Blocking action of adenosine and ATP on synaptic transmission in isolated rat brain slices

The effect of ATP and adenosine on spontaneous activity and orthodromic responses of single neurons and on global evoked potentials was investigated in surviving slices of rat neocortex, hippocampus, dentate fascia, and cerebellumin vitro. ATP and adenosine, added to the incubation medium, had a twofold action on neurons: excitatory and inhibitory. Excitation was observed only if high concentrations of the substances (10^?2, less frequently 10^?3 M) were used, and in the case of adenosine it was very weak. The excitatory effect is evidently due to the direct depolarizing action of these substances on the cell membrane. The inhibitory action of both ATP and adenosine was manifested even in low concentrations (10^?6–10^?7 M) and was expressed as inhibition of postsynaptic responses of neurons at the presynaptic level and of their spontaneous activity. Hippocampal neurons were most sensitive to these substances, cerebellar neurons least. Apamine was found to have no effect on the inhibitory action of ATP. The results do not support the view that ATP and adenosine may be classed as CNS neurotransmitters. The possible role of these drugs as neuromodulators of synaptic transmission in the CNS is discussed.     

TRPV1 agonist piperine but not olvanil enhances glutamatergic spontaneous excitatory transmission in rat spinal substantia gelatinosa neurons

We examined the effects of TRPV1 agonists olvanil and piperine on glutamatergic spontaneous excitatory transmission in the substantia gelatinosa (SG) neurons of adult rat spinal cord slices with the whole-cell patch-clamp technique. Bath-applied olvanil did not affect the frequency and amplitude of spontaneous excitatory postsynaptic current (sEPSC), and unchanged holding currents at −70 mV. On the other hand, superfusing piperine reversibly and concentration-dependently increased sEPSC frequency (half-maximal effective concentration: 52.3 μM) with a minimal increase in its amplitude. This sEPSC frequency increase was almost repetitive at an interval of more than 20 min. Piperine at a high concentration produced an inward current in some neurons. The facilitatory effect of piperine was blocked by TRPV1 antagonist capsazepine. It is concluded that piperine but not olvanil activates TRPV1 channels in the central terminals of primary-afferent neurons, resulting in an increase in the spontaneous release of l-glutamate onto SG neurons.     

Impaired episodic LH secretion in female mice with GFP in GnRH neurons

The ability to assess the activity of gonadotropin-releasing hormone (GnRH) neurons has been greatly enhanced by transgenic animal models with targeted expression of green fluorescent protein (GFP). However, it has yet to be demonstrated that the GnRH system continues to exhibit a full range of normal physiological functions in the presence of such genetic manipulation. Accordingly, we have used repetitive blood sampling via indwelling venous catheters to define LH secretory patterns in normal and transgenic mice. Transgenic females proved to be reproductively competent as defined by fecundity, appropriate cyclic changes in vaginal cytology in intact adult females, and spontaneous LH surges as well as surges in response to steroid or mating stimuli. The expression of c-fos following such steroid treatment and mating in ovariectomized transgenics was similar to the expression previously reported in nontransgenic mice. Likewise, the percentage of retrogradely labeled GnRH neurons was similar to that reported in nontransgenic mice. However, episodic LH secretion, an index of GnRH pulse generator activity, was dramatically compromised in ovariectomized female transgenics compared with C57BL6 controls of both sexes and castrated transgenic males. Taken together, these findings suggest that the GnRH pulse generator is selectively impaired in ovariectomized females in which GnRH neurons express GFP.     

Spontaneous Activity in Crustacean Neurons

Single units which discharged with regular spontaneous rhythms without intentional stimulation were observed in the ventral nerve cord by intracellular recording close to the sixth abdominal ganglion. These units were divided into two groups: group A units in which interspike intervals varied less than 10 msec.; group B units in which interspike intervals varied within a range of 10 to 30 msec. Group A units maintained "constant" interspike intervals and could not be discharged by sensory inputs, while the majority of group B units could be discharged by appropriate sensory nerve stimulation. Both group A and B units discharged to direct stimulation when the stimulating and recording electrodes were placed in the same ganglionic intersegment, and directly evoked single spikes reset the spontaneous rhythm. In group B units, presynaptic volleys reset the spontaneous rhythm of some units; but in others, synaptically evoked spikes were interpolated within the spontaneous rhythm without resetting. The phenomenon of enhancement could also be demonstrated in spontaneously active units as a result of repetitive stimulation. It is concluded that endogenous pacemaker activity is responsible for much of the regular spontaneous firing observed in crayfish central neurons, and that interaction of evoked responses with such pacemaker sites can produce a variety of effects dependent upon the anatomical relationships between pacemaker and synaptic regions.     

Contralateral input modulates the excitability of dorsal horn neurons involved in noxious signal processes. Potential role in neuronal sensitization

Wide Dynamic Range (WDR) neurons in the spinal cord receive inputs from the contralateral side that, under normal conditions, are ineffective in generating an active response. These inputs are effective when the target WDRs change their excitability conditions. To further reveal the mechanisms supporting this effectiveness shift, we investigated the weight of the excitation of the contralateral neurons on the target WDR responses. In the circuit of presynaptic (sending) and postsynaptic (receiving) neurons in crossed spinal connections the fibres that form the presynaptic neurons impinge on postsynaptic neurons can be considered the final relay of this contralateral pathway. The enhancement of the presynaptic neuron excitability may thus modify the efficacy of the contralateral input. Pairs of neurons each on a side of the spinal cord, at the L5–L6 lumbar level were simultaneously recorded in intact, anaesthetized, paralysed rats. The excitatory aminoacid NMDA and strychnine, the antagonist of the inhibitory aminoacid glycine, were iontophoretically administrated to presynaptic neurons to increase their excitability. Before and during the drug administration, spontaneous and noxious-evoked activities of the neurons were analysed. During the iontophoresis of the two substances we found that noxious stimuli applied to the receptive field of presynaptic neurons activated up to 50% of the previously unresponsive postsynaptic neurons on the opposite side. Furthermore, the neurons on both sides of the spinal cord showed significantly increased spontaneous activity and amplified responses to ipsilateral noxious stimulation. These findings indicate that the contralateral input participates in the circuit dynamics of spinal nociceptive transmission, by modulating the excitability of the postsynaptic neurons. A possible functional role of such a nociceptive transmission circuit in neuronal sensitization following unilateral nerve injury is hypothesized.     

Contralateral input modulates the excitability of dorsal horn neurons involved in noxious signal processes. Potential role in neuronal sensitization

Wide Dynamic Range (WDR) neurons in the spinal cord receive inputs from the contralateral side that, under normal conditions, are ineffective in generating an active response. These inputs are effective when the target WDRs change their excitability conditions. To further reveal the mechanisms supporting this effectiveness shift, we investigated the weight of the excitation of the contralateral neurons on the target WDR responses. In the circuit of presynaptic (sending) and postsynaptic (receiving) neurons in crossed spinal connections the fibres that form the presynaptic neurons impinge on postsynaptic neurons can be considered the final relay of this contralateral pathway. The enhancement of the presynaptic neuron excitability may thus modify the efficacy of the contralateral input. Pairs of neurons each on a side of the spinal cord, at the L5-L6 lumbar level were simultaneously recorded in intact, anaesthetized, paralysed rats. The excitatory aminoacid NMDA and strychnine, the antagonist of the inhibitory aminoacid glycine, were iontophoretically administrated to presynaptic neurons to increase their excitability. Before and during the drug administration, spontaneous and noxious-evoked activities of the neurons were analysed. During the iontophoresis of the two substances we found that noxious stimuli applied to the receptive field of presynaptic neurons activated up to 50% of the previously unresponsive postsynaptic neurons on the opposite side. Furthermore, the neurons on both sides of the spinal cord showed significantly increased spontaneous activity and amplified responses to ipsilateral noxious stimulation. These findings indicate that the contralateral input participates in the circuit dynamics of spinal nociceptive transmission, by modulating the excitability of the postsynaptic neurons. A possible functional role of such a nociceptive transmission circuit in neuronal sensitization following unilateral nerve injury is hypothesized.     

Microelectrode array-based system for neuropharmacological applications with cortical neurons cultured in vitro

Microelectrode arrays (MEAs) provide a means to investigate the electrophysiological behavior of neuronal systems through the measurements from neuronal culture preparations. Changes in activity patterns of neuronal networks are usually detected by applying neural chemicals. Because of the difficulties of fabricating the arrays, and the delicate and less reliable properties of cortical neurons, MEA-based systems with cortical neuronal networks for neurophamacological applications are technically difficult, therefore restricting their utility. Here, we report a new approach to the development of such MEA-based system with sensitive and durable MEAs conveniently fabricated and the culture conditions optimized. Upon growth differentiation, cortical neurons, cultured directly on MEAs, reach a developmentally stable and reliable activity state. With this system, we monitored the global spontaneous activities of neuronal networks and demonstrated the fine discrimination for specific substances and unique property of cortical neurons, which validated both the applicability and necessity of such system in pharmacological bioassay.     

An iontophoretic survey of opioid peptide actions in the rat limbic system: in search of opiate epileptogenic mechanisms

Iontophoretic and micropressure drug application and lesion techniques were used to investigate the cellular source of rat limbic system epileptiform responses to opioid peptides [19]. Iontophoretically applied morphine, methionine enkephalin or beta-endorphin inhibited the spontaneous or glutamate-activated firing of the great majority of single neurons in medial and lateral septum, amygdala and cingulate cortex. These inhibitions in firing were antagonized by iontophoresis of naloxone. In contrast to inhibitory effects in other limbic areas, morphine and the opioid peptides predominantly excited CA1 and CA3 pyramidal neurons in a naloxone-sensitive manner, as previously reported [36]. On rare occasions, iontophoretically applied beta-endorphin evoked repetitive waveforms similar to interictal population EPSPs or spikes. Micropressure application of opiates and peptides also excited hippocampal neurons indicating such responses were not current-induced artefacts. The possible role of the excitatory cholinergic septal hippocampal pathway in the facilitatory response of hippocampal units to the opiates was tested with iontophoretically applied atropine and scopolamine, or lesions of septal nuclei. None of these manipulations reduced the opioid-induced excitations; rather, septal lesions enhanced excitatory and epileptiform responses to the opiates. These results support the hypothesis that opiate-evoked epileptiform activity in the limbic system arises from enhanced pyramidal cell activity in the hippocampal formation, probably by a non-cholinergic mechanism.     

After-effects of repetitive stimulation in spinal neurons

Spontaneous activity of interneurons before and after repetitive stimulation at 0.1–0.5/sec was recorded in acute experiments on spinal cats and kittens. Using the dynamic selective correlation method a search was made for areas of spontaneous activity with the same distribution of action potentials in time as in the averaged evoked response to a single stimulus. In the case of some neurons portions of the background which correlate reliably in structure with the evoked response repeated at an interval equal to or a multiple of the interval of stimulation. Reproduction of the rhythm of stimulation in the spontaneous activity is intensified with an increase in the total duration of preceding stimulation with the same input and shows positive correlation with the degree of posttetanic potentiation. The facts obtained are evidence of prolonged after-processes in spinal neurons.Institute of Experimental Medicine, Academy of Medical Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 5, No. 3, pp. 272–280, May–June, 1973.     

Activity-dependent clustering of functional synaptic inputs on developing hippocampal dendrites

During brain development, before sensory systems become functional, neuronal networks spontaneously generate repetitive bursts of neuronal activity, which are typically synchronized across many neurons. Such activity patterns have been described on the level of networks and cells, but the fine-structure of inputs received by an individual neuron during spontaneous network activity has not been studied. Here, we used calcium imaging to record activity at many synapses of hippocampal pyramidal neurons simultaneously to establish the activity patterns in the majority of synapses of an entire cell. Analysis of the spatiotemporal patterns of synaptic activity revealed a fine-scale connectivity rule: neighboring synapses (<16?μm intersynapse distance) are more likely to be coactive than synapses that are farther away from each other. Blocking spiking activity or NMDA receptor activation revealed that the clustering of synaptic inputs required neuronal activity, demonstrating a role of developmentally expressed spontaneous activity for connecting neurons with subcellular precision.     

The epileptic neuron: II

Conditions under which a neuron can maintain repetitive discharges are studied with the view that some light may be shed on the problem of epilepsy. It is shown that if there are excitatory and inhibitory substances as well as binding substances in volved, then for repetitive discharges to occur, the formation of the substances must be intracellular. The geometric physical characteristics of the system are also determining factors. The role of a fatigue factor is considered in an attempt to understand the frequency and duration of mild and severe epileptic attacks.     

Responses of rabbit visual cortical neurons to intracortical electrical stimulation

Responses of rabbit visual cortical neurons to single and repetitive intracortical electrical stimulation were investigated. The stimulating electrode was located 0.7–1.2 mm away from the recording electrode. Response thresholds to single stimulation were as a rule 150–180 µA, whereas to series of stimuli they were 30–60 µA. The latent period to the first spike averaged 5–15 msec but the probability of the initial discharge was very low, namely 3–6%. With an increase in current intensity the duration of the initial inhibitory pause was increased in half of the neurons responding to it, whereas in the rest it was unchanged. After presentation of series of stimuli spontaneous activity was enhanced for a short time (4–6 sec). In about half of the cells the same kinds of discharge dynamics were observed in response to repetitive stimulation (frequency 0.25 Hz) as in responses to light, but more neurons with sensitization of discharge and fewer "habituating" neurons took part in responses to electrical stimulation. It is postulated that stimulation of a given point of the visual cortex evokes excitation of a local neuron hypercolumn and inhibition of neighboring cell columns.M. V. Lomonosov Moscow State University. Translated from Neirofiziologiya, Vol. 15, No. 4, pp. 412–419, July–August, 1983.     

静脉注射雌二醇对不同性周期雌鼠蓝斑中去甲肾上腺素能神经元放电的影响

静脉注射雌二醇可使非发情期雌鼠蓝斑(LC)中去甲肾上腺素(NE)能神经元放电增加,发情期雌鼠LC中NE能神经元放电减少。非发情期雌鼠LC中NE能神经元自发放电频率与注射雌二醇后放电频率之间存在着正相关。摘除卵巢雌鼠LC中NE能神经元放电频率比发情期、非发情期雌鼠均显著减少,但对雌二醇的反应与非发情期雌鼠相似,仍为放电增加。结果提示:静脉注射雌二醇可改变LC中NE能神经元的放电。此效应可能反映了内源性雌激素水平及LC中NE能神经元兴奋性的密切关系。     

Age-Dependent Changes in Intrinsic Neuronal Excitability in Subiculum after Status Epilepticus

Kainic acid-induced status epilepticus (KA-SE) in mature rats results in the development of spontaneous recurrent seizures and a pattern of cell death resembling hippocampal sclerosis in patients with temporal lobe epilepsy. In contrast, KA-SE in young animals before postnatal day (P) 18 is less likely to cause cell death or epilepsy. To investigate whether changes in neuronal excitability occur in the subiculum after KA-SE, we examined the age-dependent effects of SE on the bursting neurons of subiculum, the major output region of the hippocampus. Patch-clamp recordings were used to monitor bursting in pyramidal neurons in the subiculum of rat hippocampal slices. Neurons were studied either one or 2-3 weeks following injection of KA or saline (control) in immature (P15) or more mature (P30) rats, which differ in their sensitivity to KA as well as the long-term sequelae of the KA-SE. A significantly greater proportion of subicular pyramidal neurons from P15 rats were strong-bursting neurons and showed increased frequency-dependent bursting compared to P30 animals. Frequency-dependent burst firing was enhanced in P30, but not in P15 rats following KA-SE. The enhancement of bursting induced by KA-SE in more mature rats suggests that the frequency-dependent limitation of repetitive burst firing, which normally occurs in the subiculum, is compromised following SE. These changes could facilitate the initiation of spontaneous recurrent seizures or their spread from the hippocampus to other parts of the brain.     

Penis-retractor muscle of Aplysia: excitatory motor neurons

Cobalt axonal iontophoresis and intracellular recordings were used to identify a cluster of several motor neurons innervating the penis-retractor muscle of Aplysia. Intracellularly recorded motor neuron action potentials elicited direct, one-for-one, constant latency excitatory junctional potentials (ejps) in individual muscle fibers. The axons of motor neurons could be recorded extracellularly in the penis-retractor nerve and stimulation of the nerve backfired the motor neurons. Perfusion of the ganglion, the muscle, or both with solutions of either increased Mg++/decreased Ca++ or increased Ca++ sea water indicated that the presumed motor neuron impaled was not a sensory cell and that interneurons were not intercalated in the pathway. Innervation of muscle fibers was found to be functionally polyneuronal and diffuse. The ejps were found to undergo marked facilitation with repetitive motor-neuron stimulation. The motor neurons were isolated in a distinct cluster in the right pedal ganglion. Their electrical activity was characterized by spontaneous irregular action potentials and a moderate input of postsynaptic potentials.     

Acute exposure to high‐induction electromagnetic field affects activity of model peripheral sensory neurons

Exposure to repetitive low‐frequency electromagnetic field (LF‐EMF) shows promise as a non‐invasive approach to treat various sensory and neurological disorders. Despite considerable progress in the development of modern stimulation devices, there is a limited understanding of the mechanisms underlying their biological effects and potential targets at the cellular level. A significant impact of electromagnetic field on voltage‐gated calcium channels and downstream signalling pathways has been convincingly demonstrated in many distinct cell types. However, evidence for clear effects on primary sensory neurons that particularly may be responsible for the analgesic actions of LF‐EMF is still lacking. Here, we used F11 cells derived from dorsal root ganglia neurons as an in vitro model of peripheral sensory neurons and three different protocols of high‐induction magnetic stimulation to determine the effects on chemical responsiveness and spontaneous activity. We show that short‐term (<180 sec.) exposure of F11 cells to LF‐EMF reduces calcium transients in response to bradykinin, a potent pain‐producing inflammatory agent formed at sites of injury. Moreover, we characterize an immediate and reversible potentiating effect of LF‐EMF on neuronal spontaneous activity. Our results provide new evidence that electromagnetic field may directly modulate the activity of sensory neurons and highlight the potential of sensory neuron‐derived cell line as a tool for studying the underlying mechanisms at the cellular and molecular level.     

Identification and pharmacological characteristics of the two giant neurons, v-RPLN and v-VNAN, on the ventral surface in the suboesophageal ganglia of the African giant snail (Achatina fulica Férussac)

Two giant neurons, v- RPLN (ventral-right parietal large neuron) and v- VNAN (ventral-visceral noisy autoactive neuron), were identified on the ventral surface in the caudal part of the suboesophageal ganglia of the African giant snail (Achatina fulica F erussac ), and their pharmacological features to the common putative neurotransmitters and their related substances were examined. The giant neuron examined, v- RPLN , is situated in front of the exit of the right anterior pallial nerve in the right parietal ganglion. The neuron, which is 250-300 microns in diameter, one of the largest neurons in the ganglia, was usually silent without spontaneous firing. The neuron was excited by L-norepinephrine (L-NE), DL-octopamine (DL-OA), 5-hydroxytryptamine (5-HT), L-homocysteic acid (L-HCA) and erythro-beta-hydroxy-L-glutamic acid (erythro-L- BHGA ); and inhibited by dopamine (DA), GABA, acetylcholine (Ach) and its related substances. Another giant neuron examined, v- VNAN , is situated very close to the right side of the exit of the right posterior pallial nerve in the visceral ganglion. The neuron is elliptical and about 150 micron in diameter. It showed spontaneous firing highly modified by the synaptic influences. DA, 5-HT, glycine (Gly), GABA and its related substances, L-HCA, erythro-L- BHGA , and Ach and its related substances all had the direct (not via synaptic influences) excitatory effects on the neuromembrane examined. Some of them, for example, L-NE, 5-HT and Ach and its related substances caused transient excitation of the neuron, probably due to the synaptic influences, immediately after their application. No substance producing any inhibition of the neuron could be found in the present study.     

Mut-Test to detect substances suppressing spontaneous mutation due to oxidative damage

Since it has been considered that suppression of spontaneous mutation in cells is related to suppression of spontaneous carcinogenesis, it is significant to detect substances which suppress spontaneous mutation in bacterial cells such as Escherichia coli and Salmonella typhimurium in the environment. However, since the frequency of spontaneous mutation in bacteria is usually very low, generally 10(-8)-10(-10),it is difficult to determine significant suppressive ability of such substances on spontaneous mutation. A new method, Mut-Test, was developed by us, applying Luria & Delbruck fluctuation test, to detect substances which suppress spontaneous mutation using E. coli mutT mutant in which spontaneous mutation frequency due to oxidative damage is enhanced to approximately 500-1000 times of the wild type strain. Suppressive abilities of two hydroxyl radical scavengers: D(-)-mannitol and thiourea, were examined and clear positive results were obtained, suggesting that the radical scavengers are suitable as the positive control for the test. Using Mut-Test, suppressive abilities of four vitamins: L-ascorbic acid, beta-carotene, folic acid and riboflavin; 10 polyphenols: caffeic acid, ellagic acid, (-)-epicatechin, (-)-epicatechin gallate, (-)-epigallocatechin, gallic acid, pyrocatechol, pyrogallol, quercetin and tannic acid which are recognized as antimutagens, were examined. Furthermore, the concentrations for 50% of suppressive abilities of five positive samples, L-ascorbic acid, folic acid, caffeic acid, pyrocatechol and pyrogallol were compared. Negative results were obtained in nine samples, riboflavin, tannic acid, etc. suggesting that their antimutagenic effect on cells may not be related to oxidative damage in cells.     

Some characteristics of unit activity in the frontal cortex of alert monkeys

Spontaneous activity of frontal cortical neurons (middle part of sulcus principalis) and their responses to stimuli of different biological significance were studied in alert monkeys. The region studied is characterized by a lower level of spontaneous activity than the motor cortex. Fluctuations in the level of spontaneous activity are connected with changes in the experimental situation. Two types of unit responses were distinguished to repetitive stimulation: preservation of responses and habituation. The type of unit response depends on the relationship of the stimuli to the animal's motivational sphere.A. A. Ukhtomskii Physiological Research Institute, A. A. Zhdanov Leningrad State University. Translated from Neirofiziologiya, Vol. 12, No. 1, pp. 3–12, January–February, 1980.