Pharmacological analysis of slow potentials recorded in forg olfactory bulb during natural stimulation

Pharmacological agents (strychnine, picrotoxin, pentobarbital, chloralose, GABA, penicillin, morphine) were used to investigate the nature of the slow potential recorded in the frog olfactory bulb in response to natural stimulation. Three possible hypotheses were tested: 1) The slow potential is neuroglial in nature; 2) it is the analog of the dorsal-root potential of the spinal cord and reflects depolarization of primary afferents arising in the terminals of the olfactory nerve and responsible for presynaptic inhibition in the frog olfactory bulb; 3) the slow potential reflects postsynaptic processes. The results showed great similarity between changes in the slow and dorsal-root potentials of the spinal cord in response to the action of pharmacological agents. However, the slow potential is evidently a complex response and incorporates at least one other component — depolarization of the dendrites of unknown nature.     

Synaptic inhibition in an isolated nerve cell

Following the preceding studies on the mechanisms of excitation in stretch receptor cells of crayfish, this investigation analyzes inhibitory activity in the synapses formed by two neurons. The cell body of the receptor neuron is located in the periphery and sends dendrites into a fine muscle strand. The dendrites receive innervation through an accessory nerve fiber which has now been established to be inhibitory. There exists a direct peripheral inhibitory control mechanism which can modulate the activity of the stretch receptor. The receptor cell which can be studied in isolation was stimulated by stretch deformation of its dendrites or by antidromic excitation and the effect of inhibitory impulses on its activity was analyzed. Recording was done mainly with intracellular leads inserted into the cell body. 1. Stimulation of the relatively slowly conducting inhibitory nerve fiber either decreases the afferent discharge rate or stops impulses altogether in stretched receptor cells. The inhibitory action is confined to the dendrites and acts on the generator mechanism which is set up by stretch deformation. By restricting depolarization of the dendrites above a certain level, inhibition prevents the generator potential from attaining the "firing level" of the cell. 2. The same inhibitory impulse may set up a postsynaptic polarization or a depolarization, depending on the resting potential level of the cell. The membrane potential at which the inhibitory synaptic potential reverses its polarity, the equilibrium level, may vary in different preparations. The inhibitory potentials increase as the resting potential is displaced in any direction from the inhibitory equilibrium. 3. The inhibitory potentials usually rise to a peak in about 2 msec. and decay in about 30 msec. After repetitive inhibitory stimulation a delayed secondary polarization phase has frequently been seen, prolonging the inhibitory action. Repetitive inhibitory excitation may also be followed by a period of facilitation. Some examples of "direct" excitation by the depolarizing action of inhibitory impulses are described. 4. The interaction between antidromic and inhibitory impulses was studied. The results support previous conclusions (a) that during stretch the dendrites provide a persisting "drive" for the more central portions of the receptor cell, and (b) that antidromic all-or-none impulses do not penetrate into the distal portions of stretch-depolarized dendrites. The "after-potentials" of antidromic impulses are modified by inhibition. 5. Evidence is presented that inhibitory synaptic activity increases the conductance of the dendrites. This effect may occur in the absence of inhibitory potential changes.     

Connections of the second auditory cortical area with the medial geniculate body and the first auditory area

Extra — and intracellular unit responses in area AII to stimulation of geniculocortical fibers and of area AI were studied in cat immobilized with D-tubocurarine. In response to stimulation of geniculocortical fibers, antidromic mono-, di-, and polysynaptic spikes were generated by neurons in area AII. The number of antidromic responses in area AII was about half that found in area AI under the same conditions of stimulation. Most of the orthodromic responses were di- and polysynaptic. Intracellular responses also were recorded in the form of EPSPs, EPSP-IPSPs, and primary IPSPs. Stimulation of area AI evoked responses in the neurons of area AII with latent periods of 0.75–6.0, 6.1–16.0, 18.0–23.0, and 60–100 msec. Removal of the medial geniculate body led to a marked decrease in the number of responses with latent periods of 6.1–16.0 msec. Some neurons of area AII responded by spikes to stimulation of both the geniculocortical fibers and area AI. Comparison of the latent periods of responses to these two types of stimulation showed that impulses from area AI to area AII are directed both to input neurons for impulses from the medial geniculate body and to neurons at subsequent stages of the intracortical neuronal change. In response to stimulation of cortical area AI, disynaptic IPSPs appeared in many neurons of area AII. Only one IPSP with a latent period of 1.0 msec, regardable as monosynaptic, was recorded.     

Electrophysiological features of facial motoneurons in cats

Antidromic activation of facial motoneurons in cats during stimulation of different branches of the facial nerve was studied by intracellular recording. Time and amplitude characteristics of individual components of the antidromic action potentials were analyzed and fast and slow after-potentials distinguished. Correlation was found between the duration of the descending phase of the SD spike, duration of its after-hyperpolarization, and the spike conduction time along the axon. Data were obtained to show absence of a recurrent collateral pathway in motoneurons of the facial nucleus. The functional significance of the after-potentials is discussed.L. A. Orbeli Institute of Physiology, Academy of Sciences of the Armenian SSR, Erevan. Translated from Neirofiziologiya, Vol. 10, No. 3, pp. 261–270, May–June, 1978.     

Effects of bicuculline and picrotoxin on fast and slow IPSP in pyramidal neurons in hippocampal slices isolated from the rat

The effects of different concentrations of bicuculline and picrotoxin on IPSP of pyramidal neurons with high and low rates of rise ("fast" and "slow" IPSP) were investigated in hippocampal slices isolated from area CA1. These neurons generated mainly fast IPSP in response to antidromic stimulation; orthodromic stimulation produced both fast and slow IPSP, often combining into a single IPSP with two phases. Both these patterns of IPSP were recorded from the apical dendrites as well as the soma and were reversibly inhibited by bicuculline and picrotoxin. Degree of inhibition depended on dose and duration of blocker action, but slow IPSP were more resistant to this action. At the same effective concentration of bicuculline or picrotoxin, slow IPSP were inhibited later and recovered sooner after washout of blocker than fast IPSP. The difference between the inhibitory effect of blockers on fast and slow IPSP persisted under tetanic stimulation, although the progress of reduction in IPSP proceeded far more rapidly than following application of a single stimulus. The reason for this phenomenon is discussed, as well as particular features of -aminobutyric acid (GABA) receptors of pyramidal neurons mediating generation of slow IPSP.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 22, No. 1, pp. 44–54, January–February, 1990.     

Pyrethroid insecticides: Actions of deltamethrin and related compounds on insect axonal sodium channels

The actions of deltamethrin and eight other pyrethroids were tested on isolated giant axons of the cockroach Periplaneta americana, using microelectrode and oil-gap, single-fibre electrophysiological recording techniques. Deltamethrin at micromolar concentrations induced a slow progressive depolarization of the axon membrane accompanied by a gradual reduction in action potential amplitude. The deltamethrin-induced depolarization was enhanced by an increase in stimulation frequency and was reduced in the presence of the sodium channel blocking agent saxitoxin (1 × 10^?7 M).Other synthetic pyrethroids (biopermethrin and its 1S enantiomer, biotetramethrin, s-bioallethrin, bioresmethrin and its 1S enantiomer, cismethrin and kadethrin) were also studied. In contrast to the findings with deltamethrin all other compounds, apart from the 1S isomers which were inactive, induced prolonged negative (depolarizing) after-potentials. Deltamethrin appears to affect a small fraction of sodium channels which are held in a modified open-state, whereas the pyrethroids which generate large negative after-potentials appear to induce a brief alteration of the open-state sodium channels with a larger number of channels affected. Differences between the actions of pyrethroids on insect axonal sodium channels and whole insects are discussed.     

Inhibition in the olfactory bulb of the lizard

Inhibition in neurons of the lizard olfactory bulb was investigated by intracellular recording. The hyperpolarization arising in the neurons after the spike in the response to orthodromic and antidromic activation is similar in composition and reflects the development of early and late IPSPs, differing from one another in latency, duration, and mechanism of generation. The early IPSP is evidently generated by the functioning of dendrodendritic synapses, formed by dendrites of the interglomerular cell on the membrane of the apical dendrites of the secondary neurons, whereas synapses generating the late IPSP are located on the basal dendrites and are formed by endings of the granular cells. The mechanisms of generation of the early and late IPSPs in the secondary neurons are discussed. A classification of neurons of the lizard olfactory bulb is given on the basis of analysis of their intracellular activity.     

Age related changes in blood-to-brain amino acid transport and incorporation into brain protein

Blood-to-brain amino acid transport consists of at least two components: 1. a fast rate or early process, commonly measured by the intra-carotid bolus injection method and attributed to transport across the capillary endothelium and entry into the astrocytes, and, 2. a slow rate or later component measured over 2 to 15 minutes probably associated with exit from the astrocytes and entry into the neurons. Incorporation into brain protein is temporally related to the second process. In the present study the slow and fast rate transport components and the incorporation into brain protein of tyrosine (Tyr) and Valine (Val) was measured in young adult and aged male C57BL/6 mice. The results indicate that the fast rate transport component is unaffected by age while the rates of the slow process and protein turnover show an exponential decline most marked between 3 and 8 months of age. Changes in the relative incorporation of Tyr and Val suggest that brain protein metabolism is altered qualitatively as well as quantitatively in aging, in these animals.     

Electrophysiological and pharmacological properties of cultured rat thyroid cells

The electrophysiological properties of a hormone-dependent, differentiated thyroid epithelial cell strain were studied using intracellular microelectrodes. The average membrane potential of solitary, isolated cells was –78.4 ± 1.3 mV. The membrane potential depolarized 55 mV per tenfold increase in extracellular potassium concentation. Weak electrical coupling was recorded between contiguous cells. Like tyroid cells in vivo, these cells did not generate action potentials. In some cells a spontaneous, slow transition in the membrane potential from –80mV to –30 mV was accompanied by an increase in input resistance. Membrane potential transitions could be induced by perfusing cells with isotonic Hanks solutions saturated with CO₂ (pH = 5.5) or by perfusing cells with hypotonic Hanks solutions (190–290 mOsm/kg). Membrane potential transitions were due to a decreased potassium permeability. Noradrenaline elicted both a fast depolarization and a slow depolarization. The fast depolarization was due to an increase in conductance of Na⁺ channels and of Cl^– channels. Intracellular injection of Ca⁺⁺ elicited the fast depolarization. Intracellular injection of EGTA or cobalt abolished the fast depolarization. Replacemnt of extracellular Ca⁺⁺ by Mg⁺⁺ did not affect the fast depolarization. Thus, the fast depolarization was due to accumulation of intracellular Ca⁺⁺. The fast depolarization was abolished by the alpha adrenergic blocker phentolamine (10^–6 M), and was not abolished by the beta adrenergic blocker propranolol (10^–5 M).     

Role of active ion transport in reception of the generator potential of the isolated frog muscle spindle

The effect of complete replacement of sodium ions by lithium ions in Ringer's solution and of 10⁻⁴ M ouabain on the receptor potential of the isolated frog muscle spindle was investigated. Initially, under the influence of lithium ions and ouabain, the hyperpolarization phase of this potential diminished and disappeared, and later the same fate befell the static and dynamic part of its depolarization phase. The rise time of the receptor potential was increased in a solution containing lithium ions but in the solution with ouabain it remained the same as initially. No appreciable changes were found in the rate of fall of the dynamic part of the depolarization phase. On rinsing the muscle spindle in normal Ringer's solution in the experiment with lithium ions recovery was incomplete, and in the experiments with ouabain the receptor responses were not restored.     

Two Components of the Cardiac Action Potential : I. Voltage-time course and the effect of acetylcholine on atrial and nodal cells of the rabbit heart

Transmembrane potentials recorded from the rabbit heart in vitro were displayed as voltage against time (V, t display), and dV/dt against voltage (V, V or phase-plane display). Acetylcholine was applied to the recording site by means of a hydraulic system. Results showed that (a) differences in time course of action potential upstroke can be explained in terms of the relative magnitude of fast and slow phases of depolarization; (b) acetylcholine is capable of depressing the slow phase of depolarization as well as the plateau of the action potential; and (c) action potentials from nodal (SA and AV) cells seem to lack the initial fast phase. These results were construed to support a two-component hypothesis for cardiac electrogenesis. The hypothesis states that cardiac action potentials are composed of two distinct and physiologically separable "components" which result from discrete mechanisms. An initial fast component is a sodium spike similar to that of squid nerve. The slow component, which accounts for both a slow depolarization during phase 0 and the plateau, probably is dependent on the properties of a slow inward current having a positive equilibrium potential, coupled to a decrease in the resting potassium conductance. According to the hypothesis, SA and AV nodal action potentials are due entirely or almost entirely to the slow component and can therefore be expected to exhibit unique electrophysiological and pharmacological properties.     

Characteristics of cortical inhibitory neurons

Spikes were recorded extracellularly and IPSPs intracellularly from auditory cortical neurons of cats immobilized with D-tubocurarine in response to stimulation of geniculo-cortical fibers. Fibers whose stimulation induces IPSPs in auditory cortical neurons mainly have low thresholds. When two stimuli, each of which separately evoked an IPSP of maximal amplitude, were applied to them the shortest interval at which the second stimulus evoked an effect was 2.5–3 msec. This effect consisted of an increase in the duration of the integral IPSP, the amplitude of which either remained unchanged or increased under these circumstances by only 5–10%. The interval at which a separate IPSP appeared in response to the second stimulus depended on the duration of the ascending phase of the IPSP and varied from 4 to 22 msec for different neurons. The amplitude of the second IPSP in this case depended on the interval between stimuli. Under moderately deep pentobarbital anesthesia the number of neurons responding to stimulation of the geniculo-cortical fibers by spikes fell sharply but the number of neurons responding by primary IPSPs remained almost unchanged. Under very deep pentobarbital anesthesia, when spike responses of the cortical neurons completely disappeared, the IPSPs also were completely suppressed. It is concluded that inhibitory neurons of the auditory cortex are excited by thick low-threshold fibers, they have a short refractory period, and they are resistant to the narcotic action of pentobarbital.     

Kinetics of the sodium current through EDTA-modified calcium channels of the molluscan neuron somatic membrane

The kinetics of the slow current carried by sodium ions through potential-dependent calcium channels after addition of EDTA to calcium-free external solution was investigated in experiments by the intracellular dialysis method on isolatedHelix pomatia neurons. The activation kinetics of this current was similar to that of the calcium current and could be described by the use of the square of the activation variable m in Hodgkin-Huxley equations. The decay (inactivation) kinetics of the induced sodium current during prolonged depolarization is biexponential in character. It is suggested that decay of the sodium currents takes place as a result of two independent processes: potential-dependent inactivation with a time constant τ_h～1 sec, taking place as far as a certain steady-state level h_∞, and a decrease in current connected with Na⁺ accumulation inside the cell during passage of the current and a consequent change in the sodium electrochemical potential (τ_c～10 sec). It is concluded that modification of the calcium channels, so that they acquire the ability to conduct sodium, has no significant effect on the gating mechanisms responsible for opening and closing of the channels.     

Blocking action of furosemide on chloride conductance induced by acetylcholine and gaba in molluscan neuron membrane

Experiments on isolated neurons of the molluskPlanorbarius corneus under membrane voltage clamp conditions showed that furosemide (2×10^?4 to 1×10^?3 g/mg) inhibits the increase in chloride conductance evoked by iontophoretic application of acetylcholine, suberyldicholine, and gamma-aminobutyric acid (GABA). If microelectrodes filled with potassium sulfate were used in the experiments furosemide did not shift the reversal potential, but when microelectrodes filled with potassium chloride were used the reversal potential of the chloride-dependent responses became less negative. In the last case, the action of furosemide evidently was exhibited not only on passive chloride conductance of the chemoreceptive membrane, but also on active chloride transport. Furosemide had no effect on sodium- and potassium-dependent responses evoked by activation of choline receptors. Unlike D-tubocurarine, which selectively blocks acetylcholine effects, furosemide also depressed conductance evoked by GABA. In the presence of furosemide chloride-dependent responses not only were reduced in amplitude, but also developed more slowly. It is postulated that the action of furosemide is aimed not at receptors, but at chloride channels of the chemoreceptive membrane common to both acetylcholine and GABA.     

Selective Cleavage of SNAREs in Sensory Neurons Unveils Protein Complexes Mediating Peptide Exocytosis Triggered by Different Stimuli

Oligomerisation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes is required for synaptic vesicle fusion and neurotransmitter release. How these regulate the release of pain peptides elicited by different stimuli from sensory neurons has not been established. Herein, K⁺ depolarization was found to induce multiple sodium dodecyl sulfate (SDS)-resistant SNARE complexes in sensory neurons exposed to botulinum neurotoxins (BoNTs), with molecular weights ranging from 104–288 k (large) to 38–104 k (small). Isoform 1 of vesicle-associated membrane protein 1 (VAMP 1) assembled into stable complexes upon depolarisation and was required for the participation of intact synaptosome-associated protein of relative molecular mass 25 k (SNAP-25) or BoNT/A-truncated form (SNAP-25_A) in the large functional and small inactive SDS-resistant SNARE complexes. Cleaving VAMP 1 decreased SNAP-25_A in the functional complexes to a much greater extent than the remaining intact SNAP-25. Syntaxin 1 proved essential for the incorporation of intact and SNAP-25_A into the large complexes. Truncation of syntaxin 1 by BoNT/C1 caused /A- and/or /C1-truncated SNAP-25 to appear in non-functional complexes and blocked the release of calcitonin gene-related peptide (CGRP) elicited by capsaicin, ionomycin, thapsigargin or K⁺ depolarization. Only the latter two were susceptible to /A. Inhibition of CGRP release by BoNT/A was reversed by capsaicin and/or ionomycin, an effect overcome by BoNT/C1. Unlike BoNT/B, BoNT/D cleaved VAMP 1 in addition to 2 and 3 in rat sensory neurons and blocked both CGRP and substance P release. Thus, unlike SNAP-25, syntaxin 1 and VAMP 1 are more suitable targets to abolish functional SNARE complexes and pain peptide release evoked by any stimuli.     

Membrane currents induced by inflow of sodium ions into mollusk giant neurons

Membrane currents induced by inflow of sodium ions were investigated in giant neurons of the molluskHelix pomatia during tetanic stimulation or prolonged membrane depolarization under voltage clamp conditions. The membrane current thus produced consists of two components, a fast component with a reversal potential close to the potassium equilibrium potential, and a slow component only slightly dependent on membrane potential in the region from −50 to −90 mV. Addition of strophanthin K to the external solution, or replacement of sodium in the external solution by lithium or calcium abolished the slow component of the membrane current and reduced the fast component. It is concluded that the slow component appears as the result of activation of the sodium pump under electrogenic conditions, where-as the fast component arises as the result of an increase in potassium permeability, possibly coupled with intensive activity of this pump.     

Motion sensitive interneurons in the optomotor system of the fly

The three horizontal cells of the lobula plate of the blowflyCalliphora erythrocephala were studied anatomically and physiologically by means of cobalt impregnations and intracellular recordings combined with Procion and Lucifer Yellow injections. The cells are termed north, equatorial and south horizontal cell (HSN, HSE, HSS) and are major output neurons of the optic lobe. 1. The dendritic arborizations of the HSN, HSE, HSS reside in a thin anterior layer of the lobula plate and extend over the dorsal, equatorial and ventral parts of this neuropil, respectively. Due to the retinotopic organization of the optic lobe, these parts correspond anatomically to respective regions of the ipsilateral visual field. Homologue horizontal cells in both lobula plates of the same animal and in different animals are highly variable with respect to their individual dendritic branching patterns. They are extraordinarily constant, on the other hand, with regard to the position and size of their dendritic fields as well as their dendritic branching density distributions. Each cell covers about 40% of the total area of the lobula plate and shows the highest dendritic density near the lateral margin of the neuropil which subserves the frontal eye region. The axons of the horizontal cells are relatively short and large in diameter; they terminate in the posterior ventrolateral protocerebrum. 2. The horizontal cells are directionally selective motion sensitive visual interneurons responding preferentially to progressive (front to back) motion in the ipsilateral visual field with graded depolarization of their axons and superimposed action potentials. Stimulation with motion in the reverse direction leads to hyperpolarizing graded responses. The HSE and HSN are additionally activated by regressive motion in the contralateral visual field.     

Synaptic potentials of motoneurons of the masseter muscle

Postsynaptic potentials of 93 motoneurons of the masseter muscle evoked by stimulation of different branches of the trigeminal nerve were studied. Stimulation of the most excitable afferent fibers of the motor nerve of the masseter muscle evoked monosynaptic EPSPs with a latent period of 1.2–2.0 msec, changing into action potentials when the strength of stimulation was increased. A further increase in the strength of stimulation produced an antidromic action potential in the motoneurons with a latent period of 0.9 msec. In some motoneurons polysynaptic EPSPs and action potentials developed following stimulation of the motor nerve to the masseter muscle. The ascending phase of synaptic and antidromic action potentials was subdivided into IS and SD components, while the descending phase ended with definite depolarization and hyperpolarization after-potentials. Stimulation of cutaneous branches of the trigeminal nerve, and also of the motor nerve of the antagonist muscle (digastric) evoked IPSPs with a latent period of 2.7–3.5 msec in motoneurons of the masseter muscle. These results indicate the existence of functional connections between motoneurons of the masseter muscle and its proprioceptive afferent fibers, and also with proprioceptive afferent fibers of the antagonist muscle and cutaneous afferent fibers.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 1, No. 3, pp. 262–268, November–December, 1969.     

Unit responses of the second auditory area to acoustic stimulation

Responses of 116 neurons of the second auditory area to clicks were recorded extracellularly in experiments on unanesthetized cats immobilized with D-tubocurarine. Neurons with and without (54.6%) took part in the response to clicks. The unit response to a click consisted of 1 or 2 spikes or a short volley. Different neurons responded to clicks at different times. The latent period of 25.8% of all neurons recorded was 6.5–13 msec, of 70% it was 14–25 msec, and of 4.2% it was over 25 msec. Long-latency responses to clicks (40, 50, and 100 msec) also were recorded. The responding neurons were found throughout the thickness of the cortex, but more frequently in layers III and IV. No relationship was found between the depth of the neuron and its latest period. Responses consisting of EPSP, EPSP-spike, EPSP-spike-IPSP, EPSP-IPSP, and primary IPSP were recorded intracellularly from the neurons of this area. It was concluded from the results that neurons of the second auditory area can be activated by the arrival of an afferent volley along the geniculo-cortical pathway and also by the arrival of impulses from the first auditory area.     

Nonsynaptic membrane of retinal horizontal cells as a slow potential amplifier

A simplified model of the membrane of horizontal cells of the L-type is designed to reflect two principal features of these cells previously studied experimentally: 1) their hyperpolarization response to light is the result of a decrease in the EPSP that is kept constant in darkness; 2) the resistance of their nonsynaptic membrane is reduced during hyperpolarization within physiological limits (from 0 to−70 mV). The model also reproduces properties of the horizontal cells such as the low membrane potential in darkness, reversal of the response to light during depolarization beyond the zero level, mutual amplification of color signals, saturation of the response to bright light, steady-state volt-ampere characteristics in darkness and light, and the amplitude characteristic curve which often has a steep part within a certain range of membrane potentials. The presence of hysteresis loops of the volt-ampere and amplitude characteristic curves of the horizontal cells predicted by the model was confirmed experimentally on the fish retina. Analysis of the model and results obtained with it show that the nonsynaptic membrane of the horizontal cells can actively amplify slow graded potentials.