Tiziana Florio

Evidence that non-NMDA receptors are involved in the excitatory pathway from the pedunculopontine region to nigrostriatal dopaminergic neurons

SummaryExtracellular single-neuron recordings were obtained from electrophysiologically identified nigrostriatal neurons in chloral hydrate anesthetized rats, in order to test the hypothesis that excitatory amino acid receptors are involved in responses of these neurons to electrical stimulation of the pontine region where the pedunculopontine nucleus (PPN) is located. The effects of iontophoretic application of excitatory amino acids and their antagonists as well as of cholinergic antagonists were tested on the fast orthodromic excitation of nigrostriatal neurons evoked by stimulation of the PPN region. The N-methyl-D-aspartate (NMDA) receptor antagonist D-a-aminoadipic acid as well as the cholinergic receptor antagonists mecamylamine and atropine failed to suppress the synaptic excitation of nigral neurons. The NMDA receptor antagonist DL-2-amino-5-phosphonovalerate exerted a weak depressant action on the synaptic response in a few neurons only. On the contrary, the broad spectrum antagonists of excitatory amino acid receptors kynurenic acid and gamma-Dglutamyl-amino-methyl-sulphonate were found to block simultaneously both the synaptic excitation and the neuronal responses to iontophoretic pulses of glutamate while leaving unaffected the neuronal responses to local application of acetylcholine or carbachol. The competitive antagonist of non-NMDA receptors 6-cyano-2,3-dihy-droxy-7-nitro-quinoxaline suppressed the synaptic excitation at ejection currents which antagonized neuronal responses to quisqualate and kainate. These results suggest that PPN excitatory fibers synapsing onto pars compacta nigrostriatal neurons utilize an excitatory amino acid as a synaptic transmitter acting preferentially on non-NMDA receptors.

High-frequency stimulation of the subthalamic nucleus modulates the activity of pedunculopontine neurons through direct activation of excitatory fibres as well as through indirect activation of inhibitory pallidal fibres in the rat

Recent data suggest a potential role of pedunculopontine nucleus (PPN) electrical stimulation in improving gait and posture in Parkinsons disease. Because the PPN receives fibres from the subthalamic nucleus (STN), we investigated the effects of STN‐high‐frequency stimulation (HFS) on PPN neuronal activity in intact rats and in rats bearing either an ibotenate lesion of the entopeduncular nucleus (EP) or a lesion of the substantia nigra (SN). The main response of PPN neurons to STN single‐shock stimulations in the three experimental groups was a short latency (4.5 ± 2.1 ms) and brief (15.3 ± 6.5 ms) excitation. This response was maintained during 1–5 s of STN‐HFS (130 Hz, 60 µs, 100–1000 µA). In EP‐lesioned rats the percentage (75.0%) of PPN neurons showing a modulation of activity following STN‐HFS was significantly higher compared with that observed in intact (39.7%) and in SN‐lesioned rats (35.4%). Furthermore, in EP‐lesioned rats the most frequent response of PPN neurons following STN‐HFS was a 5–20 s excitation, which was present in 76.6% of responsive neurons in comparison to 15.4% and 9.1% of neurons responsive in intact and in 6‐hydroxydopamine‐lesioned rats, respectively. Neurons responsive to STN‐HFS in the three experimental groups showed either a sharp positively skewed distribution of interspike intervals or multisecond oscillations in autocorrelograms. The results support that STN‐HFS modulates the PPN through a balance of excitatory and inhibitory influences, which may be independent from the dopaminergic nigral neurons. In the absence of inhibitory EP fibres, the direct excitatory influence exerted by the STN on the PPN appears to predominate.

The pedunculopontine nucleus projection to the parafascicular nucleus of the thalamus: an electrophysiological investigation in the rat.

Summary. Extracellular electrophysiological recordings of neurons of the parafascicular nucleus of the thalamus were done in normal rats and in rats bearing lesions of either the cerebellar nuclei or the entopeduncular nucleus to investigate the functional control of the pedunculopontine nucleus on the parafascicular nucleus. A total of 97 neurons were recorded in the parafascicular nucleus in intact rats, 83 in rats bearing a chronic electrolytic lesion of the ipsilateral deep cerebellar nuclei, and 69 in rats bearing an ibotenate lesion of the ipsilateral entopeduncular nucleus. Lesions of the cerebellar nuclei or the entopeduncular nucleus were made to evaluate the participation of cerebellothalamic fibers or of polysynaptic basal ganglia circuits in the responses recorded in parafascicular neurons following electrical microstimulation of the ipsilateral pedunculopontine nucleus. Two types of excitation and one type of inhibition were the main responses observed in neurons of the parafascicular nucleus following stimulation of the pedunculopontine nucleus. The first type of excitation, observed in 49.5% of neurons recorded in normal rats, had an onset of 1.8 ± 0.6 ms, lasted 9.2 ± 0.8 ms and was able to follow high frequency stimulation over 300 Hz. The second type of excitation, observed in a smaller percentage of neurons recorded (3.1%), was a long-latency (8.3 ± 0.7 ms) activation lasting 19.0 ± 4.5 ms. It did not follow stimulation frequencies higher than 50–100 Hz. The inhibitory response was observed in 17.5% of the neurons recorded. The latency of this inhibition was 4.5 ± 1.8 ms and the duration 41.9 ± 6.8 ms. In rats bearing a lesion of the deep cerebellar nuclei or of the entopeduncular nucleus, the short-latency activation was still present in 24.1% and 31.9% of neurons recorded, respectively. However, the occurrence of the long-latency excitation rats bearing lesions of either the cerebellum or the entopeduncular nucleus increased to 12.1% and to 17.4%, respectively, while the occurrence of the inhibition rose to 22.9% and to 28.9%. These results show that an excitatory influence on the parafascicular nucleus is exerted by the pedunculopontine nucleus irrespectively of the presence of cerebellofugal fibers. This influence appears to be also independent from the integrity of basal ganglia circuits having a relay at the level of the entopeduncular nucleus. However, the variety of responses recorded suggests that the influences of the pedunculopontine nucleus on the parafascicular nucleus are by far more complex than those exerted on its basal ganglia targets such as the substantia nigra. The results are discussed according to a model of functioning of pedunculopontine fibers directed to thalamic and basal ganglia nuclei.

The function of the pedunculopontine nucleus in the preparation and execution of an externally-cued bar pressing task in the rat

In the present study the role of the pedunculopontine nucleus (PPN) in the preparation and execution of an externally-cued rewarded motor act was investigated. Animals were instructed to press down a lever at the presentation of a combined visual and acoustic signal and were required to hold down the lever until a trigger stimulus occurred after an unpredictable delay ranging from 2 to 4 s. The trigger stimulus required animals to release the lever and to press a second lever for food reinforcement. The time between instruction and trigger signals represented the preparation phase preceding movement. Unilateral ibotenic acid-induced focal degeneration of pedunculopontine neurons did not influence either reaction and movement times, or capacity of the animals to correctly respond to presentation of stimuli of behavioral significance. On the contrary, bilateral lesions increased both reaction and movement times, and dramatically reduced the percentage of correct responses. The analysis of incorrect responses suggested that the most striking deficit exhibited by the animals following the bilateral lesion was a lack of conditioned response to the signal initiating each trial. However, the animals retained the capability to respond correctly in some trials, and were able to collect the reward when delivered outside the behavioral context. Histological analysis of lesions showed that in addition to loss of neurons within the pedunculopontine region, reduction of tyrosine-hydroxylase positive neurons had occurred in the pars compacta of the substantia nigra. The data suggest that the PPN is involved in the preparation and execution of externally-cued movements, and demonstrate that its destruction mimics the main effects produced by the dopaminergic denervation of the dorsal striatum.

Influence of prelimbic and sensorimotor cortices on striatal neurons in the rat: electrophysiological evidence for converging inputs and the effects of 6-OHDA-induced degeneration of the substantia nigra

These studies were designed to investigate whether there are convergent prelimbic and sensorimotor cortical inputs onto striatal neurons in the rat and whether dopaminergic (DA) nigrostriatal fibers regulate these inputs. The influence of the nigrostriatal DA system was assessed in rats with either small or large 6-hydroxydopamine-induced lesions of the substantia nigra. In normal rats 39 out of 74 neurons (52.7%) were excited by stimulation of both the prelimbic and the sensorimotor cortex. No marked change in corticostriatal transmission was evident in rats with small 6-OHDA-induced lesions (defined as 10-35% decrease in [3H]DA uptake in striatal synaptosomes). In rats with large lesions (75-85% decrease in striatal [3H]DA uptake), however, a complete rearrangement of the corticostriatal transmission occurred. This was evident in a decrease of thresholds to obtain cortical responses, by modifications of the discharge properties of striatal neurons receiving input from cortices and by an increase in the number of neurons responding to cortical stimulation. In addition, a significantly higher percentage of striatal neurons responded to stimulation of both prelimbic and sensorimotor cortices in rats with large lesions than in rats with small lesions or in control rats. This data suggests that: (1) no functional separation of prelimbic and sensorimotor cortical inputs occurs in the rat striatum, (2) the nigrostriatal DA system exerts a focusing effect on these inputs, (3) the striatum is actively involved in the integrative processing of descending cortical information.

Targeting CXCR1 on breast cancer stem cells: signaling pathways and clinical application modelling

In breast cancer it has been proposed that the presence of cancer stem cells may drive tumor initiation, progression and recurrences. IL-8, up-regulated in breast cancer, and associated with poor prognosis, increases CSC self-renewal in cell line models. It signals via two cell surface receptors, CXCR1 and CXCR2. Recently, the IL-8/CXCR1 axis was proposed as an attractive pathway for the design of specific therapies against breast cancer stem cells. Reparixin, a powerful CXCR1 inhibitor, was effective in reducing in vivo the tumour-initiating population in several NOD/SCID mice breast cancer models, showing that the selective targeting of CXCR1 and the combination of reparixin and docetaxel resulted in a concomitant reduction of the bulk tumour mass and CSC population. The available data indicate that IL-8, expressed by tumour cells and induced by chemotherapeutic treatment, is a key regulator of the survival and self-renewal of the population of CXCR1-expressing CSC. Consequently, this investigation on the mechanism of action of the reparixin/paclitaxel combination, was based on the observation that reparixin treatment contained the formation of metastases in several experimental models. However, specific data on the formation of breast cancer brain metastases, which carry remarkable morbidity and mortality to a substantial proportion of advanced breast cancer patients, have not been generated. The obtained data indicate a beneficial use of the drug combination reparixin and paclitaxel to counteract brain tumour metastasis due to CSC, probably due to the combined effects of the two drugs, the pro-apoptotic action of paclitaxel and the cytostatic and anti-migratory effects of reparixin.

The pedunculopontine tegmental nucleus: implications for a role in modulating spinal cord motoneuron excitability.

There is evidence that deep brain stimulation (DBS) of the pedunculopontine tegmental nucleus (PPTg) improves parkinsonian motor signs. The mechanisms that mediate these effects and the modifications that occur in the PPTg in Parkinson’s disease (PD) are not fully known and are the object of current debate. The aim of this paper was to critically review available data with respect to (1) the presence of PPTg neurons linked to reticulospinal projections, (2) the involvement of these neurons in modulating spinal reflexes, and (3) the participation of fibers close to or within the PPTg region in such modulation. The PPTg neurons are distributed in a large pontotegmental region, stimulation of which can evoke activity in hindlimb, shoulder and neck muscles, and potentiate motor responses evoked by stimulation of dorsal roots. This influence seems to be carried out by fast-conducting descending fibers, which likely run in the medial reticulospinal pathway. It is yet unclear which neurotransmitters are involved and on which elements of the gray matter of the spinal cord PPTg fibers synapse. The modulation of spinal cord activity which can be achieved by stimulating the PPTg region seems to be mediated not only by PPTg neurons, but also by tecto-reticular fibers which run in the pontotegmental area, and which likely are activated during PPTg-DBS. The importance of these fibers is discussed taking into account the degeneration of PPTg neurons in PD and the benefits in gait and postural control that PPTg-DBS exerts in PD. The potential usefulness of PPTg-DBS in other neurodegenerative disorders characterized by neuronal loss in the brainstem is also considered.

Nucleolin antagonist triggers autophagic cell death in human glioblastoma primary cells and decreased in vivo tumor growth in orthotopic brain tumor model

Nucleolin (NCL) is highly expressed in several types of cancer and represents an interesting therapeutic target. It is expressed at the plasma membrane of tumor cells, a property which is being used as a marker for several human cancer including glioblastoma. In this study we investigated targeting NCL as a new therapeutic strategy for the treatment of this pathology. To explore this possibility, we studied the effect of an antagonist of NCL, the multivalent pseudopeptide N6L using primary culture of human glioblastoma cells. In this system, N6L inhibits cell growth with different sensitivity depending to NCL localization. Cell cycle analysis indicated that N6L-induced growth reduction was due to a block of the G1/S transition with down-regulation of the expression of cyclin D1 and B2. By monitoring autophagy markers such as p62 and LC3II, we demonstrate that autophagy is enhanced after N6L treatment. In addition, N6L-treatment of mice bearing tumor decreased in vivo tumor growth in orthotopic brain tumor model and increase mice survival. The results obtained indicated an anti-proliferative and pro-autophagic effect of N6L and point towards its possible use as adjuvant agent to the standard therapeutic protocols presently utilized for glioblastoma.

Unilateral lesions of the pedunculopontine nucleus do not alleviate subthalamic nucleus-mediated anticipatory responding in a delayed sensorimotor task in the rat.

Lesions of the subthalamic nucleus (STN) in the rat are known to cause anticipated movements in behavioral tasks requiring a preparatory period before the execution of externally cued conditioned movements. In the present study, we describe the effects of lesions of the pedunculopontine nucleus (PPN), a structure located on the outflow of the STN to lower brainstem and spinal motor nuclei, on the anticipatory responding caused by a unilateral lesion of the STN in a delayed sensorimotor task. Rats were instructed to keep a lever pressed down by the presentation of a composite visual and acoustic signal, and were required to hold the lever pressed until a trigger stimulus occurred after an unpredictable delay. The trigger stimulus required the animals to release the lever and to press a second lever for food reinforcement. The task was evaluated according to analysis of movement parameters and errors made by the animals during the preparative and executive phases of the conditioned movement. An ibotenate lesion was placed into the STN in either side of the brain. This lesion was followed 3 weeks later by an ibotenate lesion of the PPN ipsilaterally to the STN previously lesioned. The results indicate that the anticipatory responding induced by the STN lesion was not alleviated by the subsequent PPN lesion. However, the animals bearing the combined lesion were severely impaired in conditioned responding to salient stimuli involved in the paradigm and showed side-specific lengthening of reaction and movement times without global motor impairments. The results suggest that the anticipatory responses caused by STN lesions do not require the intervention of the PPN and that the disruption of the dopaminergic nigrostriatal pathway following the combined lesion may be responsible for impairments observed.

Dopamine denervation of specific striatal subregions differentially affects preparation and execution of a delayed response task in the rat

In the present study, the effects of unilateral or bilateral dopamine denervation of either the dorsal or ventral striatum on the preparation and execution of a delayed response task in the rat were investigated. Animals were instructed to hold a lever pressed down by the presentation of a visual and/or acoustic signal, and were required to hold the lever until a trigger stimulus occurred after an unpredictable delay ranging from 2 to 4 s. The trigger stimulus required animals to release the lever and to press a second lever for food reinforcement. The time between instruction and trigger signal represented the preparation phase preceding movement. The motor performance was evaluated by using reaction and movement times in addition to correct responses in each session. Dopaminergic denervation of either the dorsal or ventral striatum ipsilaterally to the side in which the second lever to be pressed was located did not significantly change reaction and movement times, although it reduced the percentage of correct trials. A significant increase of both reaction and movement times was recorded only after bilateral denervation of the ventral striatum. The analysis of incorrect responses indicated that dopaminergic innervation of the two striatal subregions had different functions in the correct execution of the behavioral paradigm. In the group of animals with dorsal lesions the most frequent incorrect response was represented by a lack of the conditioned response to the presentation of the instruction stimulus starting the trial. If the animals reacted properly to this signal, the performance thereafter was correct in the majority of trials. Conversely, animals with ventral lesions exhibited a large repertoire of incorrect responses throughout the paradigm, including premature release or delayed press of levers, and omission of the second lever press. Histological verification of brain coronal sections by tyrosine-hydroxylase immunoreactivity showed that the lesions were confined in either the dorsal or ventral striatum, sparing the lateral region. The data support the hypothesis that dopaminergic innervation enables the two striatal regions to differently participate in the preparation and execution of complex delayed sensorimotor tasks. Indeed, the dorsal striatum seems to be involved in the correct utilization of external sensory information for the initiation of conditioned behavior, whereas, the ventral striatum appears to be mainly concerned with the temporal expectation of impending stimuli that trigger reward-reinforced movements.