Electrophysiological properties of rat phrenic motoneurons during perinatal development

Past studies determined that there is a critical period at approximately embryonic day (E)17 during which phrenic motoneurons (PMNs) undergo a number of pivotal developmental events, including the inception of functional recruitment via synaptic drive from medullary respiratory centers, contact with spinal afferent terminals, the completion of diaphragm innervation, and a major transformation of PMN morphology. The objective of this study was to test the hypothesis that there would be a marked maturation of motoneuron electrophysiological properties occurring in conjunction with these developmental processes. PMN properties were measured via whole cell patch recordings with a cervical slice-phrenic nerve preparation isolated from perinatal rats. From E16 to postnatal day 1, there was a considerable transformation in a number of motoneuron properties, including 1) 10-mV increase in the hyperpolarization of the resting membrane potential, 2) threefold reduction in the input resistance, 3) 12-mV increase in amplitude and 50% decrease duration of action potential, 4) major changes in the shapes of potassium- and calcium-mediated afterpotentials, 5) decline in the prominence of calcium-dependent rebound depolarizations, and 6) increases in rheobase current and steady-state firing rates. Electrical coupling among PMNs was detected in 15-25% of recordings at all ages studied. Collectively, these data and those from parallel studies of PMN-diaphragm ontogeny describe how a multitude of regulatory mechanisms operate in concert during the embryonic development of a single mammalian neuromuscular system.     

Intracellular injection of acetylcholine blocks various potassium conductances in vagal motoneurons

Injection of acetylcholine into cholinergic neurons of the dorsal motor nucleus of the vagus induced membrane depolarization, an increase in input resistance, a decrease of early and late afterhyperpolarizations and a prolongation of the action potential. These effects were reversible and within 10-20 min almost complete recovery was always observed. Externally applied acetylcholine, even with doses as high as 15 mM, was not effective. Acetylcholine appeared to block voltage- and Ca2+-dependent K+ conductances. This block was manifested by the reduction of both the early and late afterhyperpolarizations and a decrease of the delayed rectification. The reversal potential for the conductance decrease was 15-30 mV negative to the resting potential. As a result of this blockade an increased Ca2+ current ensues, which is responsible for most of the prolongation of the action potential. The same responses were obtained after the injection of carbamylcholine, neostigmine and choline. However, unlike acetylcholine no sign of recovery was observed. In fact injection of neostigmine, carbamylcholine or neostigmine, together with acetylcholine, produced a delayed response which may reflect the accumulation of endogenous acetylcholine.     

Role of potassium conductances in determining input resistance of developing brain stem motoneurons

The role of potassium conductances in determining input resistance was studied in 166 genioglossal (GG) motoneurons using sharp electrode recording in brain stem slices of the rats aged 5-7 days, 13-15 days, and 19-24 days postnatal (P). A high magnesium (Mg(2+); 6 mM) perfusate was used to block calcium-mediated synaptic release while intracellular or extracellular cesium (Cs(+)) and/or extracellular tetraethylammonium (TEA) or barium (Ba(2+)) were used to block potassium conductances. In all cases, the addition of TEA to the high Mg(2+) perfusate generated a larger increase in both input resistance (R(n)) and the first membrane time constant (tau(0)) than did high Mg(2+) alone indicating a substantial nonsynaptic contribution to input resistance. With intracellular injection of Cs(+), GG motoneurons with lower resistance (<40 MOmega), on the average, showed a larger percent increase in R(n) than cells with higher resistance (>40 MOmega). There was also a significant increase in the effect of internal Cs(+) on R(n) and tau(0) with age. The largest percent increase (67%) in the tau(0) due to intracellular Cs(+) occurred at P13-15, a developmental stage characterized by a large reduction in specific membrane resistance. Addition of external Cs(+) blocked conductances (further increasing R(n) and tau(0)) beyond those blocked by the TEA perfusate. Substitution of external calcium with 2 mM barium chloride produced a significant increase in both R(n) and tau(0) at all ages studied. The addition of either intracellular Cs(+) or extracellular Ba(2+) created a depolarization shift of the membrane potential. The amount of injected current required to maintain the membrane potential was negatively correlated with the control R(n) of the cell at most ages. Thus low resistance cells had, on the average, more Cs(+)- and Ba(2+)-sensitive channels than their high resistance counterparts. There was also a disproportionately larger percent increase in tau(0) as compared with R(n) for both internal Cs(+) and external Ba(2+). Based on a model by Redman and colleagues, it might be suggested that the majority of these potassium conductances underlying membrane resistance are initially located in the distal dendrites but become more uniformly distributed over the motoneuron surface in the oldest animals.     

Denervation causes changes in electrophysiological properties in rat phrenic motoneurons

Thirty-nine male adult rats were divided into a control group and a denervation group that had been subjected to phrenicotomy 4 weeks earlier. Electrophysiological membrane properties (input resistance and rheobase) of phrenic motoneurons were measured from intracellular recordings made with glass microelectrodes. Under anesthetized and artificially ventilated conditions, the recorded motoneurons were divided into recruited (spike discharge) and non-recruited (depolarization only) types. There was a significant inverse relationship between the rheobase and input resistance in the control rats, but not in the denervated rats. In the control rats, the mean value of rheobase in the non-recruited motoneurons was significantly higher than that in the recruited motoneurons. In denervated rats, however, the mean value of rheobase in the recruited motoneurons was identical to that in the non-recruited motoneurons. The results indicated that phrenicotomy induced a de-differentiation of electrophysiological properties of the phrenic motoneurons, and that these changes might be restricted to the motoneurons innervating fast-twitch, low fatigue resistance muscle fibers.     

Bötzinger-complex expiratory neurons monosynaptically inhibit phrenic motoneurons in the decerebrate rat

We examined respiratory neurons in the Bötzinger complex of the medulla oblongata in 18 vagotomized, paralyzed, ventilated, and decerebrated rats and tested the hypothesis that bulbospinal expiratory neurons in this region monosynaptically inhibit phrenic motoneurons. First, we surveyed the types of respiratory neurons found in the Bötzinger complex; only 11 of the 98 (～11%) examined were bulbospinal, and all discharged only during late expiration (E2), usually with an augmenting discharge frequency (AUG). Then, we examined the spinal projections of 34 E2-AUG neurons using antidromic activation and found that all projected as far as the C4 or C5 segments of the spinal cord but no further caudally. Most (30, ～88%) had only unilateral projections, the majority (25, ～83%) ipsilateral, but 4 neurons (～12%) had bilateral projections. Their axons could be antidromically activated at low currents (less than 10 μA) in the dorsal-lateral part of the spinal cord at the C2–3 border; 0.5–1.2 mm (mean±SD 0.84±0.23 mm) below the dorsal surface and 0.7–1.5 mm (1.19±0.25 mm) lateral from the midline. We sought evidence for connections from bulbospinal E2-AUG neurons to 118 phrenic motoneurons by computing spike-triggered averages (STAs) of their intracellular potentials triggered by the action potentials of 38 unilaterally-projecting E2-AUG neurons. Resting phrenic motoneuron membrane potentials ranged from –40 to –75 mV (–56±8 mV) and fluctuations with the respiratory cycle from 7 to 20 mV (14±4 mV). Of the 118 STAs computed, hyperpolarizations were evident in 18 (～15%) STAs, evoked by 11 of 38 (～29%) E2-AUG neurons. Their amplitudes varied from 35 to 550 μV (105±113 μV), 10–90% fall times from 0.4 to 0.9 ms (0.63±0.17 ms), and half-amplitude widths from 1.3 to 3.2 ms (2.0±0.52 ms). Most (16/95, ～17%) of the STAs that displayed hyperpolarizations were associated with ipsilateral trigger neurons but some (2/23, ～9%) resulted from contralateral trigger neurons. We conclude that Bötzinger-complex, expiratory neurons project to the C4 and/or C5 segments of the cervical spinal cord but no further caudal. Their axons are located dorsolaterally in the upper cervical segments of the spinal cord, and they monosynaptically inhibit phrenic motoneurons during the late part of expiration.     

Postnatal rat nigrostriatal dopaminergic neurons exhibit five types of potassium conductances

1. We have investigated the electrical properties of neurons acutely dissociated from the substantia nigra zona compacta (SNZC) of the postnatal rat with whole cell patch-clamp recordings. Retrogradely labeled nigrostriatal neurons were identified with the use of rhodamine-labeled fluorescent latex microspheres. Over 90% of the rhodamine-labeled neurons in the SNZC demonstrated formaldehyde/glutaraldehyde-induced catecholamine fluorescence, indicating that they were dopaminergic (DA) neurons. 2. DA neurons had 15-20 microns ovoid or fusiform-shaped cell bodies with 2-3 thick proximal processes. Labeled neurons generated spontaneous action-potential activity in both regular and irregular patterns. These cells exhibited input resistances of 300-600 M omega and action-potential amplitudes of 60-80 mV. Locally applied dopamine inhibited the spontaneous activity of these neurons by hyperpolarizing the cells. 3. Outward currents were examined with voltage-clamp recordings using a tetrodotoxin (TTX)-containing medium. In all DA cells, depolarizing voltage commands activated several components of outward current depending on the holding potential of the cell. When cells were held at -40 mV (or more positive), voltage steps activated a sustained outward current. If the membrane potential was held more negative than -50 mV, a rapidly activating and inactivating component of outward current response could also be detected. 4. From a hyperpolarized holding potential (-90 mV) the transient outward current activated with depolarizing commands to -55 mV, peaking within 5 ms. The current inactivated with a monoexponential time constant of 53 +/- 4 (SE) ms. At more positive holding potentials (-40 mV) the steady-state inactivation of the current could be removed by applying a conditioning hyperpolarizing prepulse. In response to a fixed depolarizing voltage step, half-maximal inactivation occurred at about -65 mV. The transient current was blocked by 4-aminopyridine (4-AP). 5. The sustained outward currents were isolated by holding the cells at -40 mV. Two components of sustained outward current were distinguished by their sensitivity to the calcium channel blockers Co2+ (5 mM) and/or Cd2+ (200 microM). The current remaining in the presence of Co2+/Cd2+ was activated by depolarizing voltage commands more positive than -40 mV.(ABSTRACT TRUNCATED AT 400 WORDS)     

Excitatory interactions between phrenic motoneurons in the cat

Summary 1. Interactions between phrenic motoneurons have been analysed in anaesthetized, paralyzed cats after C3 to C7 deafferentation. Effects of electrical stimulation of the C5 phrenic axons have been studied on thin filaments dissected from the stimulated nerve. Repetitive stimulation could elicit, after the primary direct response of the stimulated axons, a secondary response named Recurrent Response, RR. 2. RRs have been obtained in 117/186 phrenic axons. They appear sporadically (mean occurrence: 3.75 RRs elicited by 100 shocks of stimulation) at a constant latency. They originate from a spinal mechanism since they persist after C2 transection and disappear after section of the ventral roots. 3. The mechanism responsible for RR shows spatial and temporal facilitation. The RR probability increases with the number of antidromically invaded motoneurons as revealed by changes either of stimulation intensity or of central respiratory drive. However, RR could be evoked in a motoneuron without an antidromic volley in its axon. 4. Systemic injections of nicotinic blocking drugs such as dihydro--erytroidin or mecamylamine decrease or suppress the occurrence of RR; therefore, cholinergic synapses are involved in the RR generating process. 5. RR are assumed to be due to direct excitatory interactions between homonymous motoneurons. Recurrent axon collaterals impinging directly on neighbouring motoneurons would link together the different motoneurons of the phrenic pool. The functional significance of this phenomenon is discussed.     

Repetitive firing properties of phrenic motoneurons in the cat

1. Using both rectangular- and ramp-shaped intracellularly injected currents, the repetitive firing properties of 23 antidromically identified phrenic motoneurons were determined in anesthetized, paralyzed, and artificially ventilated cats during hypocapnic apnea. In response to rectangular depolarizing current injections, regular repetitive firing was observed in all cells. 2. At the beginning of a rectangular current pulse, the firing pattern was characterized by high frequency of firing that rapidly adapted to a much lower steady-state value. The relationship between the reciprocal of the first interspike interval (F1-2) and injected current was described by an initial linear portion of shallow slope, followed by a much steeper segment that smoothly reached a plateau value. The plateau value of F1-2 did not change with further increase in injected current. 3. The steady-state firing frequency versus injected current relationship was represented by a line of shallow slope over the entire range of injected currents. The slope of this line ranged between 1.1 and 4.5 Hz/nA. 4. A weaker correlation between minimal firing frequency for continuous activity and the reciprocal of the after hyperpolarization duration (1/AHPdur) was found for phrenic motoneurons than exists for lumbosacral motoneurons (26). Comparison of AHP shape at different levels of repetitive firing revealed that the slopes of the ascending portions of the AHP were similar except at the higher injected currents. Further, in the same cells during natural inspiratory activity the ascending part of the AHP was similar to that observed during current injection. 5. Depolarizing current ramps (approximately 1-s duration) were injected into 11 phrenic motoneurons. Instantaneous firing frequency was directly proportional to the intensity of the instantaneous injected current and independent of the rate of change of current for the range of ramp slopes tested (5-80 nA/s). Ramp-and-hold current injections were done in three motoneurons, and the peak instantaneous firing frequency showed little adaptation during the hold maneuver. 6. During hypocapnic apnea, we mimicked the expiratory-phase inhibition and inspiratory-phase excitation of phrenic motoneurons by injecting a 1-s depolarizing current ramp that was immediately preceded by a 1-s hyperpolarizing current ramp of the same absolute peak current intensity. Compared with the effects of positive current ramps alone the spike onsets during the negative-positive current ramp paradigm were either facilitated or retarded. Various ionic mechanisms are suggested for these effects as well as their function in determining the onset of firing during natur     

Electrophysiological properties of developing phrenic motoneurons in the cat

1. Intracellular recordings were made in 427 phrenic motoneurons from kittens (in four stages of postnatal development, ranging from 2 to 14 wk) and in 72 motoneurons from adult cats. These experiments were performed to determine how the pattern of spontaneous discharge changes in phrenic motoneurons during development and how these changes might be influenced by alterations in the electrophysiological properties of these neurons. 2. The mean axonal conduction velocity increased significantly (P less than 0.0001) throughout this period of development, with the most rapid increase occurring between weeks 2 and 5 (18.5 +/- 5.4 and 32.4 +/- 5.6 m/s, respectively, mean +/- SD). 3. There was no change in the magnitude of the membrane potential, antidromic action potential, or positive overshoot; whereas there was a decrease in the half-width of the action potential from 2 (652 +/- 184 ms) to 14 (525 +/- 116 ms) wk postnatal. 4. The mean duration of the afterhyperpolarization (AHPdur) decreased from 69 +/- 20 ms at 2 wk to 60 +/- 16 ms by 9 wk, then increased to 66 +/- 18 ms by 14 wk of age and to 75 +/- 21 ms in the adult. The mean amplitude of the afterhyperpolarization (AHPamp) in the 2-wk-old group (4.9 +/- 1.8 mV) was larger than that at weeks 5 (3.9 +/- 1.7 mV) and 9 (3.9 +/- 1.6 mV), whereas the mean AHPamp of the adult (3.1 +/- 1.2 mV) was significantly smaller than the mean of any younger group. A significant negative correlation was found between AHPdur and axonal conduction velocity in all age groups studied, including the adult.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of the potassium conductances of principal cells of rat cortical collecting ducts

In this study we examined by impalement techniques properties of the macroscopic K⁺ conductances in the luminal and basolateral membrane of principal cells from isolated perfused cortical collecting ducts (CCD) of the rat. Both membranes possess a dominating K⁺ conductance. Compared to their behaviour with K⁺, both membranes appear much less permeable to NH ₄ ⁺ and Rb⁺, and the K⁺ conductances of both membranes are inhibited by these cations. In light of these findings, it is very unlikely that significant amounts of NH ₄ ⁺ , which is secreted in the CCD, cross the principal cells as NH ₄ ⁺ . Several inhibitors with known effects on K⁺ channels in patch-clamp studies have been examined. Tetraethylammonium, which inhibits the excised K⁺ channels of these cells, has no effect on the macroscopic K⁺ conductances of either membrane. Verapamil, which inhibits the K⁺ channels in the luminal membrane, acts predominantly on the basolateral membrane K⁺ conductance in the intact tubule. Therefore, some of the macroscopic properties of the K⁺ conductances are distinct from those of single channels thus far observed in patchclamp studies.Supported by DFG Schl 277/2-1 and Gr 480/10     

Structural and functional characteristics of individual phrenic motoneurons

Summary Intracellular recording and staining techniques were applied to the study of cat phrenic motoneurons. Spontaneously driven phrenic cells possessed individualistic depolarization and spiking patterns that were a function of the conduction velocity in the different motor axons. Staining of phrenic motoncurons with Procion yellow indicated that fast conducting cells with small slow-wave depolarizations were large in size while slow conducting cells with large depolarizations were small in size. This implicated differences in membrane input resistance between large and small cells, although an unequal distribution of inputs to the individual components could not be discounted.On the average, phrenic motoneurons had a smaller dendritic surface area and smaller dendritic dominance than lumbosacral motoneurons. These factors help to explain the higher membrane resistances and longer time constants of phrenic cells.Phrenic dendrites were found to project in all directions away from the cell body and form ellipsoidal receptive fields that overlapped with other phrenic fields. It is speculated that the close approximation of phrenic dendrites with one another could, in part, be responsible for the high degree of synchronization among the different phrenic units.     

Is there electrical coupling between phrenic motoneurons in cats?

The possibility of electrical coupling between phrenic motoneurons was studied in anesthetized cats. In animals with C4-C7 dorsal roots cut (spinal cord intact), no sign of short-latency increase in the firing probability was observed in one phrenic rootlet following stimulation of the other phrenic rootlet. Intracellular recording from 21 phrenic motoneurons, in cats spinalized at the C1 segment, revealed no graded, short-latency antidromic depolarizations, even when the collision technique was used. Conditioning depolarizations of the impaled motoneurons never resulted in an increase of cell firing following test antidromic activation of adjacent motoneurons. It is concluded that the short-term discharge synchronization, observed within the phrenic nucleus by other authors, must be due to the action of chemical synapses.     

Intracellular recording from phrenic motoneurons receiving respiratory drive in vitro

Stable, long-term (2-4 h) intracellular recordings were obtained from phrenic motoneurons receiving respiratory drive in an in vitro neonatal rat brainstem-spinal cord preparation. Several passive and active phrenic motoneuron properties in vitro, including resting membrane potential, inspiratory drive potentials, and threshold depolarization levels, are similar to those in the adult mammal in vivo. Manipulations of the extracellular fluid environment by the addition or washout of chemicals affecting motoneuronal activity and spinal synaptic transmission of respiratory drive did not affect the quality of the intracellular recordings. These results establish the feasibility of long-term intracellular recording from the in vitro brainstem-spinal cord preparation for studies of cellular and synaptic mechanisms underlying control of respiratory movements.     

Rat hippocampal neurons in culture: potassium conductances

Two-electrode voltage-clamp methodology was used to analyze voltage-dependent ionic conductances in 81 rat hippocampal neurons grown in culture for 4-6 wk. Pyramidal and multipolar cells with 15- to 20-micron-diameter cell bodies were impaled with two independent KCl electrodes. The cells had resting potentials of -30 to -60 mV and an average input resistance of about 30 M omega. A depolarizing command applied to a cell maintained in normal medium invariably evoked a fast (2-10 ms) inward current that saturated the current-passing capacity of the system. This was blocked in a reversible manner by application of tetrodotoxin (TTX) (0.1-1.0 microM) near the recorded cell. In the presence of TTX, a depolarizing command evoked a rapidly rising (3-5 ms), rapidly decaying (25 ms) transient outward current reminiscent of "IA" reported in molluscan neurons. This was followed by a more slowly activating (approximately 100 ms) outward current response of greater amplitude that decayed with a time constant of about 2-3 s. These properties resemble those associated with the K+ conductance, IK, underlying delayed rectification described in many excitable membranes. IK was blocked by extracellular application of tetraethylammonium (TEA) but was insensitive to 4-aminopyridine (4-AP) at concentrations that effectively eliminated IA. IA, in turn, was only marginally depressed by TEA. Unlike IK, IA was completely inactivated when the membrane was held at potentials positive to -50 mV. Inactivation was completely removed by conditioning hyperpolarization at -90 mV. A brief hyperpolarizing pulse (10 ms) was sufficient to remove 95% of the inactivation. IA activated on commands to potentials more positive than -50 mV. The inversion potential of the ionic conductance underlying IA and IK was in the range of the K+ equilibrium potential, EK, as measured by the inversion of tail currents; and this potential was shifted in a depolarizing direction by elevated [K+]0. Thus, both current species reflect activation of membrane conductance to K+ ions. Hyperpolarizing commands from resting potentials revealed a time- and voltage-dependent slowly developing inward current in the majority of cells studied. This membrane current was observed in cells exhibiting "anomalous rectification" and was therefore labeled IAR. It was activated at potentials negative to -70 mV with a time constant of 100-200 ms and was not inactivated. A return to resting potential revealed a tail current that disappeared at about EK. IAR was blocked by extracellular CS+ and was enhanced by elevating [K+]0. It thus appears to be carried by inward movement of K+ ions.(ABSTRACT TRUNCATED AT 400 WORDS)