Noradrenaline depresses facial stimulation-evoked cerebellar MLI-PC synaptic transmission via α2-AR/PKA signaling cascade in vivo in mice.

The noradrenergic fibers of the locus coeruleus, together with mossy fibers and climbing fibers, comprise the three types of cerebellar afferents that modulate the cerebellar neuronal circuit. We previously demonstrated that noradrenaline (NA) modulated synaptic transmission in the mouse cerebellar cortex via adrenergic receptors (ARs). In the present study, we investigated the effect of NA on facial stimulation-evoked cerebellar molecular layer interneuron (MLI)-Purkinje cell (PC) synaptic transmission in urethane-anesthetized mice using an in vivo cell-attached recording technique and a pharmacological method. MLI-PC synaptic transmission was induced by air-puff stimulation (duration: 60 ms) of the ipsilateral whisker pad, which exhibited positive components (P1 and P2) accompanied by a pause in simple spike activity. Cerebellar molecular layer application of NA (15 µM) decreased the amplitude and area under the curve of P1, and the pause in simple spike activity, but increased the P2/P1 ratio. The NA-induced decrease in P1 amplitude was concentration-dependent, and the half-inhibitory concentration was 10.94 µM. The NA-induced depression of facial stimulation-evoked MLI-PC GABAergic synaptic transmission was completely abolished by blockade of α-ARs or α2-ARs, but not by antagonism of α1-ARs or ß-ARs. Bath application of an α2-AR agonist inhibited MLI-PC synaptic transmission and attenuated the effect of NA on the synaptic response. NA-induced depression of MLI-PC synaptic transmission was completely blocked by a mixture of α2A- and 2B-AR antagonists, and was abolished by inhibition of protein kinase A. In addition, electrical stimulation of the molecular layer evoked MLI-PC GABAergic synaptic transmission in the presence of an AMPA receptor antagonist, which was inhibited by NA through α2-ARs. Our results indicate that NA inhibits MLI-PC GABAergic synaptic transmission by reducing GABA release via an α2-AR/PKA signaling pathway.

Corticotrophin-Releasing Factor Modulates the Facial Stimulation-Evoked Molecular Layer Interneuron-Purkinje Cell Synaptic Transmission in vivo in Mice.

Corticotropin-releasing factor (CRF) is an important neuromodulator in central nervous system that modulates neuronal activity via its receptors during stress responses. In cerebellar cortex, CRF modulates the simple spike (SS) firing activity of Purkinje cells (PCs) has been previously demonstrated, whereas the effect of CRF on the molecular layer interneuron (MLI)-PC synaptic transmission is still unknown. In this study, we examined the effect of CRF on the facial stimulation-evoked cerebellar cortical MLI-PC synaptic transmission in urethane-anesthetized mice by in vivo cell-attached recording, neurobiotin juxtacellular labeling, immunohistochemistry techniques, and pharmacological method. Cell-attached recordings from cerebellar PCs showed that air-puff stimulation of ipsilateral whisker pad evoked a sequence of tiny parallel fiber volley (N1) followed by MLI-PC synaptic transmission (P1). Microapplication of CRF in cerebellar cortical molecular layer induced increases in amplitude of P1 and pause of SS firing. The CRF decreases in amplitude of P1 waveform were in a dose-dependent manner with the EC50 of 241 nM. The effects of CRF on amplitude of P1 and pause of SS firing were abolished by either a non-selective CRF receptor antagonist, α-helical CRF-(9-14), or a selective CRF-R1 antagonist, BMS-763534 (BMS, 200 nM), but were not prevented by a selective CRF-R2 antagonist, antisauvagine-30 (200 nM). Notably, application CRF not only induced a significant increase in spontaneous spike firing rate, but also produced a significant increase in the number of the facial stimulation-evoked action potential in MLIs. The effect of CRF on the activity of MLIs was blocked by the selective CRF-R1 antagonist, and the MLIs expressed the CRF-R1 imunoreactivity. These results indicate that CRF increases excitability of MLIs via CRF-R1, resulting in an enhancement of the facial stimulation-evoked MLI-PC synaptic transmission in vivo in mice.

Nicotine modulates the facial stimulation-evoked responses in cerebellar granule cell layer in vivo in mice.

Nicotinic acetylcholine receptors are cationic channels that mediate fast excitatory transmission in the central nervous system. Several nicotinic acetylcholine receptor subunits have been detected within cerebellar granule cell layer (GCL), and activation of these receptors may have a significant influence on neuronal synaptic transmission of the cerebellum. The aim of present study was to better understand the roles of nicotinic acetylcholine receptors during the sensory stimulation-evoked synaptic transmission in the cerebellar GCL. Our results showed that cerebellar surface perfusion of nicotine significantly facilitated the cerebellar GCL field potential responses evoked by air-puff stimulation of ipsilateral whisker pad, which exhibited increases in amplitude and area under the curve (AUC) of both stimulus onset responses (N1) and stimulus offset responses (N2). The nicotine-induced increase in AUC of facial stimulation-evoked N1 was dose-dependent with a 50% effective concentration (EC50) of 32.6 µM. Application of either a selective α4ß2 nicotinic acetylcholine receptors antagonist, DHßE (1 µM) or a selective α7 nicotinic acetylcholine receptors antagonist, MLA (1 µM) alone attenuated, but not completely abolished the nicotine-induced increases in the amplitude and AUC of the facial stimulation-evoked N1. However, simultaneous blockade of α7 and α4ß2 nicotinic acetylcholine receptor subunits abolished the nicotine-induced increase in the amplitude of N1. These results indicate that nicotine activates α7 and α4ß2 nicotinic acetylcholine receptor subunits, resulting in an enhancement of facial stimulation-evoked responses in mouse cerebellar GCL. Our results suggest that nicotine modulates the sensory information processing in the cerebellar GCL through α7 and α4ß2 subunits nicotinic acetylcholine receptors.

Ethanol Modulates the Spontaneous Complex Spike Waveform of Cerebellar Purkinje Cells Recorded in vivo in Mice.

Cerebellar Purkinje cells (PCs) are sensitive to ethanol, but the effect of ethanol on spontaneous complex spike (CS) activity in these cells in vivo is currently unknown. Here, we investigated the effect of ethanol on spontaneous CS activity in PCs in urethane-anesthetized mice using in vivo patch-clamp recordings and pharmacological manipulation. Ethanol (300 mM) induced a decrease in the CS-evoked pause in simple spike (SS) firing and in the amplitude of the afterhyperpolarization (AHP) under current clamp conditions. Under voltage-clamp conditions, ethanol significantly decreased the area under the curve (AUC) and the number of CS spikelets, without changing the spontaneous frequency of the CSs or the instantaneous frequency of the CS spikelets. Ethanol-induced a decrease in the AUC of spontaneous CSs was concentration dependent. The EC50 of ethanol for decreasing the AUC of spontaneous CSs was 168.5 mM. Blocking N-methyl-D-aspartate receptors (NMDARs) failed to prevent the ethanol-induced decreases in the CS waveform parameters. However, blockade of cannabinoid receptor 1 (CB1) significantly suppressed the ethanol-induced effects on the CS-evoked pause in SS firing, amplitude of the AHP, spikelet number and the AUC of CSs. Moreover, a CB1 receptor agonist not only reduced the number of spikelets and the AUC of CSs, but also prevented the ethanol-induced inhibition of CS activity. Our results indicate that ethanol inhibits CS activity via activation of the CB1 receptor in vivo in mice, suggesting that excessive ethanol intake inhibits climbing fiber (CF)-PC synaptic transmission by modulating CB1 receptors in the cerebellar cortex.

Ethanol modulates facial stimulation-evoked outward currents in cerebellar Purkinje cells in vivo in mice.

Acute ethanol overdose can induce dysfunction of cerebellar motor regulation and cerebellar ataxia. In this study, we investigated the effect of ethanol on facial stimulation-evoked inhibitory synaptic responses in cerebellar Purkinje cells (PCs) in urethane-anesthetized mice, using in vivo patch-clamp recordings. Under voltage-clamp conditions, ethanol (300 mM) decreased the amplitude, half-width, rise time and decay time of facial stimulation-evoked outward currents in PCs. The ethanol-induced inhibition of facial stimulation-evoked outward currents was dose-dependent, with an IC50 of 148.5 mM. Notably, the ethanol-induced inhibition of facial stimulation-evoked outward currents were significantly abrogated by cannabinoid receptor 1 (CB1) antagonists, AM251 and O-2050, as well as by the CB1 agonist WIN55212-2. Moreover, the ethanol-induced inhibition of facial stimulation-evoked outward currents was prevented by cerebellar surface perfusion of the PKA inhibitors H-89 and Rp-cAMP, but not by intracellular administration of the PKA inhibitor PKI. Our present results indicate that ethanol inhibits the facial stimulation-evoked outward currents by activating presynaptic CB1 receptors via the PKA signaling pathway. These findings suggest that ethanol overdose impairs sensory information processing, at least in part, by inhibiting GABA release from molecular layer interneurons onto PCs.

Facial stimulation induces long-term depression at cerebellar molecular layer interneuron-Purkinje cell synapses in vivo in mice.

Cerebellar long-term synaptic plasticity has been proposed to provide a cellular mechanism for motor learning. Numerous studies have demonstrated the induction and mechanisms of synaptic plasticity at parallel fiber-Purkinje cell (PF-PC), parallel fiber-molecular layer interneurons (PF-MLI) and mossy fiber-granule cell (MF-GC) synapses, but no study has investigated sensory stimulation-evoked synaptic plasticity at MLI-PC synapses in the cerebellar cortex of living animals. We studied the expression and mechanism of MLI-PC GABAergic synaptic plasticity induced by a train of facial stimulation in urethane-anesthetized mice by cell-attached recordings and pharmacological methods. We found that 1 Hz, but not a 2 Hz or 4 Hz, facial stimulation induced a long-term depression (LTD) of GABAergic transmission at MLI-PC synapses, which was accompanied with a decrease in the stimulation-evoked pause of spike firing in PCs, but did not induce a significant change in the properties of the sensory-evoked spike events of MLIs. The MLI-PC GABAergic LTD could be prevented by blocking cannabinoid type 1 (CB1) receptors, and could be pharmacologically induced by a CB1 receptor agonist. Additionally, 1 Hz facial stimulation delivered in the presence of a metabotropic glutamate receptor 1 (mGluR1) antagonist, JNJ16259685, still induced the MLI-PC GABAergic LTD, whereas blocking N-methyl-D-aspartate (NMDA) receptors during 1 Hz facial stimulation abolished the expression of MLI-PC GABAergic LTD. These results indicate that sensory stimulation can induce an endocannabinoid (eCB)-dependent LTD of GABAergic transmission at MLI-PC synapses via activation of NMDA receptors in cerebellar cortical Crus II in vivo in mice. Our results suggest that the sensory stimulation-evoked MLI-PC GABAergic synaptic plasticity may play a critical role in motor learning in animals.

Cellular transplantation-based evolving treatment options in spinal cord injury.

Spinal cord injury (SCI) often represents a condition of permanent neurologic deficit. It has been possible to understand and delineate the mechanisms contributing to loss of function following primary injury. The clinicians might hope to improve the outcome in SCI injury by designing treatment strategies that could target these secondary mechanisms of response to injury. However, the approaches like molecular targeting of the neurons or surgical interventions have yielded very limited success till date. In recent times, a great thrust is put on to the cellular transplantation mode of treatment strategies to combat SCI problems so as to gain maximum functional recovery. In this review, we discuss about the various cellular transplantation strategies that could be employed in the treatment of SCI. The success of such cellular approaches involving Schwann cells, olfactory ensheathing cells, peripheral nerve, embryonic CNS tissue and activated macrophage has been supported by a number of reports and has been detailed here. Many of these cell transplantation strategies have reached the clinical trial stages. Also, the evolving field of stem cell therapy has made it possible to contemplate the role of both embryonic stem cells and induced pluripotent stem cells to stimulate the differentiation of neurons when transplanted in SCI models. Moreover, the roles of tissue engineering techniques and synthetic biomaterials have also been explained with their beneficial and deleterious effects. Many of these cell-based therapeutic approaches have been able to cause only a little change in recovery and a combinatorial approach involving more than one strategy are now being tried out to successfully treat SCI and improve functional recovery.

Effects of ethanol on sensory stimulus-evoked responses in the cerebellar molecular layer in vivo in mice.

Overdose intake of ethanol can impair cerebellar cortical neurons to integrate and transfer external information, resulting in a dysfunction of cerebellar motor regulation or cerebellar ataxia. However, the mechanisms underlying ethanol-impaired transfer of sensory information from cerebellar cortical molecular layer neurons remain unclear. In the present study, we investigated the effects of ethanol on sensory stimulation-evoked responses in the cerebellar molecular layer of urethane-anesthetized mice, by electrophysiological and pharmacological methods. Our results demonstrated that air-puff stimulation (30 ms, 50-60 psi) of the ipsilateral whisker-pad evoked field potential responses in the molecular layer of the cerebellar cortex folium Crus II, which expressed a negative component (N1) followed by a gamma-aminobutyric acid receptor A (GABAA)-mediated positive component (P1). Cerebellar surface perfusion of ethanol between 2 and 5mM did not change the latency of the evoked responses and the amplitude of N1, but enhanced the amplitude and the area under the curve of P1. Interestingly, high concentrations (>20mM) of ethanol induced a significantly decrease in the amplitude and area under the curve of P1. Furthermore, high concentration ethanol (300 mM) significantly decreased the rise in tau and tau decay value of P1, whereas low concentration ethanol (2-5mM) significantly increased these values of P1. Inhibition of GABAA receptor activity reversed P1 and also abolished the effects of ethanol on sensory stimulation-evoked responses. These results indicated that ethanol induced a bidirectional effect on the sensory stimulation-evoked GABAergic responses in the cerebellar cortical molecular layer, suggesting that acute alcohol intake impacted the sensory information processing of cerebellar cortex.