Reactivity of the cortical neurons to acetylcholine in rats

Activity of 55 neurons of the sensorimotor cerebral cortex of rats was recorded at iontophoretic application of acetylcholine. 36% of neurons exhibited an excitatory reaction, 30%--inhibitory-excitatory, 18%--inhibitory-excitatory-inhibitory and 16%--excitatory-inhibitory reactions; the type of reaction, in contrast to its expressiveness, did not depend on the the type of reaction, in contrast to its expressiveness, did not depend on the strength of phoresis current. Duration of the excitatory components entering reactions of all neurons formed a continuous series of values in the range of 1.4 to 16 s and had 2 maxima--at the 4-th and 8-th seconds. It is suggested that duration of this component of reaction reflects important functional properties of the nerve cell.     

The culture of rat submandibular ganglion neurons and their macroscopic responses to acetylcholine

The culture of rat submandibular ganglion cells is described. The neurons can be distinguished from the non-neuronal cells in the cultures by their morphology. Recording with whole-cell voltage-clamp techniques indicates that the neurons have resting potentials of about -55 mV and that the kinetics of the ionic channels opened by locally perfused acetylcholine (ACh) are very similar to those previously observed in adult submandibular ganglion neurons. The major differences observed are that the recorded cell input impedances are much higher than those recorded with microelectrodes from adult neurons and that the sensitivity of the cultured neurons to ACh is much less than that of the adult neurons. Whether the latter is due to changed receptor properties or to the presence of fewer receptors is not known.     

Effect of morphine on the sensitivity of cerebral cortical neurons to acetylcholine

Sensitivity of sensorimotor cortical neurons to microiontophoretically applied morphine and acetylcholine has been studied in the experiments on unanesthetized rabbits. The predominant reaction to morphine and acetylcholine was decrease and increase in the rate of neuronal impulse activity, respectively. There was no correlation in the responses to morphine and acetylcholine. Atropine failed to influence the morphine effect. When both drugs are simultaneously applied to neurons, morphine decreases both excitatory and inhibitory responses to acetylcholine. This effect of morphine may occur in the case when the drug is applied in doses which do not change spontaneous neuronal activity. On the contrary, excitatory effect of glutamic acid decreased only when morphine was applied in doses causing local anesthetic effect and decreasing background neuronal activity. It is suggested that morphine can exercise a modulating influence on choline receptors of cortical neurons.     

Biphasic responses to acetylcholine in mammalian reticulospinal neurons

Acetylcholine (ACh) responses were elicited by ionophoresis from neurons, located in the medial pontine reticular formation, which were antidromically identified as having axons projecting in the reticulospinal tracts. Most neurons were silent at rest and could be caused to discharge at a regular, slow rate by a constant application of glutamate. ACh altered this slow rate of firing in 28 of 29 cells but showed three different patterns of effect: approximately one-third were excited, one-third were inhibited, and one-third showed biphasic inhibition-excitation. The ACh responses were not sensitive to atropine. These observations suggest that reticulospinal neurons have ACh receptors mediating both inhibition and excitation, perhaps located on different portions of the same neuron.     

Reactivity of the cholinoceptive cortical neurons to the repeated action of acetylcholine

Three groups of sensorimotor cortical cholinoceptive neurons have been established in albino rats according to the dynamics of reactivity to repeated action of acetylcholine. There are neurons with decreased, increased or unchanged response to transmitter microintophoretic application. Dependence of the dynamics of background and evoked activity on the duration of excitatory reaction component induced by transmitter has been discovered. It was concluded that the duration of the given component is a significant and informative functional parameter of cortical neurons.     

Effects of glutamate on the serotonin-induced responses of vestibular neurons

The aim of this work was to verify whether and how spontaneous or glutamate(GLU)-induced enhancements of the neuronal firing rate modified the responsiveness of the vestibular neurons to microiontophoretic application of serotonin (5-HT). During experiments performed on anaesthetized Wistar rats the responses to 5-HT applications were studied in neurons of the lateral vestibular nucleus identified by the antidromic activation upon stimulation of the vestibulospinal tract. The magnitude (in percent) of the 5-HT induced excitatory responses decreased (hyperbolic correlation, r = 0.91) when the background mean firing rate was enhanced spontaneously or by long-lasting application of GLU. Even in high-discharging units, the response never changed its sign. The trend to a depression of the response to 5-HT in function of the background discharge was observed when either the enhancement of firing occurred spontaneously and it was induced by an application of GLU, no significant difference (F-test) being found between the two cases. It is concluded that serotoninergic afferents can exert a strong control upon the vestibular neurons when the background activity is depressed, and only a weak influence when the neuronal firing is enhanced by other excitatory afferents. It remains to verify whether the type of interference observed between GLU and 5-HT is specific or can be also detected between 5-HT and other excitatory neuromediators.     

Dynamics of the reactivity of cortical neurons to repeated electrophoretic application of acetylcholine

Dynamics of reactivity was studied in 50 neurones of the rat sensorimotor cortex to repeated acetylcholine microiontophoresis. By the parameter of response plasticity the neurones are distributed into three groups--unchanging, decreasing and increasing the excitatory component of the reaction. A connection has been established of the type and rate of tonic and evoked activities dynamics with the duration of the excitatory component of neuronal reactions to acetylcholine. The highest probability of these dynamic activity changes manifestation is observed in cholinoceptive neurones with duration of excitatory reaction components to acetylcholine equal to 3.2, 8.1 and 13.5 s.     

Fleeting activation of ionotropic glutamate receptors sensitizes cortical neurons to complement attack

Insidious attack of cortical neurons by complement has been implicated in Alzheimer's and other neurodegenerative diseases. Excitotoxicity, triggered by excessive activation of glutamate receptors, has been implicated in neuronal death following diverse insults, including ischemia and seizures. Clinical studies suggested that a minimal excitotoxic insult might sensitize neurons to complement attack. We found that fleeting activation of ionotropic glutamate receptors sensitizes neurons but not astrocytes to complement attack. The complement molecule effecting cytotoxicity was the membrane attack complex. The site within the complement cascade at which sensitization was effected was the membrane attack pathway. Sensitization mediated by glutamate receptor activation required Ca(2+)(o) and generation of reactive oxygen species. These in vitro findings predict that a fleeting excitotoxic insult could act synergistically with complement to destroy cortical neurons and accelerate neurological deterioration.     

Variability of calcium responses to agonists of glutamate receptors in hippocampal neurons

The subject of this work was to study the reasons of the variability of the calcium response amplitudes in individual neurons of the hippocampal cell culture to agonists of ionotropic glutamate receptors and the regularities of the calcium response amplitude distribution. Changes of [Ca²⁺] _i in the neurons in response to the NMDA-, AMPA-, and KA-receptor agonists were recorded using fluorescence probe Fura-2. The calcium response amplitudes (expressed as the ratio of fluorescence intensities of Fura-2 upon excitation at wave-lengths 340 and 380 nm) to short-term application of glutamate receptor agonists N-methyl-D-aspartate (NMDA), domoic acid (DA), α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), and (S)-(−)-5-fluorowillardiine (FW) were measured. Calcium responses of individual cells differed in shape and amplitude but always reproduced upon the second application of the agonist. To elucidate the nature of calcium response variability, we compared distributions of calcium response amplitudes to the NMDA-, KA-, and AMPA-receptor agonists in cultures of various ages in the presence of receptor desensitization inhibitors and different agonist concentrations. An even increase from 0.05 to 1.6 was characteristic for distributions of calcium response amplitudes. Nevertheless, in 1–3% neurons of the cell culture, calcium response amplitudes reached much higher values. The efficiency of the ligands usually increased in the following order: FW ≈ NMDA > DA. However, this regularity varied with age and depended on the presence of the receptor desensitization inhibitor. In the process of growth and differentiation of neurons in culture from 1 to 14 day in vitro, calcium response amplitude to AMPA- and KA-receptor agonists increased. Desensitization inhibitors transformed the response from pulse-like with a sharp peak into stepwise and increased the amplitude of calcium responses but did not abolish the character of even amplitude distribution. The effect of AMPA- and KA-receptor desensitization inhibitor decreased with calcium response amplitude growth in the control and approached zero in neurons with initially maximal amplitude. KA- and AMPA-receptor agonists at high concentrations possessed a property of desensitization inhibitors and transformed a transient response into a continuous one that lasted throughout the application time. Thus, the amplitude and shape of the calcium response to glutamate receptor agonists is a characteristic parameter of an individual cell.     

Visual aftereffects: cortical neurons change their tune

Recent studies of areas V1 and MT in the visual cortex show that exposure to a stimulus can change the contrast sensitivity of cells and shift their peak sensitivity to a new orientation or movement direction. In MT, these shifts can correctly predict illusory changes - visual aftereffects - in movement direction, but in V1, they are more difficult to interpret.     

Identification of the cortical neurons that mediate antidepressant responses

Our understanding of current treatments for depression, and the development of more specific therapies, is limited by the complexity of the circuits controlling mood and the distributed actions of antidepressants. Although the therapeutic efficacy of serotonin-specific reuptake inhibitors (SSRIs) is correlated with increases in cortical activity, the cell types crucial for their action remain unknown. Here we employ bacTRAP translational profiling to show that layer 5 corticostriatal pyramidal cells expressing p11 (S100a10) are strongly and specifically responsive to chronic antidepressant treatment. This response requires p11 and includes the specific induction of Htr4 expression. Cortex-specific deletion of p11 abolishes behavioral responses to SSRIs, but does not lead to increased depression-like behaviors. Our data identify corticostriatal projection neurons as critical for the response to antidepressants, and suggest that the regulation of serotonergic tone in this single cell type plays a pivotal role in antidepressant therapy.     

Effect of HgCl2 on acetylcholine,carbachol, and glutamate currents ofAplysia neurons

Summary 1. Using conventional two-microelectrode voltage-clamp techniques we studied the effects of inorganic mercury (HgCl₂) on acetylcholine-, carbachol-, and glutamate-activated currents onAplysia neurons. Hg²⁺ was applied with microperfusion.2. Acetylcholine and carbachol activated an inward, sodium-dependent current in the anterior neurons of the pleural ganglion. The medial neurons gave a biphasic current to acetylcholine and carbachol, which was outward at resting membrane potential. The faster component was Cl^– dependent and reversed at about –60 mV, while the slower component was K⁺ dependent and reversed at greater than –80 mV.3. Hg²⁺ (0.1–10 µM) caused a dramatic increase in the acetylcholine- and carbachol-induced inward current in anterior neurons and the fast Cl^– current in medial neurons. With only a 1-min preapplication of Hg²⁺, the acetylcholine- or carbachol-activated sodium or chloride currents were increased to 300% and the effect was only partly reversible. The threshold concentration was 0.1 µM Hg²⁺.4. Contrary to the effects on sodium and chloride currents, concentrations of 0.1–10 µM Hg²⁺ caused a complete and irreversible blockade of K⁺-dependent acetylcholine and carbachol currents. The block of the potassium current was relatively fast and increased with time. The concentration of HgCl₂ that gave a half-maximal blockade of the carbachol-activated potassium current was 0.89 µM. The chloride-dependent current elicited by glutamate on medial neurons was increased by HgCl₂ as well.5. These results suggest that actions at agonist-activated channels must be considered as contributing to mercury neurotoxicity. It is possible that the toxic actions of Hg²⁺ on synaptic transmission at both pre- and postsynaptic sites are important factors in the mechanism of Hg²⁺ toxicity.     

Effect of picamilon on inhibitory postsynaptic responses of cortical neurons

In experiments on immobilized anesthetized rats, we intracellularly recorded neuronal responses in the motor cortex before and after application of picamilon (PM) on the cortical surface; the responses were evoked by intracortical stimulation. Aplications of PM in the 5, 20, 50, and 100 μM concentrations noticeably increased, while that in the 10 μM concentration decreased the amplitude of IPSP in the cortical neurons. Probable mechanisms of the effect of PM on a cellular level are discussed.     

Calcium responses of circadian pacemaker neurons of the cockroach Rhyparobia maderae to acetylcholine and histamine

The accessory medulla (aMe) is the pacemaker that controls circadian activity rhythms in the cockroach Rhyparobia maderae. Not much is known about the classical neurotransmitters of input pathways to the cockroach circadian system. The circadian pacemaker center receives photic input from the compound eye, via unknown excitatory and GABAergic inhibitory entrainment pathways. In addition, neuropeptidergic inputs couple both pacemaker centers. A histamine-immunoreactive centrifugal neuron connects the ventral aMe with projection areas in the lateral protocerebrum and may provide non-photic inputs. To identify neurotransmitters of input pathways to the circadian clock with Fura-2-dependent Ca²⁺ imaging, primary cell cultures of the adult aMe were stimulated with acetylcholine (ACh), as the most prominent excitatory, and histamine, as common inhibitory neurotransmitter. In most of aMe neurons, ACh application caused dose-dependent increases in intracellular Ca²⁺ levels via ionotropic nicotinic ACh receptors. These ACh-dependent rises in Ca²⁺ were mediated by mibefradil-sensitive voltage-activated Ca²⁺ channels. In contrast, histamine application decreased intracellular Ca²⁺ levels in only a subpopulation of aMe cells via H2-type histamine receptor chloride channels. Thus, our data suggest that ACh is part of the light entrainment pathway while histamine is involved in a non-photic input pathway to the ventral circadian clock of the Madeira cockroach.     

Brain-derived neurotrophic factor enhances depolarization-evoked glutamate release in cultured cortical neurons

Brain-derived neurotrophic factor (BDNF) has been reported to play an important role in neuronal plasticity. In this study, we examined the effect of BDNF on an activity-dependent synaptic function in an acute phase. First, we found that short-term treatment (10 min) with BDNF enhanced depolarization-evoked glutamate release in cultured cortical neurons. The enhancement diminished gradually according to the length of BDNF treatment. The BDNF-enhanced release did not require the synthesis of protein and mRNA. Both tetanus toxin and bafilomycin abolished the depolarization-evoked glutamate release with or without BDNF, indicating that BDNF acted via an exocytotic pathway. Next, we investigated the effect of BDNF on intracellular Ca(2+). BDNF potentiated the increase in intracellular Ca(2+) induced by depolarization. The Ca(2+) was derived from intracellular stores, because thapsigargin completely inhibited the potentiation. Furthermore, both thapsigargin and xestospongin C inhibited the effect of BDNF. These results suggested that the release of Ca(2+) from intracellular stores mediated by the IP(3) receptor was involved in the BDNF-enhanced glutamate release. Last, it was revealed that the enhancement of glutamate release by BDNF was dependent on the TrkB-PLC-gamma pathway. These results clearly demonstrate that short-term treatment with BDNF enhances an exocytotic pathway by potentiating the accumulation of intracellular Ca(2+) through intracellular stores.     

Glucagon-like peptide 1 modulates calcium responses to glutamate and membrane depolarization in hippocampal neurons

Glucagon-like peptide 1 (GLP-1) activates receptors coupled to cAMP production and calcium influx in pancreatic cells, resulting in enhanced glucose sensitivity and insulin secretion. Despite evidence that the GLP-1 receptor is present and active in neurons, little is known of the roles of GLP-1 in neuronal physiology. As GLP-1 modulates calcium homeostasis in pancreatic beta cells, and because calcium plays important roles in neuronal plasticity and neurodegenerative processes, we examined the effects of GLP-1 on calcium regulation in cultured rat hippocampal neurons. When neurons were pre-treated with GLP-1, calcium responses to glutamate and membrane depolarization were attenuated. Whole-cell patch clamp analyses showed that glutamate-induced currents and currents through voltage-dependent calcium channels were significantly decreased in neurons pre-treated with GLP-1. Pre-treatment of neurons with GLP-1 significantly decreased their vulnerability to death induced by glutamate. Acute application of GLP-1 resulted in a transient elevation of intracellular calcium levels, consistent with the established effects of GLP-1 on cAMP production and activation of cAMP response element-binding protein. Collectively, our findings suggest that, by modulating calcium responses to glutamate and membrane depolarization, GLP-1 may play important roles in regulating neuronal plasticity and cell survival.     

Characteristics of responses of preoptic neurons to presentation of serial cortical stimuli

Responses of neurons of the medial (MPO) and lateral (LPO) preoptic region (RPO) and adjacent hypothalamic structures to serial stimuli (6–300/sec) of the prefrontal (area 8) and cingulate (area 24) cortex, piriform lobe (periamygdaloid cortex — RPA), and hippocampus (area CA3) were investigated in acute experiments on cats under ketamine anesthesia. Four main types of responses were found: excitatory, inhibitory, excitatory on-off effect, and inhibitory on-off effect. With the use of stimuli with increasing frequencies, the direction of the response remained constant, only its intensity changed. Neurons responding to presentation of serial stimuli were localized mainly in the central part of the MPO and basal part of the LPO, where the most pronounced foci of convergence were observed. During serial stimulation of cortical structures, inhibitory responses occurred considerably more often than excitatory (ratio 3.4:1). The presence of a gradient of inhibition was established from new to old (in a phylogenetic respect) brain formations in a number of stimulated structures. In the case of stimulating the neocortex (proreal gyrus), the predominance of inhibitory responses over excitatory was minimum (1.7:1); it increased (1.9:1) in the case of stimulating the intermediate cortex (cingulate gyrus), still more (4.5:1) under conditions of stimulating the paleocortex (periamygdaloid cortex), and in the case of stimulating the archicortex (10.2:1).A. M. Gorky Medical Institute, Ukrainian Ministry of Health, Donetsk. Translated from Neirofiziologiya, Vol. 23, No. 6, pp. 720–731, November–December, 1991.     

Immature cortical neurons are uniquely sensitive to glutamate toxicity by inhibition of cystine uptake

Using the N18-RE-105 neuroblastoma X retina cell line, we previously described Ca2(+)-dependent quisqualate-type glutamate toxicity caused by the inhibition of high-affinity cystine uptake, leading to glutathione depletion and accumulation of cellular oxidants. We now demonstrate that primary cultures of rat cortical neurons (E17; 24-72 h in culture), but not glia, also degenerate when exposed to culture medium with reduced cystine or containing competitive inhibitors of cystine uptake, including glutamate. At this developmental stage, neurotoxicity did not occur as a consequence of continuous exposure to glutamate receptor subtype agonists, N-methyl-D-aspartate, kainate, or 2(RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionate. However, those that inhibited neuronal cystine uptake--quisqualate, glutamate, homocysteate, beta-N-oxalyl-L-alpha,beta-diaminopropionic acid, and ibotenate--were neurotoxic. Toxicity related to quisqualate did not correlate with the development of quisqualate-stimulated phosphatidylinositol turnover. The toxic potencies of glutamate, quisqualate, and homocysteate were inversely proportional to the concentration of cystine in the medium, suggesting that they competitively inhibit cystine uptake. Autoradiographic analysis of the cellular localization of L-[35S]cystine uptake indicated that embryonic neurons have a high-affinity transport system that is sensitive to quisqualate, whereas non-neuronal cells in the same cultures have a low-affinity system that is insensitive to quisqualate but potently blocked by D-aspartate and glutamate. Exposure to glutamate or homocysteate resulted in a time-dependent depletion of the cellular antioxidant glutathione. The centrally acting antioxidant idebenone and alpha-tocopherol completely blocked the neurotoxicity resulting from glutamate exposure. We propose that competitive inhibition of cystine transport and reduction of extracellular cystine levels result in neuronal cell death due to accumulation of cellular oxidants.