OBJECTIVE: To investigate the variations in the expression of voltage-gated sodium (Na_v) channel subunits during development of rat cerebellar Purkinje neurons and their correlation with maturation of electrophysiological characteristics of the neurons. METHODS: We observed the changes in the expression levels of Na_V1.1, 1.2, 1.3 and 1.6 during the development of Purkinje neurons using immunohistochemistry in neonatal (5-7 days after birth), juvenile (12-14 days), adolescent (21-24 days), and adult (42-60 days) SD rats. Using whole-cell patch-clamp technique, we recorded the spontaneous electrical activity of the neurons in ex vivo brain slices of rats of different ages to analyze the changes of electrophysiological characteristics of these neurons during development. RESULTS: The expression of Na_V subunits in rat cerebellar Purkinje neurons showed significant variations during development. Na_V1.1 subunit was highly expressed throughout the developmental stages and increased progressively with age (P < 0.05). Na_V1.2 expression was not detected in the neurons in any of the developmental stages (P > 0.05). The expression level of Na_V1.3 decreased with development and became undetectable after adolescence (P < 0.05). Na_V1.6 expression was not detected during infancy, but increased with further development (P < 0.05). Na_V1.1 and Na_V1.3 were mainly expressed in the early stages of development. With the maturation of the rats, Na_V1.3 expression disappeared and Na_V1.6 expression increased in the neurons. Na_V1.1 and Na_V1.6 were mainly expressed after adolescence. The total Na_V protein level increased gradually with development (P < 0.05) and tended to stabilize after adolescence. The spontaneous frequency and excitability of the Purkinje neurons increased gradually with development and reached the mature levels in adolescence. The developmental expression of Na_V subunits was positively correlated with discharge frequency (r=0.9942, P < 0.05) and negatively correlated with the excitatory threshold of the neurons (r=0.9891, P < 0.05). CONCLUSION The changes in the expression levels of Na_V subunits are correlated with the maturation of high frequency electrophysiological properties of the neurons, suggesting thatmature Na_V subunit expressions is the basis of maturation of electrophysiological characteristics of the neurons.
Rats ; Animals ; Purkinje Cells/physiology* ; Rats, Sprague-Dawley ; Neurons ; Brain ; Sodium/metabolism*

Cerebellar Purkinje cells (PCs) exhibit two types of discharge activities: simple spike (SS) and complex spike (CS). Previous studies found that noradrenaline (NA) can inhibit CS and bidirectionally regulate SS, but the enhancement of NA on SS is overwhelmed by the strong inhibition of excitatory molecular layer interneurons. However, the mechanism underlying the effect of NA on SS discharge frequency is not clear. Therefore, in the present study, we examined the mechanism underlying the increasing effect of NA on SS firing of PC in mouse cerebellar cortex in vivo and in cerebellar slice by cell-attached and whole-cell recording technique and pharmacological methods. GABA_A receptor was blocked by 100 µmol/L picrotoxin in the whole process. In vivo results showed that NA significantly reduced the number of spikelets of spontaneous CS and enhanced the discharge frequency of SS, but did not affect the discharge frequency of CS. In vitro experiments showed that NA reduced the number of CS spikelets and after hyperpolarization potential (AHP) induced by electrical stimulation, and increased the discharge frequency of SS. NA also reduced the amplitude of excitatory postsynaptic current (EPSC) of parallel fiber (PF)-PC and significantly increased the paired-pulse ratio (PPR). Application of yohimbine, an antagonist of α2-adrenergic receptor (AR), completely eliminated the enhancing effect of NA on SS. The α2-AR agonist, UK14304, also increased the frequency of SS. The β-AR blocker, propranolol, did not affect the effects of NA on PC. These results suggest that in the absence of GABA_A receptors, NA could attenuate the synaptic transmission of climbing fiber (CF)-PC via activating α2-AR, inhibit CS activity and reduce AHP, thus enhancing the SS discharge frequency of PC. This result suggests that NA neurons of locus coeruleus can finely regulate PC signal output by regulating CF-PC synaptic transmission.
Action Potentials/physiology* ; Animals ; Cerebellar Cortex/metabolism* ; Cerebellum/metabolism* ; Mice ; Norepinephrine/pharmacology* ; Purkinje Cells/metabolism* ; Receptors, Adrenergic, alpha-2/metabolism* ; Receptors, GABA-A/metabolism*

Sparse labeling of neurons contributes to uncovering their morphology, and rapid expression of a fluorescent protein reduces the experiment range. To achieve the goal of rapid and sparse labeling of neurons in vivo, we established a rapid method for depicting the fine structure of neurons at 24 h post-infection based on a mutant virus-like particle of Semliki Forest virus. Approximately 0.014 fluorescent focus-forming units of the mutant virus-like particle transferred enhanced green fluorescent protein into neurons in vivo, and its affinity for neurons in vivo was stronger than for neurons in vitro and BHK21 (baby hamster kidney) cells. Collectively, the mutant virus-like particle provides a robust and convenient way to reveal the fine structure of neurons and is expected to be a helper virus for combining with other tools to determine their connectivity. Our work adds a new tool to the approaches for rapid and sparse labeling of neurons in vivo.
Animals ; Cells, Cultured ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Green Fluorescent Proteins ; genetics ; metabolism ; Immunohistochemistry ; methods ; Male ; Mice, Inbred C57BL ; Microscopy, Fluorescence ; methods ; Neurons ; cytology ; metabolism ; Purkinje Cells ; cytology ; metabolism ; Semliki forest virus ; genetics

OBJECTIVE: Propofol is an intravenously administered anesthetic that enhances γ-aminobutyric acid-mediated inhibition in the central nerve system. Other mechanisms may also be involved in general anesthesia. Propofol has been implicated in movement disorders. The cerebellum is important for motor coordination and motor learning. The aim of the present study was to investigate the propofol effect on excitatory synaptic transmissions in cerebellar cortex. METHODS: Excitatory postsynaptic currents by parallel fiber stimulation and complex spikes by climbing fiber stimulation were monitored in Purkinje cells of Wister rat cerebellar slice using whole-cell patch-clamp techniques. RESULTS: Decay time, rise time and amplitude of excitatory postsynaptic currents at parallel fiber Purkinje cell synapses and area of complex spikes at climbing fiber Purkinje cell synapses were significantly increased by propofol administration. CONCLUSION: The detected changes of glutamatergic synaptic transmission in cerebellar Purkinje cell, which determine cerebellar motor output, could explain cerebellar mechanism of motor deficits induced by propofol.
Anesthesia, General ; Anesthetics ; Animals ; Cerebellar Cortex ; Cerebellum ; Excitatory Postsynaptic Potentials ; Learning ; Movement Disorders ; Patch-Clamp Techniques ; Propofol* ; Purkinje Cells* ; Rats ; Synapses ; Synaptic Transmission

What is memory? How does the brain process the sensory information and modify an organism's behavior? Many neuroscientists have focused on the activity- and experience-dependent modifications of synaptic functions in order to solve these fundamental questions in neuroscience. Recently, the plasticity of intrinsic excitability (called intrinsic plasticity) has emerged as an important element for information processing and storage in the brain. As the cerebellar Purkinje cells are the sole output neurons in the cerebellar cortex and the information is conveyed from a neuron to its relay neurons by forms of action potential firing, the modulation of the intrinsic firing activity may play a critical role in the cerebellar learning. Many voltage-gated and/or Ca²⁺-activated ion channels are involved in shaping the spiking output as well as integrating synaptic inputs to finely tune the cerebellar output. Recent studies suggested that the modulation of the intrinsic excitability and its plasticity in the cerebellar Purkinje cells might function as an integrator for information processing and memory formation. Moreover, the intrinsic plasticity might also determine the strength of connectivity to the sub-cortical areas such as deep cerebellar nuclei and vestibular nuclei to trigger the consolidation of the cerebellar-dependent memory by transferring the information.
Action Potentials ; Automatic Data Processing ; Brain ; Cerebellar Cortex ; Cerebellar Nuclei ; Cerebellum ; Fires ; Ion Channels ; Learning* ; Memory ; Neuronal Plasticity ; Neurons ; Neurosciences ; Plastics* ; Purkinje Cells* ; Vestibular Nuclei

We reviewed the anatomical characteristics of the conduction system in the ventricles of human and ungulate hearts and then raised some questions to be answered by clinical and anatomical studies in the future. The ventricular conduction system is a 3-dimensional structure as compared to the 2-dimensional character of the atrial conduction system. The proximal part consisting of the atrioventricular node, the bundle of His and fascicles are groups of conducting cells surrounded by fibrous connective tissue so as to insulate from the underlying myocardium. Their location and morphological characters are well established. The bundle of His is a cord like structure but the left and right fascicles are broad at the proximal and branching at the distal part. The more distal part of fascicles and Purkinje system are linear networks of conducting cells at the immediate subendocardium but the intra-mural network is detected at the inner half of the ventricular wall. The papillary muscle also harbors Purkinje system not in the deeper part. It is hard to recognize histologically in human hearts but conducting cells as well as Purkinje cells are easily recognized in ungulate hearts. Further observation on human and ungulate hearts with myocardial infarct, we could find preserved Purkinje system at the subendocardium in contrast to the damaged system at the deeper myocardium. Further studies are necessary on the anatomical characteristics of this peripheral conduction system so as to correlate the clinical data on hearts with ventricular arrhythmias.
Arrhythmias, Cardiac ; Atrioventricular Node ; Bundle of His ; Connective Tissue ; Heart ; Heart Conduction System ; Humans ; Myocardial Infarction ; Myocardium ; Papillary Muscles ; Purkinje Cells ; Purkinje Fibers ; Tachycardia, Ventricular

PURPOSE: Rotenone is the most widely used neurotoxin for the making Parkinson disease (PD) animal model. The neurodegenerative disorder PD shows symptoms, such as slowness of movements, tremor at resting, rigidity, disturbance of gait, and instability of posture. We investigated whether treadmill running improves motor ability using rotenone-caused PD rats. The effect of treadmill running on PD was also assessed in relation with apoptosis of cerebellar Purkinje cells. METHODS: Treadmill running was applied to the rats in the exercise groups for 30 minutes once a day for 4 weeks, starting 4 weeks after birth. We used rota-rod test for the determination of motor coordination and balance. In this experiment, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, immunohistochemistry for calbindin, glial fibrillary acidic protein (GFAP), Iba-1, and western blot analysis for Bax and Bcl-2 were performed. RESULTS: Treadmill running enhanced motor balance and coordination by preventing the loss of Purkinje cells in the cerebellar vermis. Treadmill running suppressed PD-induced expression of GFAP-positive reactive astrocytes and Iba-1-positive microglia, showing that treadmill running suppressed reactive astrogliosis and microglia activation. Treadmill running suppressed TUNEL-positive cell number and Bax expression and enhanced Bcl-2 expression, demonstrating that treadmill running inhibited the progress of apoptosis in the cerebellum of rotenone-induced PD rats. CONCLUSIONS: Treadmill running improved motor ability of the rotenone-induced PD rats by inhibiting apoptosis in the cerebellum. Apoptosis suppressing effect of treadmill running on rotenone-induced PD was achieved via suppression of reactive astrocyte and inhibition of microglial activation.
Animals ; Apoptosis ; Astrocytes ; Blotting, Western ; Calbindins ; Cell Count ; Cerebellar Vermis ; Cerebellum ; Gait ; Glial Fibrillary Acidic Protein ; Immunohistochemistry ; Microglia ; Models, Animal ; Neurodegenerative Diseases ; Parkinson Disease* ; Parturition ; Posture ; Purkinje Cells* ; Rats* ; Rotenone ; Running ; Tremor

Spinocerebellar ataxias (SCAs) are autosomal dominant neurodegenerative disorders which disrupt the afferent and efferent pathways of the cerebellum that cause cerebellar ataxia. Spectrin beta non-erythrocytic 2 (SPTBN2) gene encodes the β-III spectrin protein with high expression in Purkinje cells that is involved in excitatory glutamate signaling through stabilization of the glutamate transporter, and its mutation is known to cause spinocerebellar ataxia type 5. Three years and 5 months old boy with delayed development showed leukodystrophy and cerebellar atrophy in brain magnetic resonance imaging (MRI). Diagnostic exome sequencing revealed that the patient has heterozygous mutation in SPTBN2 (p.Glu1251Gln) which is a causative genetic mutation for spinocerebellar ataxia type 5. With the patient's clinical findings, it seems reasonable to conclude that p.Glu1251Gln mutation of SPTBN2 gene caused spinocerebellar ataxia type 5 in this patient.
Amino Acid Transport System X-AG ; Atrophy ; Brain ; Cerebellar Ataxia ; Cerebellum ; Efferent Pathways ; Exome ; Glutamic Acid ; Humans ; Magnetic Resonance Imaging ; Male ; Neurodegenerative Diseases ; Purkinje Cells ; Spectrin ; Spinocerebellar Ataxias*

OBJECTIVETo investigate the protective effect of succinic acid (SA) on the cerebellar Purkinje cells (PCs) of neonatal rats with convulsion.

METHODSA total of 120 healthy neonatal Sprague-Dawley rats aged 7 days were randomly divided into a neonatal period group and a developmental period group. Each of the two groups were further divided into 6 sub-groups: normal control, convulsion model, low-dose phenobarbital (PB) (30 mg/kg), high-dose PB (120 mg/kg), low-dose SA (30 mg/kg), and high-dose SA (120 mg/kg). Intraperitoneal injection of pentylenetetrazole was performed to establish the convulsion model. The normal control group was treated with normal saline instead. The rats in the neonatal group were sacrificed at 30 minutes after the injection of PB, SA, or normal saline, and the cerebellum was obtained. Those in the developmental group were sacrificed 30 days after the injection of PB, SA, or normal saline, and the cerebellum was obtained. Whole cell patch clamp technique was used to record the action potential (AP) of PCs in the cerebellar slices of neonatal rats; the parallel fibers (PF) were stimulated at a low frequency to induce excitatory postsynaptic current (EPSC). The effect of SA on long-term depression (LTD) of PCs was observed.

RESULTSCompared with the normal control groups, the neonatal and developmental rats with convulsion had a significantly higher AP frequency of PCs (P<0.05), and the developmental rats with convulsion had a significantly decreased threshold stimulus (P<0.01) and a significantly greater inhibition of the amplitude of EPSC in PCs (P<0.05). Compared with the normal control groups, the neonatal and developmental rats with convulsion in the high-dose PB groups had a significantly decreased threshold stimulus (P<0.01), a significantly higher AP frequency of PCs (P<0.05), and a significantly greater inhibition of EPSC in PCs (P<0.05). Compared with the neonatal and developmental rats in the convulsion model groups, those in the high-dose SA groups had a significantly decreased AP frequency of PCs (P<0.05). The developmental rats in the low- and high-dose SA groups had a significantly higher AP threshold than those in the convulsion model group (P<0.05).

CONCLUSIONSThe high excitability of PCs and the abnormal PF-PC synaptic plasticity caused by convulsion in neonatal rats may last to the developmental period, which can be aggravated by PB, while SA can reduce the excitability of PCs in neonatal rats with convulsion and repair the short- and long-term abnormalities of LTD of PCs caused by convulsion.

Action Potentials ; drug effects ; Animals ; Animals, Newborn ; Cytoprotection ; Excitatory Postsynaptic Potentials ; drug effects ; Purkinje Cells ; drug effects ; physiology ; Rats ; Rats, Sprague-Dawley ; Seizures ; drug therapy ; physiopathology ; Succinic Acid ; pharmacology

OBJECTIVETo investigate the influence of cefuroxime sodium (CS) on the electrophysiological function of cerebellar Purkinje cells (PCs) in Sprague-Dawley rats.

METHODSPostnatal day 7 (P7) Sprague-Dawley rats were divided into early administration I and II groups (administered from P7 to P14) and late administration group (administered from P14 to P21), and all the groups received intraperitoneally injected CS. The control groups for early and late administration groups were also established and treated with intraperitoneally injected normal saline of the same volume. There were 10 rats in each group. The rats in the early administration I group and early administration control group were sacrificed on P15, and those in the early administration II group, late administration group, and late administration control group were sacrificed on P22. The whole-cell patch-clamp technique was used to record inward current and action potential of PCs on cerebellar slices, as well as the long-term depression (LTD) of excitatory postsynaptic current (EPSC) in PCs induced by low-frequency stimulation of parallel fiber (PF).

RESULTSCompared with the control groups, the early and late administration groups had a slightly higher magnitude of inward current and a slightly higher amplitude of action potential of PCs (P>0.05). All administration groups had a significantly higher degree of EPSC inhibition than the control groups (P<0.01), and the early administration II group had a significantly greater degree of EPSC inhibition than the late administration group (P<0.01).

CONCLUSIONSEarly CS exposure after birth affects the synaptic plasticity of PF-PCs in the cerebellum of young rats, which persists after drug withdrawal.

Animals ; Anti-Bacterial Agents ; pharmacology ; Cefuroxime ; pharmacology ; Excitatory Postsynaptic Potentials ; drug effects ; Neuronal Plasticity ; drug effects ; Purkinje Cells ; drug effects ; physiology ; Rats ; Rats, Sprague-Dawley