Changes in Dendritic Spine Density and Inhibitory Perisomatic Connectivity onto Medium Spiny Neurons in L-Dopa-Induced Dyskinesia.

Using bacterial artificial chromosome-double transgenic mice expressing tdTomato in D1 receptor-medium spiny neurons (MSNs) and enhanced green fluorescent protein in D2 receptor-MSNs, we have studied changes in spine density and perisomatic GABAergic boutons density in MSNs of both the D1R and D2R pathways, in an experimental model of parkinsonism (mouse injected with 6-hydroxydopamine in the medial forebrain bundle), both in the parkinsonian and dyskinetic condition induced by L-DOPA treatment. To assess changes in perisomatic GABAergic connectivity onto MSNs, we measured the number of contacts originated from parvalbumin (PV)-containing striatal "fast-spiking" interneurons (FSIs), the major component of a feed-forward inhibition mechanism that regulates spike timing in MSNs, in both cell types as well as the number of vesicular GABA transporter (VGAT) contacts. Furthermore, we determined changes in PV-immunoreactive cell density by PV immunolabeling combined with Wisteria floribunda agglutinin (WFA) labeling to detect FSI in a PV-independent manner. We also explored the differential expression of striatal activity-regulated cytoskeleton-associated protein (Arc) and c-Fos in both types of MSNs as a measure of neuronal activation. Our results confirm previous findings of major structural changes in dendritic spine density after nigrostriatal denervation, which are further modified in the dyskinetic condition. Moreover, the finding of differential modifications in perisomatic GABAergic connectivity and neuronal activation in MSNs suggests an attempt by the system to regain homeostasis after denervation and an imbalance between excitation and inhibition leading to the development of dyskinesia after exposure to L-DOPA.

Turning behaviour induced by stimulation of the 5-HT receptors in the subthalamic nucleus.

The basal ganglia, which receive a rich serotonergic innervation, have been implicated in hyperkinetic and hypokinetic disorders. Moreover, a decrease in subthalamic nucleus (STN) activity has been associated with motor hyperactivity. To address the role of subthalamic serotonergic innervation in its motor function, turning behaviour was studied in rats with stimulation of the subthalamic serotonin (5-HT) receptors by intracerebral microinjections. The intrasubthalamic administration of 5-HT induced dose-dependent contralateral turning behaviour, with a maximal effect at a dose of 2.5 microg in 0.2 microL. Similar results were observed with microinjections of other 5-HT receptor agonists: quipazine (a 5-HT2B/C/3 agonist), MK-212 (a 5-HT2B/C agonist) and m-chlorophenylbiguanidine (a 5-HT3 agonist), while microinjections of 5-HT into the zona incerta or in the previously lesioned STN were ineffective. The effect of 5-HT was blocked by coadministration of the antagonist mianserin. Stimulation of subthalamic 5-HT receptors in animals bearing a lesion of the nigrostriatal pathway did not modify the motor response, which indicates that the dopamine innervation of the nucleus is not involved in this effect. Kainic acid lesion of the substantia nigra pars reticulata (SNr) suppressed the contralateral rotations elicited by stimulation of 5-HT2B/C/3 subthalamic receptors. This suggests a role of the subthalamic-nigral pathway in the turning activity. Furthermore, the partial blockade of glutamatergic receptors in the SNr by the antagonist DNQX increased the contralateral circling elicited by stimulation of 5-HT receptors in the STN. We concluded that the activation of the 5-HT2B/C and 5-HT3 subthalamic receptors elicited contralateral turning behaviour, probably via the subthalamic-nigral pathway.

Stimulation of dental pulp with simultaneous recording of the jaw opening reflex in the rat.

The processing of nociceptive information in the central nervous system has been analysed in most studies by activation of peripheral nerves. However, the limitation of this method is the simultaneous activation of noxious and inocuous fibers. Nevertheless, the stimulation of the tooth pulp is believed to activate mainly nociceptive fibers which could be the method of choice. On the other hand, the response to nociceptive activation of the dental pulp is easily quantified by the amplitude of the jaw opening reflex, a nocifensive flexion withdrawal reflex. In this protocol we describe a technique to manufacture and implant electrodes in lower incisors of the rat and a method to prepare and insert stainless steel twisted bipolar electrodes to record the electromyographic activity of both digastric muscles, in response to nociceptive dental pulp stimulation. This approach was applied in the study of the analgesic effects of the rat's striatal stimulation.

Pharmacological involvement of the calcium channel blocker flunarizine in dopamine transmission at the striatum.

Single intrastriatal microinjections of 25, 50 and 100nmol/microl of flunarizine in normal rats produced a dose-dependent turning behavior toward the injected side when they were challenged with apomorphine (1mg/kg, s.c). This effect was seen at 1, 3 and 7 days following administration of the high dose of flunarizine, but had subsided by 24h after administration of the intermediate dose; the low dose was ineffective. However, intrastriatal injection of the high dose of flunarizine resulted in a local lesion and thereafter this dose was not used. A similar dose-response relationship was determined for nifedipine, an L-type calcium channel antagonist. Injection of this antagonist did not result in apomorphine-elicited rotational behavior, reflecting its lack of antidopaminergic action. Intrastriatal injections of haloperidol (5microg/microl), an antagonist of dopamine D(2) receptors, or the sodium channel blocker lidocaine (40microg/microl), were given in order to compare their effects to those observed with flunarizine. Intracerebral injection of haloperidol produced ipsilateral turning in response to systemic administration of apomorphine given 60min after. The same response was obtained with the injection of apomorphine 10min after the injection of intracerebral lidocaine. This effect was no longer apparent 24h after the microinjection of haloperidol and 60min after the injection of lidocaine. In rats rendered hemiparkinsionian by lesioning the nigrostriatal pathway with 6OHDA, intrastriatal microinjection of flunarizine (50nmol/microl) significantly reduced apomorphine (0.2mg/kg, s.c.)-elicited turning behavior towards the non-lesioned side. These results suggest an antidopaminergic effect of flunarizine mediated by antagonistic action of post-synaptic striatal dopamine receptors. However, an action of the drug on sodium channels may not be ruled out. These studies offer additional supporting evidence for the induction or aggravation of extrapyramidal side-effects in patients receiving flunarizine.

Striatal modulation of the jaw opening reflex.

The effect of striatal electrical and chemical conditioning stimulation (L-glutamate 80-160 nmoles/0.5 microl) on the jaw opening reflex (JOR) was studied in Sprague-Dawley male rats anesthetized with urethane. The JOR was evoked by stimulation of the tooth pulp of lower incisors. This response was suppressed by transection of the dental root, which indicates according with the bibliography, a specific activation of the pulp nerves. Three type of responses were obtained on the evoked JOR by conditioning stimulation of the striatum; being the main one the suppression of the reflex elicited by tooth pulp activation. A second type of response was an increase of the tooth-JOR amplitude. This effect was observed more frequently with glutamate stimulation rather than with electrical activation of the striatum. A third response was observed with chemical stimulation but not by electrical stimulation of the striatum. This was a triphasic response which consisted in an increase followed by an inhibition and a late increase of the tooth-JOR amplitude. A biphasic effect, an increase prior to a decrease of the JOR amplitude, was also recorded with a minor frequency. The distribution of effective sites for electrical and chemical stimulation within the striatum are mainly similar located in the rostral aspect of the nucleus, with the inhibitory sites in the middle of the nucleus and intermingled with the excitatory ones. The complex responses (tri/biphasic) were observed ventrally and caudally in the nucleus. On the basis of the results mentioned above, one could assume that the striatum is related to the modulation of the JOR evoked probably by nociceptive stimulation. However, activation of other type of fibers could not be ruled out.

Substantia nigra pars reticulata units in 6-hydroxydopamine-lesioned rats: responses to striatal D2 dopamine receptor stimulation and subthalamic lesions.

In order to increase our understanding of Parkinson's disease pathophysiology, we studied the effects of intrastriatally administered selective dopamine receptor agonists on single units from the substantia nigra pars reticulata of 6-hydroxydopamine (6-OHDA)-lesioned rats with or without an additional subthalamic nucleus lesion. Nigral pars reticulata units of 6-OHDA-lesioned rats were classified into two types, showing regular and bursting discharge patterns, respectively ('non-burst' and 'burst' units). Non-burst and burst units showed distinct responses to intrastriatal quinpirole (the former were excited and burst units inhibited). Furthermore, subthalamic nucleus lesions significantly decreased the number of nigral units showing a spontaneous bursting pattern, and reduced the proportion of units that responded to quinpirole. In contrast, subthalamic lesions did not alter the proportion of nigral units that responded to SKF38393, although the lesions changed some response features, e.g. response type and magnitude. Burst analysis showed that quinpirole did not modify the discharge pattern of burst units, whereas SKF38393 produced a shift to regular firing in 62% of the burst units tested. In conjunction, our results support that: (i) the subthalamic nucleus has an important influence on output nuclei firing pattern; (ii) striatal D2 receptors have a strong influence on nigral firing rate, and a less relevant role in controlling firing pattern; (iii) burst and non-burst units differ in their response to selective stimulation of striatal dopamine receptors; (iv) the effects of striatal D2 receptors on nigral units are mainly, though not exclusively, mediated by the subthalamic nucleus; and (v) nigral responses to SKF38393 involve the subthalamic nucleus.