir system. To bypass the problem of transfection efficiency, we used a glioma cell line stably expressing the HSVtk gene, which was also fully characterized from gene to protein. We also designed a more clinically relevant experimental protocol, consisting of late GCV delivery on large tumor formations. In short-term studies, histological examination revealed a significant decrease in tumor volume in GCV-treated animals from day 1 or from day 10 after cell inoculation. We observed that late GCV delivery is as efficient as early delivery, probably because GCV can reach tumor cells more easily when neoangiogenesis occurs. In long-term experiments, the survival of treated rats bearing 15-day tumors was improved by 60% compared with C6 control animals. Surprisingly, a 30% survival rate was observed in C6TK control animals. Nuclear magnetic resonance imaging demonstrated, in all surviving animals, a complete regression of tumors without mass effect. These results clearly demonstrate that the HSVtk/GCV system remains a potent therapeutic strategy, even when tested in large tumors, in contrast with the microscopic tumor formations previously reported.

Estradiol modulation of GNRH-stimulated LH release in rat anterior pituitary cells: involvement of dihydropyridine-sensitive calcium channels.

Although enhancement of GnRH-stimulated luteinizing hormone (LH) release by estradiol (E2) has been established, it is not known at what stages of the process of transduction E2 acts. We investigated the release of LH in response to GnRH and to Bay K 8644, an activator of L-type calcium channels, in a culture of pituitary cells obtained from ovariectomized females, these cells having being treated or not with E2 (OVX + E2 and OVX). We studied the effects of D600, an antagonist of T- and L-type calcium channels, and PN 200-110, an antagonist of L-type calcium channels. The effects of the latter were studied in protein kinase C-depleted cells in order to investigate the possible phosphorylation of these channels. D600 caused a decrease in GnRH-stimulated LH release in OVX and OVX + E2 cells. However, this decrease was greater in OVX + E2 cells, suggesting that at least one type of calcium channels may be involved as a result of treatment with E2. We confirmed the involvement of L-type calcium channels in the action of GnRH since the GnRH-stimulated LH release was enhanced in the presence of Bay K 8644 in OVX cells. Bay K 8644 alone increased basal LH in a dose-dependent manner only in OVX + E2 cells. PN 200-110 induced a decrease of GnRH-stimulated LH release only in OVX + E2 cells. These results suggest that L-type calcium channels are activated in E2-treated cells. The dose-dependent decrease caused by PN 200-110 in OVX + E2 cells disappeared in the OVX + E2 PKC-depleted cells. This result was confirmed with Bay K 8644 and suggests a phosphorylation of dihydropyridine-sensitive calcium channels by protein kinase C.

Gonadotropin-releasing hormone-induced changes of intracellular pH in pituitary gonadotrophs: influence of estradiol.

Using the pH indicator, seminaphtorhodafluor, we studied the effects of GnRH on intracellular pH (pHi) in single gonadotroph cells, obtained from 3-week ovariectomized rats, treated or not with estradiol (E2) (OVX + E2, OVX). In a majority of cells (77.7% for OVX cells and 93.7% for OVX + E2 cells), GnRH induced acidification. A biphasic change of pHi, acidification followed by alkalinization, was observed in about 44% of the cells tested. In E2-treated cells, amplitude of acidification and duration of alkalinization were increased. Acidification and Ca2+ mobilization were related in time with a short delay (4-5 sec.). Depolarization with KCl and ionomycin, a Ca2+ ionophore, induced acidification. Taken together these observations suggest that acidification was caused by [Ca2+]i increase. When the Na+/H+ exchanger was blocked by amiloride or in Na(+)-free medium, GnRH-induced alkalinization was inhibited. Alkalinization disappeared completely when the cells were depleted in protein kinase C (PKC). Nevertheless, acute application of phorbol myristate acetate, known to activate PKC, was not sufficient to induce alkalinization. We conclude that PKC is necessary but not sufficient for alkalinization. In contrast, the GnRH response can be mimicked by a simultaneous application of phorbol myristate acetate and KCl. To further explore the putative role of pHi in the secretory process, LH release was studied. Using Na(+)-free medium or amiloride, we show that basal LH was not dependent upon the Na+/H+ exchanger activity. Conversely, GnRH-induced LH release was significantly decreased; this decrease was greater in E2-treated cells but prevented by bicarbonate. These data show that pHi and the Na+/H+ exchanger play an important role in the stimulus secretion coupling process of gonadotrophs. E2, which is an important factor in the regulation of gonadotropic hormone release, participates also in the pHi variations.

Transient but not oscillating component of the calcium mobilizing response to gonadotropin-releasing hormone depends on calcium influx in pituitary gonadotrophs.

Gonadotropin-releasing hormone (GnRH)-stimulated changes in the cytosolic free Ca2+ concentration ([Ca2+]i) were studied in gonadotrophs cultured from 3-week ovariectomized rat pituitaries. One animal was used per cell preparation. [Ca2+]i was monitored in individual gonadotrophs by dual emission microspectrofluorimetry, using Indo-1 as the intracellular fluorescent Ca2+ probe. A short stimulation with GnRH evoked a complex concentration-dependent Ca2+ response in individual gonadotrophs. 0.1-1 nM GnRH triggered a series of sinusoidal-like [Ca2+]i oscillations superimposed upon a modest slow [Ca2+]i rise--the oscillating response mode--while 10-100 nM GnRH caused a biphasic increase in [Ca2+]i consisting of a monophasic transient and oscillations--the transient/oscillating response mode. Despite the consistency of Ca2+ responses, an inter-preparation heterogeneity of [Ca2+]i oscillations frequency was noticed. Moreover, we observed that, within a given cell preparation, the frequency of [Ca2+]i oscillations was independent of GnRH concentration whereas both peak [Ca2+]i and area under the [Ca2+]i versus time curve were concentration-dependent. Thus, in gonadotrophs, the presence of the GnRH signal would lead to [Ca2+]i oscillations, while the amplitude of the [Ca2+]i responses would code for the concentration of agonist. Both transient and oscillating components of GnRH responses depended on releasing activity of Ca(2+)-sequestering pools in as much as GnRH responses were unaffected by brief removal of external Ca2+, but suppressed by chelating intracellular free Ca2+ with BAPTA. However, prolonged exposure to a Ca(2+)-free medium suppressed the transient component while leaving the oscillating component unaffected. We therefore propose that gonadotrophs employ Ca(2+)-sequestering pools, whose maintenance depends on a slow Ca(2+)-entry, to give an amplitude-coded Ca2+ rise in response to a short GnRH stimulation.