n-Ping Chu

Sensory stimulus evokes inhibition rather than excitation in cerebellar Purkinje cells in vivo in mice.

Cerebellar Purkinje cells (PC) response precisely to tactile stimulus via granule cells, however, the interaction between sensory evoked synaptic input and the resulting pattern of output spikes in cerebellar cortex is unclear. In this study, we used electrophysiological recording and pharmacological methods to investigate the cerebellar PC in response to natural stimulus on ipsilateral whisker pad in urethane-anesthetized mice. We found that air-puff stimulus on ipsilateral whisker pad evoked neither complex spikes nor simple spike firing, but indeed evoked a strong GABA(A) receptor-mediated inhibition in PCs in cerebellar cortex folium Crus II. Field potential recordings from both molecular layer and PC layer showed that air-puff stimulus evoked a sequence of parallel fiber volley followed by a GABA(A) receptor-mediated inhibition, which completely blocked by AMPA receptor antagonist, NBQX. Cell-attached recordings showed that air-puff stimulus evoked a pause of simple spike firing, GABA(A) receptor antagonist abolished the pause, revealed the tactile stimulus-evoked spike firing in PCs. These results indicated that natural stimulus of whisker pad neither evoked complex spikes, nor fired simple spikes, but induced inhibition in PCs, suggesting that the interneuron network are rapid activated and involved in controlling the spread of sensory information processing in mouse cerebellar cortex folium Crus II.

Synaptic Responses Evoked by Tactile Stimuli in Purkinje Cells in Mouse Cerebellar Cortex Crus II In Vivo

Background Sensory stimuli evoke responses in cerebellar Purkinje cells (PCs) via the mossy fiber-granule cell pathway. However, the properties of synaptic responses evoked by tactile stimulation in cerebellar PCs are unknown. The present study investigated the synaptic responses of PCs in response to an air-puff stimulation on the ipsilateral whisker pad in urethane-anesthetized mice. Methods and Main Results Thirty-three PCs were recorded from 48 urethane-anesthetized adult (6–8-week-old) HA/ICR mice by somatic or dendritic patch-clamp recording and pharmacological methods. Tactile stimulation to the ipsilateral whisker pad was delivered by an air-puff through a 12-gauge stainless steel tube connected with a pressurized injection system. Under current-clamp conditions (I = 0), the air-puff stimulation evoked strong inhibitory postsynaptic potentials (IPSPs) in the somata of PCs. Application of SR95531, a specific GABAA receptor antagonist, blocked IPSPs and revealed stimulation-evoked simple spike firing. Under voltage-clamp conditions, tactile stimulation evoked a sequence of transient inward currents followed by strong outward currents in the somata and dendrites in PCs. Application of SR95531 blocked outward currents and revealed excitatory postsynaptic currents (EPSCs) in somata and a temporal summation of parallel fiber EPSCs in PC dendrites. We also demonstrated that PCs respond to both the onset and offset of the air-puff stimulation. Conclusions These findings indicated that tactile stimulation induced asynchronous parallel fiber excitatory inputs onto the dendrites of PCs, and failed to evoke strong EPSCs and spike firing in PCs, but induced the rapid activation of strong GABAA receptor-mediated inhibitory postsynaptic currents in the somata and dendrites of PCs in the cerebellar cortex Crus II in urethane-anesthetized mice.

Properties of 4 Hz stimulation-induced parallel fiber–Purkinje cell presynaptic long-term plasticity in mouse cerebellar cortex in vivo

Cerebellar parallel fiber–Purkinje cell (PF–PC) long‐term synaptic plasticity is important for the formation and stability of cerebellar neuronal circuits, and provides substrates for motor learning and memory. We previously reported both presynaptic long‐term potentiation (LTP) and long‐term depression (LTD) in cerebellar PF–PC synapses in vitro. However, the expression and mechanisms of cerebellar PF–PC synaptic plasticity in the cerebellar cortex in vivo are poorly understood. In the present study, we studied the properties of 4 Hz stimulation‐induced PF–PC presynaptic long‐term plasticity using in vivo the whole‐cell patch‐clamp recording technique and pharmacological methods in urethane‐anesthetised mice. Our results demonstrated that 4 Hz PF stimulation induced presynaptic LTD of PF–PC synaptic transmission in the intact cerebellar cortex in living mice. The PF–PC presynaptic LTD was attenuated by either the N‐methyl‐D‐aspartate receptor antagonist, D‐aminophosphonovaleric acid, or the group 1 metabotropic glutamate receptor antagonist, JNJ16259685, and was abolished by combined D‐aminophosphonovaleric acid and JNJ16259685, but enhanced by inhibition of nitric oxide synthase. Blockade of cannabinoid type 1 receptor activity abolished the PF–PC LTD and revealed a presynaptic PF–PC LTP. These data indicate that both endocannabinoids and nitric oxide synthase are involved in the 4 Hz stimulation‐induced PF–PC presynaptic plasticity, but the endocannabinoid‐dependent PF–PC presynaptic LTD masked the nitric oxide‐mediated PF–PC presynaptic LTP in the cerebellar cortex in urethane‐anesthetised mice.

Propofol depresses cerebellar Purkinje cell activity via activation of GABAA and glycine receptors in vivo in mice

Propofol is an intravenous sedative-hypnotic agen, which causes rapid and reliable loss of consciousness. Under in vitro conditions, propofol activates GABAA and glycine receptors in spinal cord, hippocampus and hypothalamus neurons. However, the effects of propofol on the cerebellar neuronal activity under in vivo conditions are currently unclear. In the present study, we examined the effects of propofol on the spontaneous activity of Purkinje cells (PCs) in urethane-anesthetized mice by cell-attached recording and pharmacological methods. Our results showed that cerebellar surface perfusion of propofol (10-1000 μM) induced depression of the PC simple spike (SS) firing rate in a dose-dependent manner, but without significantly changing the properties of complex spikes (CS). The IC50 of propofol for inhibiting SS firing of PCs was 144.5 μM. Application of GABAA receptor antagonist, SR95531 (40 μM) or GABAB receptor antagonist, saclofen (20 μM), as well as glycine receptor antagonist, strychnine (10 μM) alone failed to prevent the propofol-induced inhibition of PCs spontaneous activity. However, application the mixture of SR95531 (40 μM) and strychnine (10 μM) completely blocked the propofol-induced inhibition of PC SS firing. These data indicated that cerebellar surface application of propofol depressed PC SS firing rate via facilitation of GABAA and functional glycine receptors activity in adult cerebellar PCs under in vivo conditions. Our present results provide a new insight of the anesthetic action of propofol in cerebellar cortex, suggesting that propofol depresses the SS outputs of cerebellar PCs which is involved in both GABAA and glycine receptors activity.

Ethanol attenuates sensory stimulus-evoked responses in cerebellar granule cells via activation of GABAA receptors in vivo in mice

Acute alcohol intoxication affects cerebellar motor regulation possibly by altering the transfer and integration of external information in cerebellar cortical neurons, resulting in a dysfunction of cerebellar motor regulation or a cerebellar atexia. However, the synaptic mechanisms of ethanol induced impairments of sensory information processing in cerebellar cortical neurons are not fully understand. In the present study, we used electrophysiological and pharmacological methods to study the effects of ethanol on the sensory stimulation-evoked responses in cerebellar granule cells (GCs) in vivo in urethane anesthetized mice. Air-puff stimulation of the ipsilateral whisker-pad evoked stimulus-on (P1) and stimulus-off responses (P2) in GCs of cerebellar Crus II. Cerebellar surface perfusion of ethanol did not alter the onset latency of the sensory stimulation-evoked responses, but reversible reduced the amplitude of P1 and P2. The ethanol-induced reduction of the GCs sensory responses was concentration-dependent. In the presence of ethanol, the mean half-width, area under curve, rise Tau and decay Tau of P1 were significantly decreased. Blockade of gamma-aminobutyric acid type A (GABA(A)) receptors activity induced an increase in amplitude of P1, and abolished the ethanol induced inhibition of the GCs sensory responses. These results indicate that ethanol inhibits the tactile evoked responses in cerebellar GCs through enhancement of GABA(A) receptors activity.

Corticotrophin-releasing factor inhibits neuromedin U mRNA expressing neuron in the rat hypothalamic paraventricular nucleus in vitro

In the present study, we examined the effects of corticotrophin-releasing factor (CRF) on neuromedin U (NMU) mRNA-expressing neurons in the rat paraventricular nucleus (PVN) by whole-cell patch-clamp recordings and single-cell reverse transcription-multiplex polymerase chain reaction (single-cell RT-mPCR) techniques. In total, of 116 PVN putative parvocellular neurons screened for NMU mRNA, 14.7% (17/116) of them expressed NMU mRNA. The electrophysiological properties observed in the NMU mRNA-expressing neurons were generation of a low-threshold Ca(2+) spike (LTS) and robust low voltage-activated (T-type) Ca(2+) currents. Under current-clamp conditions, CRF (100 nM) induced a reversible decrease in spike firing and significantly diminished the LTS in 88.2% (15/17) of NMU mRNA-expressing neurons. Extracellular application of 1 μM α-helical CRF-(9-14) (α-hCRF), a selective CRF receptor antagonist, completely blocked the CRF-induced decrease in spike firing in the NMU mRNA-expressing neurons. Under voltage-clamp conditions, CRF (100 nM) significantly decreased the peak value of the T-type Ca(2+) currents by 35.6±7.8%. These findings suggest that CRF decreases neuronal excitability and diminishes T-type Ca(2+) currents in a population of rat PVN NMU phenotype neurons in vitro.

Dynamic properties of sensory stimulation evoked responses in mouse cerebellar granule cell layer and molecular layer.

Sensory information coming from climbing fiber and mossy fiber-granule cell pathways, generates motor-related outputs according to internal rules of integration and computation in the cerebellar cortex. However, the dynamic properties of sensory information processing in mouse cerebellar cortex are less understood. Here, we studied the dynamic properties of sensory stimulation-evoked responses in the cerebellar granule cell layer (GCL) and molecular layer (ML) by electrophysiological recordings method. Our data showed that air-puff stimulation (5-10 ms in duration) of the ipsilateral whisker pad evoked single-peak responses in the GCL and ML; whereas a duration of stimulation ≥30 ms in GCL and ≥60 ms in ML, evoked double-peak responses that corresponded with stimulation-on and -off responses via mossy fiber pathway. The highest frequency of stimulation train for evoking GCL responses was 33 Hz. In contrast, the highest frequency of stimulation train for evoking ML responses was 4 Hz. These results indicate that the cerebellar granule cells transfer the high-fidelity sensory information from mossy fibers, which is cut-off by molecular layer interneurons (MLIs). Our results suggest that the MLIs network acts as a low-pass filter during the processing of high-frequency sensory information.

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.

N-methyl-D-Aspartate Receptors Contribute to Complex Spike Signaling in Cerebellar Purkinje Cells: An In vivo Study in Mice

N-methyl-D-aspartate receptors (NMDARs) are post-synaptically expressed at climbing fiber-Purkinje cell (CF-PC) synapses in cerebellar cortex in adult mice and contributed to CF-PC synaptic transmission under in vitro conditions. In this study, we investigated the role of NMDARs at CF-PC synapses during the spontaneous complex spike (CS) activity in cerebellar cortex in urethane-anesthetized mice, by in vivo whole-cell recording technique and pharmacological methods. Under current-clamp conditions, cerebellar surface application of NMDA (50 μM) induced an increase in the CS-evoked pause of simple spike (SS) firing accompanied with a decrease in the SS firing rate. Under voltage-clamp conditions, application of NMDA enhanced the waveform of CS-evoked inward currents, which expressed increases in the area under curve (AUC) and spikelet number of spontaneous CS. NMDA increased the AUC of spontaneous CS in a concentration-dependent manner. The EC50 of NMDA for increasing AUC of spontaneous CS was 33.4 μM. Moreover, NMDA significantly increased the amplitude, half-width and decay time of CS-evoked after-hyperpolarization (AHP) currents. Blockade of NMDARs with D-(-)-2-amino-5-phosphonopentanoic acid (D-APV, 250 μM) decreased the AUC, spikelet number, and amplitude of AHP currents. In addition, the NMDA-induced enhancement of CS activity could not be observed after α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors were blocked. The results indicated that NMDARs of CF-PC synapses contributed to the spontaneous CS activity by enhancing CS-evoked inward currents and AHP currents.

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.