Alterations in CA1 pyramidal neuronal intrinsic excitability mediated by Ih channel currents in a rat model of amyloid beta pathology.

Amyloid beta (Aß) accumulation plays an important role in the pathogenesis of Alzheimer's disease (AD) by changing the neuronal excitability. However, the cellular mechanisms by which accumulation of Aß affects intrinsic neuronal properties are not well understood. The effect of bilateral intra-frontal cortex Aß (1-42) peptide injection on the intrinsic excitability of hippocampal CA1 pyramidal neurons with particular focus on the contribution of hyperpolarization-activated (Ih) channel currents was examined using whole-cell patch-clamp recording. Passive avoidance memory impairment and morphological changes in rats receiving intra-frontal Aß treatment were observed, which was associated with significant changes both in passive and active intrinsic electrical membrane properties of CA1 pyramidal neurons. Electrophysiological recording showed a significant decrease in neuronal excitability associated with an augmentation in the first spike after-hyperpolarization (AHP) amplitude. In addition, the depolarizing sag voltage was altered in neurons recorded from Aß-treated group. In voltage-clamp condition, a hyperpolarizing activated inward current sensitive to ZD7288 and capsaicin was significantly increased in neurons from Aß-treated rats. The Ih current density was increased and the activation curve was shifted toward less negative potential in the Aß-treated group as compared to control group. The enhancing effect of Aß treatment on Ih current was confirmed by showing upregulation of the mRNA of HCN1 channel in the CA1 pyramidal layer of hippocampi. These findings suggest the contribution of Ih and possibly TRPV1 channel currents to the changes induced by Aß treatment in the intrinsic membrane properties, which, in turn, may provide therapeutic targets for treatment of AD.

Repeated transcranial magnetic stimulation prevents kindling-induced changes in electrophysiological properties of rat hippocampal CA1 pyramidal neurons.

The mechanisms underlying antiepileptic or antiepileptogenic effects of repeated transcranial magnetic stimulation (rTMS) are poorly understood. In this study, we investigated the effect of rTMS applied during rapid amygdala kindling on some electrophysiological properties of hippocampal CA1 pyramidal neurons. Male Wistar rats were kindled by daily electrical stimulation of the basolateral amygdala in a semi-rapid manner (12 stimulations/day) until they achieved stage-5 seizure. One group (kindled+rTMS (KrTMS)) of animals received rTMS (1Hz for 4min) 5min after termination of daily kindling stimulations. Twenty four hours following the last kindling stimulation electrophysiological properties of hippocampal CA1 pyramidal neurons were investigated using whole-cell patch-clamp technique. Amygdala kindling significantly depolarized the resting membrane potential and increased the input resistance, spontaneous firing activity, number of evoked spikes and half-width of the first evoked spike. Kindling also decreased the first-spike latency and amplitude significantly. Application of rTMS during kindling somehow prevented the development of seizures and protected CA1 pyramidal neurons of hippocampus against deleterious effect of kindling on both passive and active neuronal electrophysiological properties. Interestingly, application of rTMS alone enhanced the excitability of CA1 pyramidal neurons significantly. Based on the results of our study, it may be suggested that rTMS exerts its anticonvulsant effect, in part, through preventing the amygdala kindling-induced changes in electrophysiological properties of hippocampal CA1 pyramidal neurons. It seems that rTMS exerts protective effects on the neural circuits involved in spreading the seizures from the focus to other parts of the brain.

Cerebellar Purkinje cells fire paroxysmal depolarization shift (PDS)-like events in response to epileptogenic drugs.

OBJECTIVE: Cerebellar Purkinje cells (PCs) fire burst of Na(+) spikes riding on a Ca(2+) spike which basically involves the same ionic channels and currents establishing the paroxysmal depolarization shift (PDS) discharges. METHODS: Intracellular recordings were taken from somata of PCs to explore effects of the epileptogenic drugs of pentylenetetrazol (PTZ), bicuculline methiodide (BCC) and 4-aminopyridine (4-AP) on the firing behavior of these cells. RESULTS: PCs showed spontaneous PDS-like events in presence of these drugs. Generally, PTZ and BCC-induced PDSs were similar in shape and properties but were remarkably different from 4-AP-induced PDSs. Blockade of glutamate transmission inhibited generation of PDSs by PTZ and BCC but it did not affect discharge of PDSs induced by 4-AP. Careful analysis of PDS discharges revealed that they have remarkable differences with normal and 4-AP-induced spontaneous activity. DISCUSSION: Data presented here indicate that PDS discharges in PCs are induced either by the imbalance between excitatory and inhibitory synaptic transmission or by the suppression of 4-AP-sensitive currents.

Maternal exposure to the CB1 cannabinoid agonist WIN 55212-2 produces robust changes in motor function and intrinsic electrophysiological properties of cerebellar Purkinje neurons in rat offspring.

The cerebellum, which controls coordinated and rapid movements, is a potential target for the deleterious effects of drugs of abuse including cannabis (i.e. marijuana, cannabinoids). Prenatal exposure to cannabinoids has been documented to cause abnormalities in motor and cognitive development, but the exact mechanism of this effect at the cellular level has not been fully elucidated. Previous studies indicate that cannabinoids are capable of modulating synaptic neurotransmission. In addition to altering synaptic activity, cannabinoid exposure may also change intrinsic neuronal properties. In the present study several different approaches including behavioral assays, extracellular field potential recordings and whole-cell patch clamp recordings, were used to address whether maternal exposure to the CB1 cannabinoid receptor agonist WIN 55-212-2 (WIN) affects the intrinsic electrophysiological properties of Purkinje neurons. WIN treatment of pregnant rats produced a significant decrease in the rearing frequency, total distance moved and mobility of the offspring, but significantly increased the time of the righting reflex, the grooming frequency and immobility. Neuromotor function, as assessed in the grip test and balance beam test, was also significantly impaired in prenatally WIN-treated group. Prenatal exposure to WIN increased the amplitude of population spikes (PS) recorded from the cerebellar Purkinje cell layer of offspring following synaptic blockage. WIN treatment of pregnant rats also profoundly affected the intrinsic properties of Purkinje neurons in offspring. This treatment increased the firing regularity, firing frequency, amplitude of afterhyperpolarization (AHP), the peak amplitude of action potential and the first spike latency, but decreased significantly the time to peak and duration of action potentials, the instantaneous firing frequency, the rate of rebound action potential and the voltage "sag" ratio. These results raise the possibility that maternal exposure to cannabinoids may profoundly affect the intrinsic membrane properties of cerebellar Purkinje neurons of offspring by altering the membrane excitability through modulation of intrinsic ion channels.

Profound alterations in the intrinsic excitability of cerebellar Purkinje neurons following neurotoxin 3-acetylpyridine (3-AP)-induced ataxia in rat: new insights into the role of small conductance K+ channels.

Alterations in the intrinsic properties of Purkinje cells (PCs) may contribute to the abnormal motor performance observed in ataxic rats. To investigate whether such changes in the intrinsic neuronal excitability could be attributed to the role of Ca(2+)-activated K(+) channels (K(Ca)), whole cell current clamp recordings were made from PCs in cerebellar slices of control and ataxic rats. 3-AP induced profound alterations in the intrinsic properties of PCs, as evidenced by a significant increase in both the membrane input resistance and the initial discharge frequency, along with the disruption of the firing regularity. In control PCs, the blockade of small conductance K(Ca) channels by UCL1684 resulted in a significant increase in the membrane input resistance, action potential (AP) half-width, time to peak of the AP and initial discharge frequency. SK channel blockade also significantly decreased the neuronal discharge regularity, the peak amplitude of the AP, the amplitude of the afterhyperpolarization and the spike frequency adaptation ratio. In contrast, in ataxic rats, both the firing regularity and the initial firing frequency were significantly increased by the blockade of SK channels. In conclusion, ataxia may arise from alterations in the functional contribution of SK channels, to the intrinsic properties of PCs.

The nociceptive and anti-nociceptive effects of white mineral trioxide aggregate.

AIM: To assess the nociceptive and antinociceptive effects of white mineral trioxide aggregate (WMTA) using the orofacial formalin test in rats. METHODOLOGY: Rats (n = 10 in each group) were separately injected into the ipsilateral upper lip with either 40 microL of a 2.5% formalin solution and eugenol (50 mg kg(-1)) or WMTA (5, 10 and 20 mg dissolved in 0.2 mL saline) alone. In a second experiment to evaluate antinociception effects, 15 min prior to formalin injection, rats were pre-treated with either white ProRoot MTA (20 mg dissolved in 0.2 mL saline) or eugenol. The time each rat spent rubbing the injected site with its paw, as an index of nociception, was recorded for a period of 45 min. RESULTS: Administration of 40 microL white ProRoot MTA (5, 10 and 20 mg per 0.2 mL) alone did not produce any significant nociceptive response. Moreover, prior treatment with WMTA caused significant (P < 0.001) inhibition of formalin-induced nociception. Injection of eugenol (50 mg kg(-1)) provoked the first phase of a nociceptive response, although its intensity was reduced compared with that produced by formalin. Pre-treatment with eugenol significantly (P < 0.0001) inhibited the induction of nociception by formalin. Comparison of the behavioural responses observed in WMTA and eugenol-treated rats alone or in combination with formalin revealed that WMTA did not only induce pain behaviour but also prevented formalin-induced nociception. CONCLUSION: White mineral trioxide aggregate, when compared with eugenol, was more effective in treating nociceptive pain in the orofacial formalin test.

Physiological role of dendrotoxin-sensitive K+ channels in the rat cerebellar Purkinje neurons.

To understand the contribution of potassium (K+) channels, particularly alpha-dendrotoxin (D-type)-sensitive K+ channels (Kv.1, Kv1.2 or Kv1.6 subunits), to the generation of neuronal spike output we must have detailed information of the functional role of these channels in the neuronal membrane. Conventional intracellular recording methods in current clamp mode were used to identify the role of alpha-dendrotoxin (alpha-DTX)-sensitive K+ channel currents in shaping the spike output and modulation of neuronal properties of cerebellar Purkinje neurons (PCs) in slices. Addition of alpha-DTX revealed that D-type K+ channels play an important role in the shaping of Purkinje neuronal firing behavior. Repetitive firing capability of PCs was increased following exposure to artificial cerebrospinal fluid (aCSF) containing alpha-DTX, so that in response to the injection of 0.6 nA depolarizing current pulse of 600 ms, the number of action potentials insignificantly increased from 15 in the presence of 4-AP to 29 action potentials per second after application of DTX following pretreatment with 4-AP. These results indicate that D-type K+ channels (Kv.1, Kv1.2 or Kv1.6 subunits) may contribute to the spike frequency adaptation in PCs. Our findings suggest that the activation of voltage-dependent K+ channels (D and A types) markedly affect the firing pattern of PCs.

Comparison of neurotoxicity of root canal sealers on spontaneous bioelectrical activity in identified Helix neurones using an intracellular recording technique.

AIM: To evaluate the neurotoxic effects of two endodontic sealers, AH-26 and Roth 801, on firing excitability and action potential configuration of F1 neural cells in the suboesophageal ganglia of Helix aspersa. METHODOLOGY: A conventional intracellular current clamp technique was used to study the blocking effects of AH-26 and Roth 801 on ionic currents underlying the action potential of F1 nerve cells. The sealers were prepared according to the manufacturers' directions and were applied to the bathing media in two ways: invasive (0.05 mL of total mixture of each sealer was applied at a distance of 3 mm from the cell), or gradual (0.05 mL of the extract of each dissolved mixture of sealers in normal Ringers solution was perfused). RESULTS: When applied in an invasive mode, both sealers reduced the duration, the amplitude of action potentials and the amplitude of after-hyperpolarization potentials significantly and led to dramatic changes in action potential configuration. In the gradual mode of application, AH-26 showed a biphasic action; it first increased the excitability and then decreased the action potential parameters, while Roth 801 exhibited solely blocking effects. CONCLUSIONS: Both sealers had significant inhibitory effects on excitability of F1 neuronal cells.

The effect of mebudipine and dibudipine, two new Ca2+ channel blockers, in comparison with nifedipine on Ca2+ spikes of F1 neuronal soma membrane in Helix aspersa.

Mebudipine and dibudipine are two new dihydropyridine (DHP) Ca2+ channel blockers that have been synthesized by Mahmoudian et al. (1997). In previous studies, they showed considerable relaxant effect on vascular and ileal smooth muscles. These two compounds also reduced the contraction force of rat left atrium (20, 22). In the present study, we attempted to compare the inhibitory actions of these new DHPs and nifedipine on the high threshold Ca2+ spikes of F1 neuronal soma membrane in the subesophageal ganglia of Helix aspersa, using current-clamp method. At a concentration of 1 microM, two new DHP compounds (mebudipine and dibudipine) were tested for their L-type Ca2+ channel blocker activity. Both compounds reversibly reduced the peak amplitude of action potential and after hyperpolarization potential and markedly decreased the duration of Ca2+ spikes. The most potent of these DHPs was mebudipine. Neither the two new DHPs nor nifedipine changed the resting membrane potential in a statistically significant way.

Two kinds of transient outward currents, I(A) and I(Adepol), in F76 and D1 soma membranes of the subesophageal ganglia of Helix aspersa.

Transient outward currents were characterized with twin electrode voltage clamp techniques in isolated F76 and D1 neuronal membranes (soma only) of Helix aspersa subesophageal ganglia. In this study, in addition to the transient outward current (A-current, I(A)) described by Connor and Stevens (1971b), another fast outward current, referred to as I(Adepol) here, is described for the first time. This is similar to the current component characterized in Aplysia (Furukawa, Kandel & Pfaffinger, 1992). The separation of these two current components was based on activation and steady-state inactivation curves, holding potentials and sensitivity to 4-aminopyridine (4-AP). In contrast to I(A), I(Adepol) did not require hyperpolarizing conditioning pulses to remove inactivation; it was evoked from a holding potential of -40 mV, at which I(A) is completely inactivated. I(Adepol) shows noticeable activation at around -5 mV, whereas I(A) activates at around -50 mV. The time courses of I(Adepol) activation and inactivation were similar but slower than I(A). It was found that I(Adepol) was more sensitive than I(A) to 4-AP. 4-AP at a concentration of 1 mm blocked I(Adepol) completely, whereas 5-6 mm 4-AP was needed to block I(A) completely. This current is potentially very important because it may, like other A currents, regulate firing frequency but notably, it does not require a period of hyperpolarization to be active.

Effect of calcium and calcium channel blockers on transient outward current of F76 and D1 neuronal soma membranes in the subesophageal ganglia of Helix aspersa.

Twin-electrode voltage-clamp techniques were used to study the effect of calcium and calcium channel blockers on the transient outward current in isolated F76 and D1 neurones of Helix aspersa subesophageal ganglia in vitro (soma only preparation with no cell processes). On lowering extracellular Ca(2+) concentration from 10 to 2 mm or removing extracellular calcium from the bathing medium, the threshold for this current shifted in a negative direction by 11. 5 and 20 mV, respectively. On the other hand, increasing the extracellular Ca(2+) concentration from 10 to 20 and to 40 mm shifted the steady-state inactivation curves in positive directions on the voltage axis by 7 and 15 mV, respectively. Upon application of calcium channel blockers, Co(2+), La(3+), Ni(2+) and Cd(2+), transient potassium current amplitude was reduced in a voltage-dependent manner, being more effective at voltages close to the threshold. The current was elicited even at a holding potential of -34 mV. The specific calcium channel blockers, amiloride and nifedipine did not shift the activation and steady-state inactivation curves and did not reduce the transient outward current amplitude. It was concluded that the transient outward current is not dependent on intracellular Ca(2+) but that it is modulated by Ca(2+) and di- and trivalent ions extracellularly. The effects of these ions are very unlikely to be due to a surface charge effect because the addition of La(3+) (200 microm) completely reverses the shift in a hyperpolarizing direction when the extracellular Ca(2+) concentration was reduced from 10 to 1 mm and additionally shifts the kinetics further still in a depolarizing direction. The responses seen here are consistent with a specific effect of di- and trivalent ions on the transient outward current channels leading to a modification of gating.

Morphological and electrophysiological features of F76 and D1 neurones of the sub-oesophageal ganglia of Helix aspersa in vitro and in culture.

Identified neurones F76 and D1 of the suboesophageal ganglia of Helix aspersa were studied in the isolated ganglia in vitro and in culture. The neurones were examined electrophysiologically with current clamp and morphologically either with intracellular injections of Lucifer Yellow or biocytin. These nerve cells had very similar resting membrane potentials and responses to injected current. The projections of D1 and F76 have been characterised, with both neurones having two main axons. The F76 neurones project to the left pallial, right pallial, anal, and visceral nerves as well as to the left and right pleural ganglia. The D1 neurones have similar projections except that they do not project to the anal and visceral nerves. The bilateral symmetry to the pallial nerves and pleural ganglia is discussed. These cells were also studied electrophysiologically after mechanical isolation and culture. F76 and D1 neurones were separated by dissection (no enzymes) and cultured in three ways. In normal snail Ringer they remained viable for up to two weeks with no development. In Ringer preincubated with a ganglia or containing endothelial growth factor, neurite outgrowths were seen. Membrane potentials were significantly lower in cultured neurones than in vitro and the after hyperpolarization never went below resting in cultured cells but it did in vitro.