Albert J. Berger

Modulation of neonatal rat hypoglossal motoneuron excitability by serotonin

The effects of 5-HT on neonatal rat hypoglossal motoneurons (HMs) were studied in two in vitro slice preparations. Serotonin caused either reversible depolarization or the generation of an inward current (I5-HT) in every cell tested. I5-HT persisted after synaptic blockade. In most of the cells tested, the magnitude of I5-HT was independent of membrane potential (-50 to -120 mV), and 5-HT had little effect on input resistance or slope conductance. In addition, 5-HT significantly reduced the amplitude of the post-spike medium-duration afterhyperpolarization. This reduction probably contributed to the resulting increase in the slope of the relationship describing the steady-state firing frequency response to injected current (f-I) observed in the presence of 5-HT. Thus, 5-HT increases the excitability of neonatal HMs via at least two different postsynaptic mechanisms.

Distribution of carotid sinus nerve afferent fibers to solitary tract nuclei of the cat using transganglionic transport of horseradish peroxidase

The distribution of carotid sinus nerve (CSN) afferent fibers to the solitary tract and its various nuclei has been determined using transganglionic transport of horseradish peroxidase (HRP). The ipsilateral solitary tract was labeled and, in addition, the heaviest labeling was seen dorsomedial within the medial solitary nucleus, as well as just lateral to the tractus solitarius in the lateral solitary nucleus. Bilateral labeling was present caudal to the obex in both the medial and commissural nuclei. The paucity of reaction product within the ventrolateral solitary nucleus supports previous neurophysiological studies suggesting carotid chemoreceptors do not make monosynaptic contacts with respiratory neurons within that nucleus.

Inhibition of N- and P-type calcium currents and the after-hyperpolarization in rat motoneurones by serotonin.

1. We investigated the effects of serotonin (5‐hydroxytryptamine, 5‐HT) on whole‐cell barium currents through calcium channels in visualized neonatal rat hypoglossal motoneurones (HMs) in a thin brainstem slice preparation. 2. High voltage‐activated (HVA) currents were elicited by depolarizing voltage steps from ‐70 to 0 mV; low voltage‐activated (LVA) currents were evoked using steps to between ‐30 and ‐40 mV from hyperpolarized potentials (< ‐80 mV). 5‐HT (1.0 microM) inhibited HVA currents by at least 10% in 70% of HMs tested (n = 99); in those responsive neurones, 5‐HT decreased HVA current by 22 +/‐ 1.3% (mean +/‐ S.E.M.). In contrast, 5‐HT had no effect on LVA current amplitude in HMs (n = 7). 3. Calcium current inhibition was mimicked by 5‐carboxamidotryptamine maleate (5‐CT), a 5‐HT1 receptor agonist, and by R(+)‐8‐hydroxydipropylaminotetralin hydrobromide (8‐OH‐DPAT), a specific 5‐HT1A agonist; N‐(3‐trifluoromethylphenyl) piperazine hydrochloride (TFMPP), a 5‐HT1B agonist, was without effect. The effect of 5‐HT was blocked by the 5‐HT1A antagonist 1‐(2‐methoxyphenyl)‐4‐[4‐(2‐phthalimido)butyl]piperazine hydrobromide (NAN‐190) but not by ketanserin, a 5‐HT2A/2C antagonist. Although R(‐)‐1‐(2,5‐dimethoxy‐4‐iodophenyl)‐2‐aminopropane hydrochloride (DOI), a 5‐HT2A/2C agonist, mimicked the current inhibition by 5‐HT, it was ineffective in the presence of NAN‐190. These data indicate that 5‐HT1A receptors mediate calcium current inhibition by 5‐HT. 4. Following application of either omega‐conotoxin‐GVIA (omega‐CgTX) or omega‐agatoxin‐IVA (omega‐Aga‐IVA), to block N‐ and P‐type components of calcium current, the 5‐HT‐sensitive current was reduced; 5‐HT had no effect on the current remaining after application of both toxins. Thus, 5‐HT inhibits both N‐ and P‐type calcium currents in neonatal HMs. 5. Inhibition of HVA current by 5‐HT was irreversible, and subsequent applications of 5‐HT were occluded, when GTP gamma S was substituted for GTP in the pipette. In addition, inhibition of HVA current by 5‐HT was relieved following depolarizing prepulses. These data indicate that inhibition of calcium channels by 5‐HT is mediated by G proteins. 6. Under current clamp, both 5‐HT and 8‐OH‐DPAT decreased the amplitude of the after‐hyperpolarization (AHP) that followed action potentials, indicating involvement of a 5‐HT1A receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Postnatal changes in rat hypoglossal motoneuron membrane properties.

Intracellular recording techniques were used to characterize changes that take place in rat hypoglossal motoneuronal excitability from early postnatal stages to adulthood. This study focused primarily on the first two weeks of postnatal life, when major changes in the maturation of the neuromuscular system take place. Neonatal hypoglossal motoneurons were identified by their location within the hypoglossal nucleus and by their characteristic electrophysiology. These criteria were supported by antidromic activation and intracellular staining of retrogradely labeled hypoglossal motoneurons. Action potential duration decreased progressively during postnatal development. The reduction was primarily due to a more rapid repolarization, suggesting developmental changes in voltage-dependent potassium conductances. The duration of the calcium-dependent afterhyperpolarization decreased by half during the first two weeks of postnatal life. Changes in subthreshold responses included a decrease in input resistance and an increase in the degree of hyperpolarizing sag and inward rectification with age. Rheobase current was negatively correlated with input resistance, and increased progressively during postnatal development. Membrane time constant decreased almost four-fold over the first two postnatal weeks, suggesting that membrane resistivity is not constant. This decrease in membrane resistivity could account for a large fraction of the change in input resistance and rheobase with age. Thus, the early postnatal development of the rat includes systematic changes in the electrophysiological properties of motoneurons innervating tongue muscles. Some of these modifications are not easily explained by a mere change in neuronal surface area but likely involve changes in the density of expressed ion channels.

Activation of adenosine A1 and A2 receptors differentially modulates calcium channels and glycinergic synaptic transmission in rat brainstem

Multiple types of calcium channels are responsible for calcium influx that triggers transmitter release in the mammalian CNS. To test the contribution of each calcium channel type on synaptic modulation, we recorded calcium currents from somata of presynaptic interneurons and unitary glycinergic postsynaptic currents in the rat brainstem. In interneuron somata, A1 receptor activation inhibited predominantly N-type (omega-conotoxin GVIA-sensitive) and, to a lesser extent, P-type (omega-agatoxin IVA-sensitive) channels. At the presynaptic terminal, N- and P-type channels mediated synaptic transmission. omega-CgTx occluded synaptic inhibition by A1 receptor activation, suggesting that synaptic inhibition was mediated predominantly by N-type channel inhibition. A2 receptor activation facilitated synaptic transmission, probably through potentiation of P-type channels at the presynaptic terminal.

Neuromodulation of hypoglossal motoneurons: cellular and developmental mechanisms.

Hypoglossal motoneurons (HMs) in the caudal brainstem have a respiratory-related activity pattern and contribute to control of upper airway resistance. In this review, we focus primarily on signalling mechanisms utilized by neurotransmitters to enhance HM excitability. In particular, we consider: (1) the membrane depolarization induced by a number of different putative transmitters [thyrotropin-releasing hormone (TRH), serotonin (5-HT), norepinephrine (NE)]; and (2) the inhibition of a calcium-dependent spike after hyperpolarization (AHP) by 5-HT and its effect on firing behavior. Potential functional consequences on HM behavior of these different neurotransmitter effects is discussed. In addition, we describe postnatal changes in transmitter effects and suggest potential cellular mechanisms to explain those developmental changes. Most of the data discussed are derived from in vitro electrophysiological recordings performed in preparations from neonatal and adult rats.

Repetitive firing properties of developing rat brainstem motoneurones.

1. The repetitive firing properties of neonatal and adult rat hypoglossal motoneurones (HMs) were investigated in a brainstem slice preparation. Neonatal HMs could be classified into two main groups: (1) neurones with a decrementing or adapting firing pattern (type D); exhibiting an early and a late phase; and (2) neurones with an incrementing or accelerating firing pattern (type I). 2. The pattern of repetitive firing changed markedly during development. While most HMs recorded from young rats (< postnatal day (P) 4) were type D, the majority of HMs recorded during the second postnatal week were type I. In adults (> P21), nearly all HMs had a decrementing firing pattern, characterized by a brief period of adaptation and high steady‐state firing rates. 3. The calcium‐dependent after‐hyperpolarization (AHP) was shortest in type I neonatal HMs, and decreased in amplitude during trains of action potentials (APs). In type D neurones, these same trains caused a slight enhancement of AHP amplitude. In adult HMs, with a decrementing firing pattern, trains of APs also caused summation of the AHP. 4. Type D neonatal HMs showed a progressive prolongation of the AP during repetitive firing. In contrast, type I neonatal HMs had almost no change in AP duration. In adult HMs the AP was short and experienced only a modest increase in duration during fast repetitive firing. 5. The function relating steady‐state firing frequency to injected current (f‐I curve) was linear. The mean steady‐state f‐I slope was significantly higher in neonates than in adults (approximately 30 vs. approximately 20 Hz nA‐1), and was weakly correlated with input resistance. The f‐I slope was negatively correlated with AHP duration in neonatal HMs only. In addition, for a given AHP duration the slope was higher in neonatal HMs. 6. Two threshold behaviours were observed among neonatal HMs: (a) a progressive rhythmic firing threshold, and (b) a sudden transition from subthreshold to regular repetitive firing. Current threshold for repetitive firing was strongly correlated with cell input conductance. Type I neonatal HMs had higher minimal steady firing rates (fmin) than type D HMs. In neonates, fmin was strongly correlated with AHP duration. Adult HMs showed a weaker correlation between these two parameters, and fmin was higher than predicted by AHP duration. 7. In summary, HMs responded to depolarizing current pulses with different firing patterns during postnatal development.(ABSTRACT TRUNCATED AT 400 WORDS)

Serotonin-mediated excitation of recurrent laryngeal and phrenic motoneurons evoked by stimulation of the raphe obscurus

Short-latency averaged responses in the recurrent laryngeal nerve (RLN) and C5 phrenic nerve to electrical stimulation (2.5-80 microA; 2.5-160 Hz; 150 microseconds pulse duration) of the medullary nucleus raphe obscurus (RO) were investigated in anesthetized, paralyzed and artificially ventilated cats. The response evoked in RLN by stimulation within RO was excitatory and consisted of a single peak. Characteristics of this response in RLN were compared with those of the delayed excitatory response in C5 phrenic nerve, which we previously showed to be elicited by stimulation within RO. Mean latency to onset for the excitatory response in RLN was 5.7 +/- 0.3 ms, while the delayed excitatory response in C5 phrenic nerve occurred at 7.0 +/- 0.3 ms. The excitatory response in both nerves could be evoked when stimulation was applied during inspiration as well as during expiration. The stimulus threshold varied between 2.5 and 5 microA for evoking the inspiratory-phase response in each nerve. The magnitude of this response in RLN and in C5 phrenic nerve was directly related to current intensity and was dependent upon stimulus frequency. Intravenous administration of the serotonin receptor antagonist, methysergide (0.1-2.4 mg/kg) caused significant dose-related reductions in the response in each nerve. In summary, characteristics of the evoked response in RLN and phrenic nerve are similar in several ways. Both responses are: (1) excitatory in nature, (2) elicited at small stimulus currents, (3) affected similarly by increasing stimulus current and frequency, (4) elicited by stimulation during inspiration and expiration, and (5) mediated at least in part by activation of pathways using serotonin as a neurotransmitter.

Intracellular pathways regulating ciliary beating of rat brain ependymal cells

1 The mammalian brain ventricles are lined with ciliated ependymal cells. As yet little is known about the mechanisms by which neurotransmitters regulate cilia beat frequency (CBF). 2 Application of 5‐HT to ependymal cells in cultured rat brainstem slices caused CBF to increase. 5‐HT had an EC50 of 30 μM and at 100 μM attained a near‐maximal CBF increase of 52.7 ± 4.1 % (mean ± s.d.) (n= 8). 3 Bathing slices in Ca2+‐free solution markedly reduced the 5‐HT‐mediated increase in CBF. Fluorescence measurements revealed that 5‐HT caused a marked transient elevation in cytosolic Ca2+ ([Ca2+]c) that then slowly decreased to a plateau level. Analysis showed that the [Ca2+]c transient was due to release of Ca2+ from inositol 1,4,5‐trisphosphate (IP3)‐sensitive stores; the plateau was probably due to extracellular Ca2+ influx through Ca2+ release‐activated Ca2+ (CRAC) channels. 4 Application of ATP caused a sustained decrease in CBF. ATP had an EC50 of about 50 μM and 100 μM ATP resulted in a maximal 57.5 ± 6.5 % (n= 12) decrease in CBF. The ATP‐induced decrease in CBF was unaffected by lowering extracellular [Ca2+], and no changes in [Ca2+]c were observed. Exposure of ependymal cells to forskolin caused a decrease in CBF. Ciliated ependymal cells loaded with caged cAMP exhibited a 54.3 ± 7.5 % (n= 9) decrease in CBF following uncaging. These results suggest that ATP reduces CBF by a Ca2+‐independent cAMP‐mediated pathway. 5 Application of 5‐HT and adenosine‐5′‐O‐3‐thiotriphosphate (ATP‐γ‐S) to acutely isolated ciliated ependymal cells resulted in CBF responses similar to those of ependymal cells in cultured slices suggesting that these neurotransmitters act directly on these cells. 6 The opposite response of ciliated ependymal cells to 5‐HT and ATP provides a novel mechanism for their active involvement in central nervous system signalling.

Development of inhibitory synaptic transmission to motoneurons.

The inhibitory effects of the neurotransmitters glycine and gamma-aminobutyric acid (GABA) on motoneurons and their role in mediating the timing of motor output have been understood for some years. Recent work, however, has revealed that these neurotransmitters function very differently in developing motor circuits. Most strikingly, both GABA and glycine depolarize neonatal motoneurons, and, in many instances, provide excitatory drive to developing motor networks. Additionally, the relative contributions of GABA and glycine to inhibitory synaptic transmission in a circuit or, indeed, within the same synapse, change with postnatal development. Here, we review three fundamental properties of inhibitory neurotransmission that are altered postnatally and may be important in shaping the unique behaviors of these synapses early in development.