A comparative study of secondary metabolites profiling and biological activity of Smyrnium olusatrum L. leaf, flower and fruit.

Essential oil (EO) composition of Smyrnium olusatrum was characterised by high proportion of furanosesquiterpenes (51.66-69.35%). The leaf methanolic extract composition was found to be rich with Quercetin-O-hexoside (39.78%). Apigenin 6,8-di-Chexoside represent the major component of flower (18.2%) and fruits (18.82%). Flower extract exhibited the highest contents of total phenolic (48.97 mg GAE/g) and flavonoid (52.63 mg RE/g). The ß-carotene and lycopene contents were in the order of 4.55-26.14 mg/100g, and 8.00-49.45 mg/100g, respectively. Methanolic extracts and EOs of different organs were found to possess antioxidant activities, as determined by scavenging effect, chelating activity and ß-carotene-linoleic acid model system. Furthermore, Fruit S. olusatrum EO exhibited a potent inhibitory activity against Acetylcholinesterase, while the methanolic extract showed a weaker activity. The methanolic extract displayed inhibitory effects on α-amylase, whereas the EOs was not as efficient in inhibiting this enzyme. The observed level of biological activities varied depending on the specific extracts and organs studied.

Reinforcing effect of tramadol in the rat.

Tramadol is one of the most commonly prescribed analgesic opioids in various pharmacopeias. Tramadol has been linked to abuse in recent clinical investigations. However, the behavioral effects and neural substrates of the drug have not been well characterized in preclinical studies. As a result, the present study investigated the effects of tramadol on behavioral sensitizations in rats. Its impacts on cellular and molecular alterations in the brain were also investigated. In conditioned place preference (CPP) paradigm, tramadol induced behavioral as well as motor sensitizations. These effects were dramatically reduced by intraperitoneal administration of naltrexone, an opioid receptor antagonist. Tramadol caused changes in several molecular markers (pERK1/2, Δ-FosB, PKCγ, PKMζ GAD67) in the anterior cingulate cortex, which could indicate an increase in excitation within this structure. Tramadol is demonstrated in the present study to be a reinforcing drug in rats, as it increased both behavioral and motor sensitizations. Tramadol's effects are most likely due to the high levels of excitation it causes in the brain, which is mostly caused by the activation of opioid receptors.

Naltrexone promotes mechanical allodynia in humans and rats.

Mechanical allodynia has been studied in chronic naltrexone-treated people (N.T.P.) and rats (N.T.R.). After persistent naltrexone administration, patients acquired static and dynamic mechanical allodynia, as measured by von Frey filament (vFf) and brush stimulations. Pregabalin and levodopa administrations in N.T.P. significantly reduced allodynic behaviour, albeit these molecules did not completely stop it. As evidenced by the deployment of the vFf, subchronic treatment with Naltrexone delivered peripherally or intrathecally induced allodynic behaviour in rats. Increased expressions of two pain markers, pERK1/2 and PKCγ, in the spinal dorsal horn laminae were associated with naltrexone-induced allodynic behaviour. After vFf stimulation, pERK1/2 expression was substantially higher (p < 0.001) in superficial spinal dorsal horn laminae than in non-stimulated or naive non-stimulated rats. In addition, when compared to control rats, N.T.R. showed a substantial (p < 0.001) increase in PKCγ expression. PKCγ expression was found to be strong in lamina IIi and laminae III-IV. A cellular mechanism is proposed for the naltrexone effect. In both people and rats, Naltrexone induces static mechanical allodynia, according to this study.

Behavioral, Cellular and Molecular Responses to Cold and Mechanical Stimuli in Rats with Bilateral Dopamine Depletion in the Mesencephalic Dopaminergic Neurons.

Pain is the major non-motor symptom in Parkinson's disease (PD). Preclinical studies have mostly investigated mechanical pain by considering the decrease in a nociceptive threshold. Only a few studies have focused on thermal pain in animal models of PD. Therefore, the goal of this study was to assess the thermal nociceptive behavior of rats subjected to 6-hydroxydopamine (6-OHDA) administration, which constitutes an animal model of PD. Thermal plate investigation demonstrated significant thermal sensitivity to cold temperatures of 10 °C and 15 °C, and not to higher temperatures, in 6-OHDA-lesioned rats when compared with sham. 6-OHDA-lesioned rats also showed cold allodynia as demonstrated by a significant difference in the number of flinches, latency and reaction time to acetone stimulus. Ropinirole administration, a dopamine receptor 2 (D2R) agonist, blocked the acetone-induced cold allodynia in 6-OHDA-lesioned rats. In addition, mechanical hypersensitivity and static allodynia, as demonstrated by a significant difference in the vocalization threshold and pain score respectively, were noticed in 6-OHDA-lesioned rats. Acetone stimulus induced a significant increase in extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) phosphorylation, a pain process molecular marker, in the spinal dorsal horn (SDH), the insular and cingulate cortices in 6-OHDA-lesioned rats when compared to sham. In 6-OHDA-lesioned rats, there was a significant augmentation in the expression of both protein kinase C gamma (PKCγ) and glutamate decarboxylase 67 (GAD67) in the SDH. This highlighted an increase in excitation and a decrease in inhibition in the SDH. Overall, the present study demonstrated a clear cold thermal hypersensitivity, in addition to a mechanical one, in 6-OHDA-lesioned rats.

Nigrostriatal dopamine depletion promoted an increase in inhibitory markers (parvalbumin, GAD67, VGAT) and cold allodynia.

Pain constitutes the major non-motor symptom in Parkinson's disease (PD). Its mechanism is still poorly understood although an increase in excitation or a decrease in inhibition have been reported in preclinical studies. The aim of this study was to investigate gamma aminobutyric acid (GABA) inhibition in the 6-hydroxydopamine (6-OHDA) PD rat model. Therefore, the expression of three inhibitory markers parvalbumin, glutamate decarboxylase 67 (GAD67) and vesicular GABA transporter (VGAT) was evaluated, besides cold allodynia, in bilateral 6-OHDA lesioned rat. There was a significant increase in the expression of the three markers labeling within the spinal dorsal horn (SDH) of 6-OHDA lesioned rats. In parallel, there was also an increase of the excitatory marker protein kinase C gamma (PKCγ) . PKCγ cells have a crucial role in pain chronicity and are regulated by GABAergic influences. Central dopamine depletion induced an increase in excitation as reveled by an increase in cFOS expression upon acetone stimulus and the presence of cold allodynia. In addition, dopamine depletion induced increased expression in inhibitory markers, which may reflect a disinhibition or a decreased inhibition in 6-OHDA lesioned rats.

Electrical Synapses are Involved in Orofacial Neuropathic Pain.

Accumulated evidences suggest important roles of glial GAP-junctions in pain. However, only a few studies have explored the role of neuronal GAP-junctions or electrical synapses in neuropathic pain (NP). Therefore, the present study explores the role of connexin 36 (Cx36) in NP using the chronic constriction injury of the infraorbital nerve (CCI-IoN) model in rat. A significant increase in Cx36 labeling was observed in the medullary dorsal horn (MDH) of CCI-IoN-lesioned compared to sham rats. The expression of Cx36 in CCI-IoN-lesioned rats revealed a rostroventral gradient of punctuate labeling within lamina IIo of the MDH. Cx36-positive somata and processes were also observed in MDH laminae IIi and III-V. These somata were mostly of the Gamma aminobutyric acid (GABA) and occasionally Glycine transporter 2 (GlyT2) cell subtypes. Moreover the GABA cell subtypes are highly coupled in lamina IIo as revealed by the intense Cx36 staining in this lamina. Pharmacological Cx36 blockade by intracisternal administration of mefloquine decreased significantly the mechanical allodynia observed in CCI-IoN-lesioned rats. Altogether, our findings demonstrated that Cx36 play an important role in mechanical allodynia by coupling GABA cells. Increasing cell coupling by enhancing Cx36 expression favors neuropathic pain while disrupting this coupling alleviates it. This mechanism may constitute a novel target for the treatment of orofacial mechanical allodynia.

Striatal changes underlie MPEP-mediated suppression of the acquisition and expression of pramipexole-induced place preference in an alpha-synuclein rat model of Parkinson's disease.

Impulsive-compulsive disorders in Parkinson's disease patients have been described as behavioural or substance addictions including pathological gambling or compulsive medication use of dopamine replacement therapy. A substantial gap remains in the understanding of these disorders. We previously demonstrated that the rewarding effect of the D2/D3 agonist pramipexole was enhanced after repeated exposure to L-dopa and alpha-synuclein mediated dopaminergic nigral loss with specific transcriptional signatures suggesting a key involvement of the glutamatergic pathway. Here, we further investigate the therapeutic potential of metabotropic glutamate receptor 5 antagonism in Parkinson's disease/dopamine replacement therapy related bias of reward-mediated associative learning. We identified protein changes underlying the striatal remodelling associated with the pramipexole-induced conditioned place preference. Acquisition and expression of the pramipexole-induced conditioned place preference were abolished by the metabotropic glutamate receptor 5 antagonist 2-methyl-6-phenylethynyl (pyridine) (conditioned place preference scores obtained with pramipexole conditioning were reduced by 12.5% and 125.8% when 2-methyl-6-phenylethynyl (pyridine) was co-administrated with pramipexole or after the pramipexole conditioning, respectively). Up-regulation of the metabotropic glutamate receptor 5 was found in the dorsomedial-striatum and nucleus accumbens core. Activation of these two brain sub-regions was also highlighted through FosB immunohistochemistry. Convergent molecular and pharmacological data further suggests metabotropic glutamate receptor 5 as a promising therapeutic target for the management of Parkinson's disease/dopamine replacement therapy related reward bias.

Pramipexole induced place preference after L-dopa therapy and nigral dopaminergic loss: linking behavior to transcriptional modifications.

RATIONALE: Impulsive-compulsive disorders (ICD) in patients with Parkinson's disease (PD) have been described as behavioral or substance addictions including hypersexuality, gambling, or compulsive medication use of the dopamine replacement therapy (DRT). OBJECTIVES: A remaining challenge is to understand the neuroadaptations leading to reward bias in PD patients under DRT. METHODS: To this end, the appetitive effect of the D2/D3 agonist pramipexole was assessed after chronic exposure to L-dopa in an alpha-synuclein PD rat model. RESULTS: Association of progressive nigral loss and chronic L-dopa was required to observe a pramipexole-induced place preference. This behavioral outcome was inhibited by metabotropic glutamate receptor 5 (mGluR5) antagonism while transcriptional profiling highlighted regulations potentially related to the context of psychostimulant addiction. CONCLUSION: This study provides evidences strongly suggesting that PD-like lesion and L-dopa therapy were concomitant factors involved in striatal remodeling underlying the pramipexole-induced place preference. Molecular and pharmacological data suggest a key involvement of the glutamatergic pathway in this behavioral outcome.

mGlu5 receptor antagonist blocks bromocriptine-induced conditioned place preference in bilateral mesolimbic-lesioned rat.

Dopamine dysregulation syndrome (DDS) has been attributed to both dopamine replacement therapies (DRT) and the mesencephalic dopaminergic lesion. The DRT reinforcement effect is due to its action on the reward system, particularly on the nucleus accumbens (NAc). This nucleus receives two major projections, a glutamatergic from the prefrontal cortex and a dopaminergic from the posterior ventral tegmental area (pVTA). The latter modulate the former within the NAc. pVTA has been demonstrated to be implicated in the motivational effect of bromocriptine (dopamine 2 receptor (D2R) agonist) in bilateral pVTA-lesioned animals. Therefore the potential implication of the metabotropic glutamate receptor 5 (mGluR5) antagonist (MTEP: 3-((2-Methyl-1,3-thiazol-4-yl)ethynyl)pyridine) on bromocriptine-induced conditioned place preference (CPP) was explored. Results showed that the administration of the MTEP blocked completely the bromocriptine-induced CPP in bilateral pVTA-lesioned rats. Both the CPP acquisition and expression were abolished. These effects are due, at least to an increase of the glutamate concentration and that of mGlu5 receptor expression in the NAc shell of the pVTA-lesioned animals. Altogether these data demonstrated the importance of the mGlu5 receptor in the bromocriptine induced-reinforcement and that DDS is probably due to DRT effect on this glutamate receptor.

Nigrostriatal dopaminergic depletion increases static orofacial allodynia.

BACKGROUND: This study investigated mesencephalic dopamine depletion effects on static mechanical allodynia (SMA) elicited by chronic constriction of the infraorbitary nerve (CCI-IoN). METHODS: Dopamine depletion (6-OHDA administration into the medial forebrain bundle) effects on CCI-IoN-induced SMA were explored using behavioral (nocifensive behavior score upon non-noxious stimuli using von Frey filament), pharmacological (bromocriptine injections) and immunohistochemical (PKCγ and pERK1/2) techniques. RESULTS: The central dopamine depletion increased significantly the SMA score. Intraperitoneal and intracisternal injections of bromocriptine alleviated the allodynic behavior observed in both CCI-IoN and CCI-IoN + 6-OHDA animal groups. At the cellular level, dopamine depletion induced a significant increase in PKCγ expression in the medullary dorsal horn (MDH) in rat with CCI-IoN + 6-OHDA when compared to sham animals (CCI-IoN only). Similarly, after static non-noxious stimuli, the expression of pain marker proteins pERK1/2 within the MDH revealed significantly a higher number of positive cells in CCI-IoN + 6-OHDA rats when compared to the CCI-IoN group. CONCLUSION: This study demonstrates that nigrostriatal dopamine depletion exacerbates the neuropathic pain resulting from CCI-IoN. This effect is probably due to an action through descending pain inhibitory systems which increased pain sensitization at the MDH level. It demonstrates also an analgesic effect elicited by D2R activation at the segmental level.

PKCγ-positive neurons gate light tactile inputs to pain pathway through pERK1/2 neuronal network in trigeminal neuropathic pain model.

AIMS: To explore the possible relationship between protein kinase C gamma (PKCγ) and phosphorylated forms of extracellular signal-regulated kinases 1/2 (pERK1/2) in the rat medullary dorsal horn and the facial hypersensitivity indicative of dynamic mechanical allodynia (DMA) following chronic constriction of the infraorbital nerve (CCI-IoN). METHODS: A well-established rat model of trigeminal neuropathic pain involving CCI-IoN was used. Facial mechanical hypersensitivity was tested with non-noxious dynamic mechanical stimulation (air-puff), and the medullary dorsal horn was examined immunohistochemically using PKCγ and pERK1/2 as pain markers. Statistical analysis was performed using Student t test or one-way analysis of variance (ANOVA). RESULTS: Increased PKCγ and pERK1/2 expressions within the medullary dorsal horn were associated with DMA following CCI-IoN. A segmental network composed of PKCγ-positive cells located in medullary dorsal horn laminae II/III, contacting more superficially located pERK1/2-expressing cells, was identified. Ultrastructural analysis confirmed the presence of PKCγ to pERK1/2-positive cells. Moreover, intracisternal administration of the selective PKCγ inhibitor KIG31-I blocked both the DMA and pERK1/2 expression in a dose-dependent manner. Although the number of pERK1/2-positive cells was significantly elevated with air-puff stimulation, DMA rats not receiving air-puff stimulation showed significant pERK1/2 expression, suggesting they were experiencing spontaneous pain. CONCLUSION: PKCγ cells in the medullary dorsal horn may be involved in DMA following CCI-IoN through the activation of pERK1/2-expressing cells, which then may relay non-nociceptive information to lamina I cells in the medullary dorsal horn.

Lesion of the dopaminergic nigrostriatal pathway induces trigeminal dynamic mechanical allodynia.

BACKGROUND: Pain constitutes the major non motor syndrome in Parkinson's disease (PD) and includes neuropathic pain; however current drug therapies used to alleviate it have only limited efficacy. This is probably due to poor understanding of the mechanisms underlying it. AIMS: We investigated a major class of trigeminal neuropathic pain, dynamic mechanical allodynia (DMA), in a rat model of PD and in which a bilateral 6-hydroxy dopamine (6-OHDA) injection was administered to produce a lesion of the nigrostriatal dopaminergic pathway. RESULTS AND DISCUSSION: Lesioned animals presented significant DMA in the orofacial area that occurred from 4 days to 5 weeks post-injury. To investigate a segmental implication in the neuropathic pain induced by dopamine depletion, the expression of the isoform gamma of the protein kinase C (PKCg) and phosphorylated extracellular signal-regulated kinases 1/2 (pERK1/2) was explored in the medullary dorsal horn (MDH). There was a high increase in PKCg expression in the III and IIi laminae of the MDH of lesioned-animals compared to shams. pERK1/2 expression was also significantly high in the ipsilateral MDH of lesioned rats in response to non-noxious tactile stimulus of the orofacial region. Since pERK1/2 is expressed only in response to nociceptive stimuli in the dorsal spinal horn, the current study demonstrates that non-noxious stimuli evoke allodynic response. Intraperitoneal and intracisternal administrations of bromocriptine, a dopamine 2 receptor (D2R) agonist, significantly decreased DMA compared to control rats injected with saline. These data demonstrate for the first time that nigrostriatal dopaminergic depletion produces trigeminal neuropathic pain that at least involves a segmental mechanism. In addition, bromocriptine was shown to have a remarkable analgesic effect on this neuropathic pain symptom.

Anterior ventral tegmental area dopaminergic neurons are not involved in the motivational effects of bromocriptine, pramipexole and cocaine in drug-free rats.

Dopamine dysregulation syndrome in Parkinson's disease has been attributed to dopamine replacement therapies and/or a lesion of the dopaminergic system. Dopaminergic neuronal loss targets the substantia nigra and the ventral tegmental area (VTA). We hypothesize that dopamine replacement therapy is responsible for the potential reinforcement effect in Parkinson's disease, by acting on the neuronal reward circuitry. We previously demonstrated that the posterior (p) VTA, which projects to the nucleus accumbens (NAc), is implicated in the motivational effect of dopamine receptor agonists in 6-OHDA bilateral pVTA-lesioned drug-free animals. In the present study we investigated the implication of the anterior (a) VTA in the potential reinforcement effect of dopamine receptor agonists. Using the conditioned place preference (CPP) behavioral paradigm, we investigated the motivational effects of dopamine receptor agonists (bromocriptine and pramipexole), and cocaine in rats with a 6-OHDA bilateral lesion of the aVTA. Bromocriptine and pramipexole did not induce a significant CPP at 1mg/kg in both sham and bilateral 6-OHDA-lesioned rats. However bromocriptine induced CPP only at a dose of 3mg/kg in both animal groups. Moreover cocaine, which is known to increase dopamine release, induced reinforcing effects in both 6-OHDA-lesioned and sham rats. Our data show a lack of involvement of aVTA dopamine neurons in the motivational effects of bromocriptine, pramipexole and cocaine.

Differential behavioral reinforcement effects of dopamine receptor agonists in the rat with bilateral lesion of the posterior ventral tegmental area.

Dopamine dysregulation syndrome in Parkinson's disease has been attributed to dopamine replacement therapies and/or a lesion of the dopaminergic system. The dopaminergic neuronal loss targets the substantia nigra and the ventral tegmental area (VTA). We hypothesize that dopamine replacement therapy is responsible for the potential reinforcement effect in Parkinson's disease by acting on the neuronal reward circuitry. Therefore this study was designed to explore the potential motivational effect of dopamine replacement therapy in bilateral VTA-lesioned animals. The posterior (p)VTA, which project to the nucleus accumbens (NAc) constitutes the major dopamine neuronal circuitry implicated in addictive disorders. Using the conditioned place preference (CPP) behavioral paradigm, we investigated the motivational effects of dopamine receptor agonists, and cocaine in rat with a 6-OHDA bilateral lesion of the pVTA. Amongst the dopamine receptor agonists used in this study only the D2R and D3R agonists (bromocriptine, PD128907 and pramipexole), induced a significant CPP in pVTA-lesioned animals. Dopamine receptor agonists did not induce behavioral sensitization in sham animals. Moreover, confocal D2R immunostaining analysis showed a significant increase in the number of D2R per cell body in the NAc shell of pVTA lesioned rats compared to sham. This result correlated, for the first time, the dopamine receptor agonists effect with DR2 overexpression in the NAc shell of pVTA-lesioned rats. In addition, cocaine, which is known to increase dopamine release, induced behavioral sensitization in sham group but not in dopamine deprived group. Thus, the later result highlighted the importance of pVTA-NAc dopaminergic pathway in positive reinforcements. Altogether these data suggested that the implication of the dopamine replacement therapy in the appearance of dopamine dysregulation syndrome in Parkinson's disease is probably due to both neuronal degeneration in the posterior VTA and dopamine receptor sensitization in the dopamine depleted NAc.

Motivational properties of D2 and D3 dopamine receptors agonists and cocaine, but not with D1 dopamine receptors agonist and L-dopa, in bilateral 6-OHDA-lesioned rat.

Dopamine dysregulation syndrome in Parkinson's disease (PD) has been attributed to dopamine replacement therapy (DRT). We hypothesize that DRT can induce a potential rewarding effect in an animal model of PD. Using the conditioned place preference (CPP) paradigm, we investigated the motivational effects of L-dopa, dopamine receptor agonists (DRAs), and cocaine in rat with a bilateral 6-OHDA lesion of the nigrostriatal dopaminergic pathway. In 6-OHDA animals, D1 receptors agonist (SKF81297) revealed significantly a conditioned place aversion (CPA) at 3 mg/kg and 9 mg/kg doses. D2 receptors agonist (bromocriptine) induced both CPP and CPA at 1 mg/kg and 10 mg/kg doses respectively. D3 receptors agonist (PD128907) induced a CPP only at 1 mg/kg, comparable to that of cocaine. Sham animals revealed biphasic CPP curves, with significant dose effect, for the intermediate dose of the 3 DRAs. However, L-dopa induced no significant effect while cocaine induced CPP in both lesioned and sham animals. In conclusion, this study confirms the predominant roles of D2R class, and most specifically D3R subtypes, in rewarding properties of DRT.

Is otospiralin inner ear specific? Evidence for its expression in mouse brain.

The small protein otospiralin has initially been identified as an inner ear specific molecule. However, compelling evidence from high throughput sequencing projects suggested that otospiralin is likely expressed in the central nervous system. Here, we tested this hypothesis using a combination of molecular biology, immunological, and histological techniques, and found that otospiralin is expressed in numerous regions of the central nervous system in mouse. In situ hybridization and immunohistochemistry revealed that otospiralin is widely expressed in neuronal cell bodies and glia. Ultrastructural observations in the cerebral cortex located the small protein in close proximity to membranous organelles in perikarya, the inner face of post-synaptic neuronal membranes, and in astrocytic processes. These results are in agreement with the predicted structure of the protein which revealed a single N-terminal transmembrane helix domain followed by a C-terminus cytosolic tail. Interestingly, 2 weeks after a mechanical trauma in the cerebral cortex, otospiralin expression increased in reactive astrocytes located within the vicinity of the site of injury, but not in neurons. Collectively, our observations suggest that otospiralin is possibly involved in signaling pathways, and could play a role in repair mechanisms subsequent to an injury in the central nervous system.

Insular cortex representation of dynamic mechanical allodynia in trigeminal neuropathic rats.

Dynamic mechanical allodynia is a widespread symptom of neuropathic pain for which mechanisms are still poorly understood. The present study investigated the organization of dynamic mechanical allodynia processing in the rat insular cortex after chronic constriction injury to the infraorbital nerve (IoN-CCI). Two weeks after unilateral IoN-CCI, rats showed a dramatic bilateral trigeminal dynamic mechanical allodynia. Light, moving stroking of the infraorbital skin resulted in strong, bilateral upregulation of extracellular-signal regulated kinase phosphorylation (pERK-1/2) in the insular cortex of IoN-CCI animals but not sham rats, in whose levels were similar to those of unstimulated IoN-CCI rats. pERK-1/2 was located in neuronal cells only. Stimulus-evoked pERK-1/2 immunopositive cell bodies displayed rostrocaudal gradient and layer selective distribution in the insula, being predominant in the rostral insula and in layers II-III of the dysgranular and to a lesser extent, of the agranular insular cortex. In layers II-III of the rostral dysgranular insular cortex, intense pERK also extended into distal dendrites, up to layer I. These results demonstrate that trigeminal nerve injury induces a significant alteration in the insular cortex processing of tactile stimuli and suggest that ERK phosphorylation contributes to the mechanisms underlying abnormal pain perception under this condition.

Expression of Rho GTPases Rho-A and Rac1 in the adult and developing gerbil cerebellum.

Rho GTPases proteins are essential for cytoskeletal reorganization and play important roles in the development of neuronal dendrites and axons. Several studies have implicated two members of the Rho GTPase family Rho-A and Rac1 activities in the neuronal polarization and the formation of axons and dendrites. In order to correlate cellular expressions of Rho-A and Rac1 with neuronal polarity (axons versus dendrite formation) in the central nervous system, the cerebellum and immunochemical techniques have been chosen. In the adult cerebellar cortex differential pattern of distribution between Rho-A and Rac1 was observed. While Rac1 expression was restricted to Purkinje cell (somata, dendrites and axons), Rho-A was ubiquitously distributed within the cerebellar cortex. Rac1 was localized in the Purkinje cell dendritic arborization (largest and tiny dendrites) and in their axons. This pattern of distribution was also observed during the postnatal development and followed the dendritic morphogenesis of Purkinje cell. Rho-A was highly expressed in the adult Purkinje cells somata, in cells of the granular layer, in glia within the white matter and in axons. Intense staining was observed in Bergmann glia cell bodies and processes. In the developing cerebellum, Rho-A was highly present in cells of the external and internal granule layers and in the Purkinje cell layer. Bergmann glia cell bodies and processes had the most intense staining during the development. The present study reveals a high expression of Rac1 and Rho-A during Purkinje cell neurites outgrowth period which occurred after birth in the cerebellum. In addition Rho-A is highly expressed in granule cell progenitor cells present in the external granular layer and therefore may play an important role in granule cell progenitor migration.

Ryanodine receptors and BK channels act as a presynaptic depressor of neurotransmission in cochlear inner hair cells.

Ryanodine receptors (RyRs) are known to contribute to the regulation of free cytosolic calcium concentration. This family of intracellular calcium channels plays a significant role in calcium-induced-calcium-release (CICR), and have been implicated in calcium-dependent processes requiring exquisite spatio-temporal regulation. In order to characterize the importance of these intracellular calcium channels in cochlear physiology, we perfused the guinea pig cochlea with antagonistic concentrations of ryanodine. The distortion products of the cochlear microphonic and the compound action potential of the auditory nerve were reversibly inhibited by ryanodine (IC(50)=27.3 microm, Hill coefficient=1.9), indicating an action at the cochlear amplifier. Single auditory nerve fibre recordings showed that ryanodine slightly increased spontaneous firing rates by 22%, suggesting an excitatory effect of ryanodine. This paradoxical effect could be explained by an inhibitory action of ryanodine on presynaptic BK channels of inner hair cells (IHC). Indeed, perfusing iberiotoxin also increased the spontaneous firing activity of the auditory nerve fibres. Furthermore, whole-cell patch-clamp recordings demonstrated that ryanodine inhibits BK currents at the IHC level. Conversely, immunohistochemistry demonstrated a strong expression of RyR in IHCs and, more particularly, below the cuticular plate where membranous BK channels are highly expressed. Overall, the study demonstrated a key role for RyR and CICR in signal transduction at the IHCs. We therefore propose that coupled RyR--BK channels act to suppress the fast neurotransmission in IHCs.