Tonic Neurons Research Articles

Substance P reconfigures the neural respiratory network in ventrolateral medulla in the neonatal rat brainstem in vitro

The effects of SP on the respiratory activity in the rat (0–4 days old) neonatal brainstem-spinal cord preparation were investigated. Respiratory activity was recorded from C4 ventral roots and intra-cellularly, from three types of respiration-related neurons: pre-inspiratory (Pre-I or biphasic E, n = 19), three subtypes of inspiratory (InspI-III, n = 18), expiratory (Exp, n = 11), and tonic neurons (n = 11) in the ventrolateral medulla (VLM). There were two types of respiratory rhythm in respiration-related neurons and in integrated C4 nerve activity under normal experimental conditions, with marked similarities to normal eupneic respiration and sighs. These two types of bursts were similar to patterns described recently in rhythmic brainstem slices in neonatal mice [1]. In control, the frequency of eupneic-like bursts varied in a range of 7–11 bursts per minute, whereas the frequency of the sigh-like activity was 10–30 times slower at 0.2–0.6 bursts per minute. Bath application of SP (10 nM-1 μM) caused a biphasic effect on eupneic-like respiratory frequency. There was a pronounced dose-dependent decline of burst rate (by 48% from control level of 9.35 ± 1.8 (mean ± SEM) bursts per minute, n = 28) after the onset the SP application (phase P1), followed by a weaker dose-dependent increase (by 17.5%) in burst rate (phase P2) (P < 0.001, two-way ANOVA). The biphasic effect of SP on inspiratory burst rate was associated with sustained membrane depolarization (in a range of 0.5–13 mV) of respiration-related and tonic neurons. C4 bursting before and after SP application was synchronized with neuronal activity of respiration-related neurons. Unlike the biphasic effect shown in the eupneic-like activity, the sigh-like burst frequency was considerably and gradually increased. This elevated sigh-like activity could reset normal respiration rhythm, so that in some cases the respiration activity was represented exclusively by the sigh-like pattern. As with the first pattern, this frequency increase depended on SP concentration (P < 0.001, one-way ANOVA), though the range of the changes was an order bigger as compared to normal respiratory pattern, and reached 650%. There was significant correlation (R = 0.537, P < 0.0001) between the eupneic-like frequency decrease during P1 and the sigh-like rhythm increase, and P1 phase corresponded in time to maximum slope of increase in sigh-like frequency. In turn, the P2 phase in the eupneic-like bursting coincided in time with maximum sigh-like rhythm developed during SP application. Thus, the biphasic effect of SP on eupneic-like respiratory activity was superimposed on a monotonic increase of the sigh-like pattern frequency. Our results suggest that a) a single respiratory network in the VLM might reproduce or generate two different respiratory patterns; b) within this respiratory network SP exerts a general excitatory effect on respiration-related and tonic neurons; c) the transient changes in neuronal activity in the VLM produced by SP may underlie the biphasic effect in the eupneic-like bursting, as well as the changes in the sigh-like pattern frequency, and d)SP can transform the particular type of the interaction of eupneic-like and sigh-like patterns, which compose the normal respiratory activity in the neonatal brainstem, and thus, reconfigure the neural network controlling respiratory rhythm generation.

Integral-differential calculus computations by short-term potentiation and depression in NTS-pontine pathways

Neural integrators are a class of neural networks that afford extended temporal summation of neural activity or, equivalently, low-pass frequency filtering of neurotransmission [1]. In the respiratory system, a biphasic neural integrator in the carotid chemoreflex response is demonstrated by the classic short-term potentiation (STP) and afterdischarge of phrenic activity [2]. Recently, a new class of neural networks called neural differentiators, which afford high-pass frequency filtering of afferent inputs through short-term depression (STD) of neurotransmission, has been identified in the vagal-carotid control of the expiratory off-switch [3]. The differentiator response is characterized by: 1) a habituation-like adaptation to the vagal Hering-Breuer (HB) reflex lengthening or carotid chemore-flex shortening of expiratory duration; 2) secondary adaptive effects in the form of pontine-mediated and NMDA receptor-dependent post-stimulus rebound and recovery of these vagal or carotid [4](or hypoxia [5]) mediated responses. To elucidate the mechanism of the habituation of the HB reflex, we obtained extracellular field potentials (FPs) in the medial and commissural regions of the nucleus tractus solitarius (NTS) evoked by electrical vagal stimulation (1 min) in urethane-anesthetized, paralyzed and mechanically ventilated rats (n =17) in vivo. FPs from vagal A-fibers (stimulus current, 20–70 μA, response latency, 4–5 ms) revealed a stimulus frequency-dependent (20–80 Hz) STD similar to the synaptic accommodation observed in vitro [6] and the habituation of HB reflex in vivo [4]. FPs from C-fibers (150–350 μA, 20–45 ms) also demonstrated a frequency-dependent (5–20 Hz) STD during afferent activation but their amplitude remained depressed for >50 s post-stimulation. Microinjection of the NMDA receptor antagonist D-APV abolished the short-term memory following C-fiber activation but not the frequency-dependent STD with A- or C-fibers. The characteristics of the vagal A-and C-fiber mediated responses are reminiscent of the type I and type II NTS neurons found in vitro [7]. To elucidate the mechanism of the pontine-mediated adaptation of the HB reflex, we obtained extracellular recordings of pontine neuronal activity simultaneously with phrenic nerve recording during electrical vagal stimulation (1.5×T, 80 Hz, 1 min). Neurons with tonic and respiratory modulated activity were found within or near the parabrachialis (PB) and Kolliker-Fuse (KF) complex. Most tonic neurons in KF (n = 11) were depressed during and/or shortly after vagal stimulation that elicited the characteristic biphasic adaptation of the HB reflex in the phrenic activity, whereas most tonic neurons in medial PB (n = 22) were not affected. Activity of respiratory modulated neurons (n = 4) recorded in KF remained in phase with phrenic activity during vagal stimulation. Results showed that vagal input induced STD of tonic neurotransmission in KF. These findings support a dual-process nonassociative learning model of integral-differential calculus computations in the brain [8] through activity-dependent STP and STD of neurotransmission in primary and secondary afferent pathways.

Effects of Lead Ions on the Synaptic Responses of Neurons of the Rat Sympathetic Ganglion

Using intracellular recording, we studied the effects of lead ions on the synaptic responses of neurons of the superior cervical ganglion (SCG) of the rat. Three groups of SCG neurons were found; they differed from each other in directions of the changes evoked by Pb2+ applications and in their sensitivity to this agent. In two groups of the phasic SCG neurons, Pb2+ decreased the amplitude of EPSP evoked by stimulation of the cervical sympathetic nerve with EC50 of (2.2 ± 0.2) · 10-6 M and (3.55 ± 0.29) · 10-5 M, respectively. In the tonic neurons, 2 · 10-6 M Pb2+ increased the EPSP amplitude by 27 ± 2%, on average, as compared with the control values. We postulate that the specificity of responses of these three groups of SCG neurons to the action of Pb2+ is determined by the different subunit composition of the nicotinic cholinoreceptors in the membrane of these cells.

Electrophysiological properties of lumbosacral preganglionic neurons in the neonatal rat spinal cord

The electrophysiological properties of parasympathetic preganglionic neurons (PGN) in L6 and S1 spinal cord slices from neonatal rats were studied using the patch clamp techniques. PGN were identified by retrograde axonal transport of a fluorescent dye (Fast Blue) injected intraperitoneally before the experiment. PGN in the intermediolateral region of the spinal cord were divided into two classes (tonic PGN and phasic PGN) on the basis of firing properties during prolonged (300 ms) depolarizing current pulses. Tonic neurons exhibited a prolonged discharge (average maximum: 5.6); whereas phasic PGN fired on average only 1.4 spikes during depolarizing pulses. PGN were usually oval in shape. The mean long axis of tonic PGN (20.7±0.5 μm) was significantly ( P<0.05) larger than that of phasic PGN (16.7±0.3 μm). Tonic and phasic PGN had similar resting membrane potentials, thresholds for spike activation, input resistances and action potential durations. The duration of the after-hyperpolarization (AHP) in tonic PGN (200.5±11.9 ms) was longer than in phasic PGN (137.6±9.8 ms). 4-aminopyridine (4-AP, 0.5 mM) reduced the threshold for spike activation in tonic and phasic PGN. 4-AP also unmasked tonic firing in phasic PGN (average maximum: 5.5 spikes during 300 ms depolarizing current pulses) and increased firing frequency by 19% in tonic PGN. These data indicate that the different discharge patterns of parasympathetic PGN are dependent in part on differences in the expression of 4-AP-sensitive K + channels. The two types of PGN may provide an innervation to different targets in the pelvic viscera.

Anatomical characteristics of tonic and phasic postganglionic neurons in guinea pig bronchial parasympathetic ganglia

Anatomical characteristics of principal parasympathetic ganglia neurons on the guinea pig primary bronchus were analyzed, and the procedure for localizing the ganglia without the aid of staining for in vitro physiological studies is described. The neurons were tightly packed within a perineural sheath, and the cell bodies formed a homogeneous population based on size and shape. By using intracellular electrophysiological recordings, unstained neurons within these ganglia were characterized with suprathreshold depolarizing stimuli as having either accommodating action potential patterns (phasic neurons) or repetitive action potential patterns (tonic neurons). After determining whether a cell was tonic or phasic, it was injected with either horseradish peroxidase or Neurobiotin for characterization of its dendrites. There were no differences between tonic and phasic neurons, and both exhibited the following: (1) dendrites were multiple and branching; (2) all processes (axon and dendrites) arose from a circumscribed area on the somatic surface; (3) the initial direction of the processes was usually toward the center of the ganglion, creating a very dense intraganglionic neuropil; (4) tapering processes (presumed dendrites) extended beyond the border of the perineural sheath; and (5) many processes terminated with bouton-like swellings near the somatic surfaces of neighboring neurons within the same ganglion. Electron microscopic examination of dendritic and cell body membranes revealed that greater than 90% of the synapses occurred on dendrites. Based on immunohistochemical staining, all neurons were calbindin negative. These results indicate a relatively homogeneous population of neurons in bronchial parasympathetic ganglia displaying dendritic characteristics compatible with complex integrative properties.

Anatomical characteristics of tonic and phasic postganglionic neurons in guinea pig bronchial parasympathetic ganglia

Anatomical characteristics of principal parasympathetic ganglia neurons on the guinea pig primary bronchus were analyzed, and the procedure for localizing the ganglia without the aid of staining for in vitro physiological studies is described. The neurons were tightly packed within a perineural sheath, and the cell bodies formed a homogeneous population based on size and shape. By using intracellular electrophysiological recordings, unstained neurons within these ganglia were characterized with suprathreshold depolarizing stimuli as having either accommodating action potential patterns (phasic neurons) or repetitive action potential patterns (tonic neurons). After determining whether a cell was tonic or phasic, it was injected with either horseradish peroxidase or Neurobiotin for characterization of its dendrites. There were no differences between tonic and phasic neurons, and both exhibited the following: (1) dendrites were multiple and branching; (2) all processes (axon and dendrites) arose from a circumscribed area on the somatic surface; (3) the initial direction of the processes was usually toward the center of the ganglion, creating a very dense intraganglionic neuropil; (4) tapering processes (presumed dendrites) extended beyond the border of the perineural sheath; and (5) many processes terminated with bouton-like swellings near the somatic surfaces of neighboring neurons within the same ganglion. Electron microscopic examination of dendritic and cell body membranes revealed that greater than 90% of the synapses occurred on dendrites. Based on immunohistochemical staining, all neurons were calbindin negative. These results indicate a relatively homogeneous population of neurons in bronchial parasympathetic ganglia displaying dendritic characteristics compatible with complex integrative properties. J. Comp. Neurol. 419:439–450, 2000. © 2000 Wiley-Liss, Inc.

Projections and firing properties of down eye-movement neurons in the interstitial nucleus of Cajal in the cat.

To clarify the role of the interstitial nucleus of Cajal (INC) in the control of vertical eye movements, projections of burst-tonic and tonic neurons in and around the INC were studied. This paper describes neurons with downward ON directions. We examined, by antidromic activation, whether these down INC (d-INC) neurons contribute to two pathways: a commissural pathway to the contralateral (c-) INC and a descending pathway to the ipsilateral vestibular nucleus (i-VN). Stimulation of the two pathways showed that as many as 74% of neurons were activated antidromically from one of the pathways. Of 113 d-INC neurons tested, 44 were activated from the commissural pathway and 40 from the descending pathway. No neurons were activated from both pathways. We concluded that commissural and descending pathways from the INC originate from two separate groups of neurons. Tracking of antidromic microstimulation in the two nuclei revealed multiple low-threshold sites and varied latencies; this was interpreted as a sign of existence of axonal arborization. Neurons with commissural projections tended to be located more dorsally than those with descending projections. Neurons with descending projections had significantly greater eye-position sensitivity and smaller saccadic sensitivity than neurons with commissural projections. The two groups of INC neurons increased their firing rate in nose-up head rotations and responded best to the rotation in the plane of contralateral posterior/ipsilateral anterior canal pair. Neurons with commissural projections showed a larger phase lag of response to sinusoidal rotation (54.6 +/- 7.6 degrees ) than neurons with descending projections (45.0 +/- 5.5 degrees ). Most neurons with descending projections received disynaptic excitation from the contralateral vestibular nerve. Neurons with commissural projections rarely received such disynaptic input. We suggest that downward-position-vestibular (DPV) neurons in the VN and VN-projecting d-INC neurons form a loop, together with possible commissural loops linking the bilateral VNs and the bilateral INCs. By comparing the quantitative measures of d-INC neurons with those of DPV neurons, we further suggest that integration of head velocity signals proceeds from DPV neurons to d-INC neurons with descending projections and then to d-INC neurons with commissural projections, whereas saccadic velocity signals are processed in the reverse order.

Detection of auditory signals by frog inferior collicular neurons in the presence of spatially separated noise.

Detection of auditory signals by frog inferior collicular neurons in the presence of spatially separated noise. J. Neurophysiol. 80: 2848-2859, 1998. Psychophysical studies have shown that the ability to detect auditory signals embedded in noise improves when signal and noise sources are widely separated in space; this allows humans to analyze complex auditory scenes, as in the cocktail-part effect. Although these studies established that improvements in detection threshold (DT) are due to binaural hearing, few physiological studies were undertaken, and very little is known about the response of single neurons to spatially separated signal and noise sources. To address this issue we examined the responses of neurons in the frog inferior colliculus (IC) to a probe stimulus embedded in a spatially separated masker. Frogs perform auditory scene analysis because females select mates in dense choruses by means of auditory cues. Results of the extracellular single-unit recordings demonstrate that 22% of neurons (A-type) exhibited improvements in signal DTs when probe and masker sources were progressively separated in azimuth. In contrast, 24% of neurons (V-type) showed the opposite pattern, namely, signal DTs were lowest when probe and masker were colocalized (in many instances lower than the DT to probe alone) and increased when the two sound sources were separated. The remaining neurons demonstrated a mix of these two types of patterns. An intriguing finding was the strong correlation between A-type masking release patterns and phasic neurons and a weaker correlation between V-type patterns and tonic neurons. Although not decisive, these results suggest that phasic units may play a role in release from masking observed psychophysically. Analysis of the data also revealed a strong and nonlinear interaction among probe, masker, and masker azimuth and that signal DTs were influenced by two factors: 1) the unit's sensitivity to probe in the presence of masker and 2) the criterion level for estimating DT. For some units, it was possible to examine the interaction between these two factors and gain insights into the variation of DTs with masker azimuth. The implications of these findings are discussed in relation to signal detection in the auditory system.

A neural model of the saccade generator in the reticular formation

A neural model is developed of the neural circuitry in the reticular formation that is used to generate saccadic eye movements. The model simulates the behavior of identified cell types—such as long-lead burst neurons, short-lead excitatory and inhibitory burst neurons, omnipause neurons, and tonic neurons—under many experimental conditions. Simulated phenomena include: saccade staircases, duration and amplitude of cell discharges for saccades of variable amplitude, component stretching to achieve straight oblique saccades, saturation of saccade velocity after saturation of saccade amplitude in response to high stimulation frequencies, trade-offs between saccade velocity and duration to generate constant saccade amplitude, conservation of saccade amplitude in response to sufficiently brief stimulation of omnipause neurons, and high velocity smooth eye movements evoked by high levels of electrical stimulation of the superior colliculus. Previous saccade generator models have not explained this range of data. These models have also invoked mechanisms for which no neurophysiological evidence has been forthcoming, such as resetable integrators, perfect integrators, or target position movement commands. The present model utilizes only known reticular formation neurons. It suggests that a key part of the feedback loop within the saccade generator is realized by inhibitory feedback from short-lead to long-lead burst neurons, in response to excitatory feedforward signals from long-lead to short-lead burst neurons. When this property is combined with opponent interactions between agonist and antagonist muscle-controlling neurons, and motor error, or vector, inputs from the superior colliculus and other saccade-controlling brain regions, all of the above data can be explained. Taken together, these components generate a saccade reset cycle whereby activation of long-lead burst neurons inhibits omnipause neurons and thereby disinhibits short-lead excitatory burst neurons. The excitatory short-lead burst neurons can then respond to excitatory inputs from the long-lead burst neurons. Outputs from the excitatory short-lead burst neurons are integrated by the tonic cells while they also inhibit the long-lead burst neurons via inhibitory burst interneurons. When this inhibition is complete, the omnipause neurons are disinhibited. The omnipause neurons can then, once again, inhibit the short-lead burst neurons, whose inhibition of the long-lead burst neurons is thereby removed. The saccadic cycle can then begin again. In response to sustained electrical input, this cycle generates a staircase of identical saccades whose properties match the data much better than the staircases proposed by alternative models. A comparative analysis of the hypotheses and predictive capabilities of other saccade generator models is provided.

Ca2+ and K+ currents regulate accommodation and firing frequency in guinea pig bronchial ganglion neurons.

Intracellular microelectrode recordings were obtained from neurons located in adult guinea pig bronchial parasympathetic ganglia in situ to determine the calcium and potassium currents regulating repetitive action potential activity and firing rates by these neurons. Neurons in these ganglia respond to prolonged suprathreshold depolarizing current steps with either a burst of action potentials at the onset of the stimulus (accommodating or phasic neurons) or repetitive action potentials throughout the stimulus (nonaccommodating or tonic neurons). Instantaneous and adapted firing rates during prolonged threshold and suprathreshold stimuli were lower in tonic than in phasic neurons, indicating a longer interspike interval between repetitive action potentials in tonic neurons. In tonic neurons, blockade of A-type current with 4-aminopyridine increased accommodation; 4-aminopyridine or apamin decreased the interspike interval in tonic neurons. Calcium-free buffer, cadmium ions, or omega-conotoxin GVIA also increased accommodation in tonic neurons but did not affect the interspike interval; nifedipine or verapamil did not affect the tonic firing pattern. Accommodation in phasic neurons could be decreased by a conditioning hyperpolarization step of the resting potential, which could be subsequently blocked by 4-aminopyridine or calcium-free buffer. Accommodation in phasic neurons could also be decreased by apamin or barium ions: the repetitive action potentials observed during these treatments could be reversed by cadmium ions or calcium-free buffer. These results indicate that tonic and phasic neurons in guinea pig bronchial parasympathetic ganglia have similar types of calcium currents, but potassium channels may ultimately regulate the accommodation pattern, the firing rate, and, consequently, the output from these neurons.

Electrical characteristics and responses to jejunal distension of neurons in Remak's juxta-jejunal ganglia of the domestic fowl.

1. Remak's nerve is a ganglionated nerve trunk found only in birds that runs parallel to the gut from the duodenal-jejunal junction to the cloaca. We report the first electrophysiological characterization of these neurons and their responses to gut distension. 2. A segment of chicken jejunum with attached Remak's nerve was pinned in an electrophysiological chamber. Neurons in Remak's ganglia were impaled with microelectrodes. The adjacent segment of gut was distended with fluid. 3. One hundred and thirty neurons were characterized into three electrophysiological classes: (i) tonic neurons (74%) fired action potentials spontaneously (frequency 3.5 Hz) and continuously (up to 40 Hz) throughout a depolarizing current pulse; (ii) AD neurons (22%) fired a brief burst of action potentials (1-10), which were followed by a prolonged after-depolarization (AD) of duration 2.8 +/- 0.3 s; and (iii) phasic neurons (4%) fired an initial burst of action potentials followed by an after-hyperpolarization (duration, 520.0 +/- 32.0 ms). Tetrodotoxin (1 microM) abolished action potentials in tonic and AD neurons as well as the after-depolarization. 4. Spontaneous fast excitatory postsynaptic potentials (FEPSPs) occurred in all classes of neurons; they were not observed, however, in ganglia isolated from the jejunum. 5. Intracellular injection of biocytin revealed that neurons could be characterized into four morphological classes. Tonic neurons, which had long and extensive dendritic trees, were Remak's Type I, II and IV neurons. AD neurons also comprised Remak's type II neurons. Phasic neurons were Remak's Type III neurons. Most neurons had axons that projected orally along Remak's nerve. 6. Distension of the jejunum evoked FEPSPs and action potentials in tonic neurons, and repetitive bursts of action potentials (1-4) followed by an after-depolarization in AD neurons. All responses to distension were blocked by hexamethonium (300 microM) and tetrodotoxin (1 microM). 7. In conclusion, neurons in Remak's juxta-jejunal nerve appear to regulate gut motility. Three distinct electrophysiological classes of neurons were observed, all of which appear to be activated by distension sensitive cholinergic intestinofugal neurons in the jejunum.

Correlation of electrophysiology, neurochemistry and axonal projections of guinea-pig sphincter of Oddi neurones.

Sphincter of Oddi (SO) ganglia are comprised of two main types of neurones based either on their electrical or neurochemical properties. This study investigated whether any correlation exists between the electrical and neurochemical properties of these cells. SO neurones were characterized electrically as either Tonic or Phasic cells, labelled with neurobiotin, fixed, and processed for beta-nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-DA) staining and choline acetyltransferase immuno-reactivity to identify whether electrically characterized neurones were nitrergic or cholinergic. A total of 119 cells were analysed in this manner; 45% of cells were Tonic and 37% were Phasic. An equivalent number of Tonic (58.1%, 18/31) and Phasic cells (60%, 21/35) were choline acetyltransferase (ChAT) positive. Three of 34 Phasic cells were NADPH-DA positive, whereas 11/33 Tonic cells were NADPH-DA positive. In none of the preparations was ChAT immunoreactivity and NADPH-DA reactivity ever observed in the same neurone. Calretinin immunoreactivity was present in a subpopulation of both Tonic and Phasic neurones. No correlation was observed between the direction of axon projections and the electrophysiological or neurochemical properties of the cell. These results suggest that there is a lack of correlation between the electrical properties and the neurochemical content of SO neurones. Various explanations for these findings are discussed.

Guinea pig pancreatic neurons: morphology, neurochemistry, electrical properties, and response to 5-HT.

The morphology, neurochemistry, and electrical properties of guinea pig pancreatic neurons were determined. The majority of neurons expressed choline acetyltransferase (ChAT) immunoreactivity; however, ChAT-negative neurons were also found. Both cholinergic and noncholinergic neurons expressed nitric oxide synthase (NOS) immunoreactivity. Three types of pancreatic neurons were distinguished. Phasic neurons fired action potentials (APs) at the onset of depolarizing current pulse, tonic neurons spiked throughout the duration of a suprathreshold depolarizing pulse, and APs could not be generated in nonspiking neurons, even though they did receive synaptic input. APs were tetrodotoxin sensitive, and all types of neurons received fast and slow excitatory postsynaptic potentials (EPSPs). Fast EPSPs had cholinergic and noncholinergic components. The majority of pancreatic neurons appeared to innervate the acini. NOS- and/or neuropeptide Y-immunoreactive phasic and tonic neurons were found. Microejection of 5-hydroxytryptamine (5-HT) caused a slow depolarization that was inhibited by the 5-HT1P antagonist N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide and mimicked by the 5-HT1P agonist 6-hydroxyindalpine. A pancreatic 5-HT transporter was located, and inhibition of 5-HT uptake by fluoxetine blocked slow EPSPs in 5-HT-responsive neurons by receptor desensitization.

Synaptic physiology and mitochondrial function in crayfish tonic and phasic motor neurons.

Phasic and tonic motor neurons of crustaceans differ strikingly in their junctional synaptic physiology. Tonic neurons generally produce small excitatory postsynaptic potentials (EPSPs) that facilitate strongly as stimulation frequency is increased, and normally show no synaptic depression. In contrast, phasic neurons produce relatively large EPSPs with weak frequency facilitation and pronounced depression. We addressed the hypothesis that mitochondrial function is an important determinant of the features of synaptic transmission in these neurons. Mitochondrial fluorescence was measured with confocal microscopy in phasic and tonic axons and terminals of abdominal and leg muscles after exposure to supravital mitochondrial fluorochromes, rhodamine-123 (Rh123) and 4-diethylaminostyryl-N-methylpyridinium iodide (4-Di-2-Asp). Mitochondria of tonic axons and neuromuscular junctions had significantly higher mean Rh123 and 4-Di-2-Asp fluorescence than in phasic neurons, indicating more accumulation of the fluorochromes. Mitochondrial membrane potential, which is responsible for Rh123 uptake and is related to mitochondrial oxidative activity (the production of ATP by oxidation of metabolic substrates), is likely higher in tonic axons. Electron microscopy showed that tonic axons contain approximately fivefold more mitochondria per microm2 cross-sectional area than phasic axons. Neuromuscular junctions of tonic axons also have a much higher mitochondrial content than those of phasic axons. We tested the hypothesis that synaptic fatigue resistance is dependent on mitochondrial function in crayfish motor axons. Impairment of mitochondrial function by uncouplers of oxidative phosphorylation, dinitrophenol or carbonyl cyanide m-chlorophenylhydrazone, or by the electron transport inhibitor sodium azide, led to marked synaptic depression of a tonic axon and accelerated depression of a phasic axon during maintained stimulation. Iodoacetate, an inhibitor of glycolysis, and chloramphenicol, a mitochondrial protein synthesis inhibitor, had no significant effects on either mitochondrial fluorescence or synaptic depression in tonic or phasic axons. Collectively, the results provide evidence that mitochondrial oxidative metabolism is important for sustaining synaptic transmission during maintained stimulation of tonic and phasic motor neurons. Tonic neurons have a higher mitochondrial content and greater oxidative activity; these features are correlated with their greater resistance to synaptic depression. Conversely, phasic neurons have a lower mitochondrial content, less oxidative activity, and greater synaptic fatigability.

Tachykinin actions on deep dorsal horn neurons in vitro: an electrophysiological and morphological study in the immature rat.

To assess whether functional neurokinin receptors exist in the deep dorsal horn of the rat, the actions of the selective neurokinin-1 receptor (NK1R) agonist [Sar9,Met(O2)11]substance P ([Sar9,Met(O2)11]SP), the neurokinin-2 receptor (NK2R) agonists [beta-Ala8]NKA(4-10) and GR64349 and the neurokinin-3 receptor (NK3R) agonist senktide were examined intracellularly in vitro. [Sar9,Met(O2)11]SP (1-4 microM) and senktide (1-2 microM) elicited slow depolarizations (<10 mV) associated with increased synaptic activity and cell firing. [beta-Ala8]NKA(4-10) (10-20 microM) and GR64349 (0.25-10 microM) caused small depolarizations (<2.0 mV) and no firing. Neurons were categorized as either 'tonic' or 'phasic' depending on their firing response to direct current step depolarizations. Tonic neurons, which, unlike phasic neurons, display no spike firing accommodation, generated a significantly larger depolarization to the NK1R and NK3R agonists. The putative contribution of these receptors to primary afferent-mediated synaptic transmission was assessed by testing the NK1R antagonist GR82334 (1 microM), the NK2R antagonist MEN10,376 (1 microM) and the NK3R antagonist [Trp7,beta-Ala8]NKA(4-10) (1 microM) against the dorsal root-evoked excitatory postsynaptic potential (DR-EPSP). GR82334 and [Trp7,beta-Ala8]NKA(4-10) significantly reduced (P < or = 0.05) the duration but not the amplitude of the DR-EPSP. MEN10,376 (1 microM) had no effect on DR-EPSP amplitude or duration. Morphological detail was obtained for seven biocytin-filled deep dorsal horn neurons tested with [Sar9,Met(O2)11]SP. Five neurons responded to the NK1R agonist, and two of these had dorsally directed dendrites into the substantia gelatinosa. The other three [Sar9,Met(O2)11]SP-sensitive neurons had dendrites within deeper laminae. These data support the existence of functional NK1Rs and NK3Rs in the deep dorsal horn which may be involved in mediating sensory afferent inputs from nociceptors.

The effects of catechol on various membrane conductances in lumbar sympathetic postganglionic neurones of the guinea-pig.

The effects of catechol on membrane properties in lumbar sympathetic postganglionic neurones isolated from guinea-pigs were studied in vitro in current and voltage clamp using single intracellular microelectrodes. Neurones with properties characteristic of two previously described classes of neurone (phasic and tonic) were studied. Catechol (3-12 mM) produced a few mV depolarization and a dose-dependent increase in membrane resistance which were both larger in tonic than in phasic neurones. In the presence of catechol, both phasic and tonic neurones fired only a single action potential at the beginning of a maintained depolarizing current step. In both neurone types, catechol reduced action potential amplitude and slowed its time course. The peak of the afterhyperpolarization became delayed and reduced in amplitude, particularly in tonic neurones. The time constant of inactivation of IA was reduced by catechol without change in the voltage sensitivity of activation or inactivation: IC50 was 3 mM in phasic and 4 mM in tonic neurones. Catechol also blocked a slow voltage-activated K+ current (resembling ID) that was present in many tonic neurones. Catechol did not modify the slow calcium-activated potassium current (gKCa1) or the anomalous rectifier; neither did it appear to affect the fast calcium-activated potassium current (IC) or the delayed rectifier. Catechol did not change the overall rate of spontaneous synaptic activity nor enhance the release of quanta of ACh from preganglionic terminals evoked by nerve stimulation. We conclude that, in addition to blocking IA, catechol blocks the slow ID-like current in sympathetic neurones. It also has a profound effect on the action potential probably by increasing inactivation of voltage-dependent Na+ channels. The change from tonic to phasic discharge in tonic neurones cannot be attributed solely to its effects on IA.

システムニューロサイエンス教育講座 ｌ０．垂直眼球運動の神経機構 Ｃａｊａｌ間質核の役割

Lesions of the interstitial nucleus of Cajal (INC) in cats and monkeys result in severe impairment of the vestibulo-ocular reflex (VOR) in the pitch plane and vertical optokinetic reflex, and impairment of the ability to hold an eccentric vertical eye position after saccadic movement. The INC region of alert animals contains many burst-tonic and tonic neurons whose activity is closely correlated to vertical eye movement, not only during spontaneous saccades, but also during pitch VOR, smooth pursuit, and optokinetic eye movements. Although this activity is closely related to these conjugate vertical eye movements, it is different from oculomotor motoneuron activity. Because the INC region alone cannot produce eye-position signals, the INC is an essential component but not sole component of the velocity-to-position integrator for vertical eye movement.The INC and closely surrounding midbrain reticular formation contain a class of cells that have properties required for vertical burster-driving neurons. Chemical deactivation of these areas produces selective impairment of generation of vertical fast eye movement. These results indicate that the INC and nearby reticular formation may also be involved in generating vertical fast eye movements.

Saccade-related burst neurons with torsional and vertical on-directions in the interstitial nucleus of Cajal of the alert monkey.

The interstitial nucleus of Cajal (iC) is known to be the neural integrator for vertical and torsional eye movements. Burst-tonic neurons are thought to be the neural substrate for this function. Until now, the iC has not been specifically considered to play a part in saccade generation. The aim of this study was to characterize saccade-related burst neurons in the iC during torsional and vertical eye movements. Saccade-related burst neurons were recorded in the iC of macaque monkeys during fast phases of torsional and vertical vestibular nystagmus, spontaneous and visually guided eye movements, and in light and darkness. Burst neurons in the iC (n = 85) were found intermingled between burst-tonic and tonic neurons. They were not spontaneously active, showed no eye position sensitivity, and responded during saccades and quick phases of nystagmus with a burst of activity whose duration was closely correlated with saccade amplitude and hence saccade duration (correlation coefficients up to 0.9). Latency in the on-direction was, on average, 10.4 ms (range 5-23 ms); it decreased with different saccade directions and became negative in the off-direction. In a horizontal-vertical coordinate system, on-direction of the majority of neurons was either upward (n = 52) or downward (n = 33). There was no horizontal on-direction. Burst neurons of different vertical on-directions were found intermingled throughout the iC. In the vertical-torsional plane, on-direction always showed an ipsiversive torsional component, i.e., a clockwise (positive) torsion for neurons in the right iC and a counterclockwise (negative) torsional component when recorded in the left iC. The findings indicate that saccade-related burst neurons in the iC control coordinate axes for vertical and torsional quick eye rotations. As in the rostral interstitial nucleus of the medial longitudinal fasciculus (riMLF), burst neurons in the iC encode vertical saccades with an ipsitorsional direction with similar burst characteristics. It is suggested that iC burst neurons play a part in the local feedback loop of the reciprocal iC-riMLF projections.

Tonic activity during visuo-oculomotor behavior in the monkey superior colliculus

The purpose of this study was to examine whether the superior colliculus is involved in intermediary cognitive processes such as memory, movement preparation, and peripheral attention. To answer this question, we recorded single cell activities in the superior colliculus of monkeys trained to perform a series of visuo-oculomotor tasks: delayed saccade task (SACD), saccade task with overlap target (SACO), and attention task (ATT). We recorded 141 neurons showing tonic activities related to the tasks. Depending on the predominance of the activities among the three tasks, we classified the tonic neurons into four types: (1) visuomotor (greater activity in SACO), (2) mnemonic motor (SACD dominant), (3) attention (ATT), and (4) nonspecific. Among 108 neurons recorded in the intermediate layer, 13 were of a visuomotor type, 15 were of a mnemonic motor type, and 13 were of an attention type. The other 67 neurons were of a non-specific type. Of the 33 neurons in the superficial layer, many neurons were of the non-specific type. These results suggested that the tonic activities in the superior colliculus are related to memory of the target location, preparation of saccades and peripheral attention.

Tachykininergic synaptic transmission in the coeliac ganglion of the guinea-pig.

1. The responses of coeliac ganglion neurones of the guinea-pig to electrical stimulation of the mesenteric nerves and applications of tachykinin receptor agonists were investigated by use of intracellular recording techniques. 2. Ganglion neurones were classified into three groups based on firing patterns in response to a depolarizing current pulse: phasic (38% of the population), tonic (39%) and atypical (23%). In the majority of phasic neurones (91%) a long after-hyperpolarization (LAH) lasting 5-8 s followed action potentials induced by a train of depolarizing current pulses. In contrast, LAH was rarely observed in tonic neurones (5%). 3. In most of tonic neurones (90%) slow excitatory post-synaptic potentials (e.p.s.ps) lasting 3-10 min were evoked by repetitive electrical stimulation of the mesenteric nerves. Prolonged depolarizations were also evoked in most tonic neurones by applications of substance P (SP), neurokinin A (NKA) or senktide, a tachykinin NK3 receptor agonist. 4. In most of phasic neurones (73%), mesenteric nerve stimulation did not induce an obvious depolarization but induced a prolonged inhibition of LAH lasting 3-10 min. Bath-applied tachykinin receptor agonists similarly induced an inhibition of LAH without causing depolarization in most of the phasic neurones. 5. GR 71251 (5 microM), a tachykinin NK1 receptor antagonist, partially depressed the nerve-evoked slow e.p.s.ps in tonic neurones and the nerve-evoked LAH inhibition in phasic neurones. 6. Capsaicin (0.1-5 microM) induced a prolonged depolarization in tonic neurones and an inhibition of LAH in phasic neurones. 7. A mixture of peptidase inhibitors potentiated the depolarization and the LAH inhibition evoked by nerve stimulation, SP and NKA, but not those evoked by senktide. 8. It is concluded that tonic neurones respond to repetitive mesenteric nerve stimulation preferentially with slow e.p.s.ps and that phasic neurones respond preferentially with LAH inhibition. The present study further suggests that SP and NKA, released from axon collaterals of primary afferent neurones, produce slow e.p.s.ps in tonic neurones and the LAH inhibition in phasic neurones via NK1 receptors.