Persistent inward currents in rat ventral horn neurones

Throughout the mammalian spinal cord, interneurones have been shown to exhibit distinct firing patterns in response to a step of injected current. In this study of ventral horn interneurones in a thick slice preparation of the lumbar cord of 11–19-day-old-rats, four distinct firing patterns were observed and classified as repetitive-firing, repetitive/burst, initial-burst or single-spiking. The hypothesis that a persistent sodium current was the predominant determinant of cell firing behaviour was investigated. A slow voltage ramp was used to assess persistent inward currents (PICs). Cells with repetitive-firing patterns had significantly larger PICs than cells displaying repetitive/burst, initial-burst or single-spiking patterns. Repetitive-firing, repetitive/burst and initial-burst-firing cells were reduced to a single-spiking pattern with the application of riluzole, which also markedly reduced the persistent sodium current. Persistent sodium current was found to account for most of the PIC with only a small contribution from L-type calcium current. These results suggest that the persistent sodium current plays a major role in determining firing patterns in these cells.     

Responses of ventral respiratory neurones in the rat to vagus stimulation and the functional division of expiration.

In anaesthetized rats, extracellular and intracellular recordings were taken from 106 respiratory neurones in the intermediate region of the nucleus ambiguus. We observed unprovoked shortening of expiratory time accompanied, in all classes of respiratory neurone, by the elimination of the changes in membrane potential that were characteristic of stage II expiration. The demonstration of the elimination of stage II expiration in both the rat and cat strongly supports the functional division of expiration into stage I expiration (post-inspiration) and stage II expiration. In order to identify the neurones in the rat that receive inputs from vagal afferents and modulate the central respiratory rhythm, we examined whether any respiratory neurones responded to stimulation of the vagus nerve. Some post-inspiratory and stage II expiratory neurones responded. The short latency (< 2 ms) of four of the responses indicates that some vagal afferents act on post-inspiratory neurones via a disynaptic pathway. While repetitive stimulation of the vagus nerve could inhibit the phrenic rhythm, it appears that most inspiratory neurones in the intermediate region of the nucleus ambiguous complex are not directly involved in integrating the information from vagal afferents with the central respiratory rhythm.     

Neurotensin excitation of rat ventral tegmental neurones.

1. Whole-cell patch-clamp recordings were made from ventral tegmental area neurones in rat midbrain slices in vitro. In principal cells, which are presumed to contain dopamine, neurotensin (< or = 1 microM) caused an inward current at -60 mV in thirty of forty-seven neurones and had no effect on the remainder. In secondary neurones, neurotensin caused an inward current in twelve of thirty-three cells. 2. The inward current evoked by neurotensin reached a maximum amplitude of about 80 pA, and declined over several minutes when the application was discontinued. The current was most commonly accompanied by a decrease in membrane conductance and reversed polarity at a strongly hyperpolarized potential; this reversal potential was less negative in a higher extracellular potassium concentration. Neurotensin also caused an inward current even in potassium-free internal and external solutions; this current was accompanied by a conductance increase, reversed close to 0 mV and was inhibited by reduction of the extracellular sodium concentration (from 150 to 20 mM). 3. The inward current was associated with a large increase in noise; this persisted in calcium-free solutions but was inhibited by low sodium concentration. The increase in noise was more prominent at hyperpolarized potentials. The amplitude of the unitary current underlying the increase in noise was estimated from the ratio of the variance to the mean as about 1.5 pA at -100 mV. 4. When the recording was made with an electrode containing guanosine 5'-thio-triphosphate, the steady inward current evoked by neurotensin did not reverse when the application was discontinued. When the recording electrode contained pertussis toxin, the action of neurotensin was not different although outward currents evoked by dopamine and baclofen declined with time. 5. It is concluded that neurotensin excites ventral tegmental area neurones by activating a pertussis toxin-insensitive guanosine nucleotide-binding protein. This leads to a reduction in membrane potassium conductance and an increase in membrane sodium conductance, the relative contribution of which varies from cell to cell.     

Correlations between the firing of supraoptic neurones in slices of rat brain

Summary These experiments were an attempt to study the possible interactions between cells of the supraoptic nucleus. In isolated brain slices pairs of supraoptic neurones were recorded simultaneously either with a single electrode or with two electrodes and cross-correlograms produced. Correlations were demonstrated in 22 of the 82 pairs studied and were found to be more common between closely neighbouring pairs of cells. Ten of the correlations indicated that the spikes of one cell followed spikes in the other cell. The correlations of another 10 pairs indicated that the cells were coactivated. In only 2 pairs was there a correlation indicative of an inhibitory connexion. That these correlations could result either from synaptic connexions within the nucleus, or from coactivation of cells from an extranuclear site is discussedBeit Memorial Fellow     

Postnatal developmental changes in activation profiles of the respiratory neuronal network in the rat ventral medulla

Two putative respiratory rhythm generators (RRGs), the para-facial respiratory group (pFRG) and the pre-Bötzinger complex (preBötC), have been identified in the neonatal rodent brainstem. To elucidate their functional roles during the neonatal period, we evaluated developmental changes of these RRGs by optical imaging using a voltage-sensitive dye. Optical signals, recorded from the ventral medulla of brainstem–spinal cord preparations of neonatal (P0–P4) rats ( n = 44), were analysed by a cross correlation method. With development during the first few postnatal days, the respiratory-related activity in the pFRG reduced and shifted from a preinspiratory (P0–P1) to an inspiratory (P2–P4) pattern, whereas preBötC activity remained unchanged. The μ-opioid agonist [ d -Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) augmented preinspiratory activity in the pFRG, while the μ-opioid antagonist naloxone induced changes in spatiotemporal activation profiles that closely mimicked the developmental changes. These results are consistent with the recently proposed hypothesis by Janczewski and Feldman that the pFRG is activated to compensate for the depression of the preBötC by perinatal opiate surge. We conclude that significant reorganization of the respiratory neuronal network, characterized by a reduction of preinspiratory activity in the pFRG, occurs at P1–P2 in rats. The changes in spatiotemporal activation profiles of the pFRG neurones may reflect changes in the mode of coupling of the two respiratory rhythm generators.     

Presynaptic delta opioid receptors differentially modulate rhythm and pattern generation in the ventral respiratory group of the rat

The specific role of the Delta opioid receptor (DOR), in opioid-induced respiratory depression in the ventral respiratory group (VRG) is largely unknown. Here, we sought to determine (1) the relationship between DOR-immunoreactive (ir) boutons, bulbospinal and functionally identified respiratory neurons in the VRG and (2) the effects of microinjection of the selective DOR agonist, D-Pen 2,5-enkephalin (DPDPE), into different subdivisions of the VRG, on phrenic nerve discharge and mean arterial pressure. Following injections of retrograde tracer into the spinal cord or intracellular labelling of respiratory neurons, in Sprague-Dawley rats, brainstem sections were processed for retrograde or intracellular labelling and DOR-ir. Bulbospinal neurons were apposed by DOR-ir boutons regardless of whether they projected to single (cervical or thoracic ventral horn) or multiple (cervical and thoracic ventral horn) targets in the spinal cord. In the VRG, a total of 24 +/- 5% (67 +/- 13/223 +/- 49) of neurons projecting to the cervical ventral horn, and 37 +/- 3% (96 +/- 22/255 +/- 37) of neurons projecting to the thoracic ventral horn, received close appositions from DOR-ir boutons. Furthermore, DOR-ir boutons closely apposed six of seven intracellularly labelled neurons, whilst the remaining neuron itself possessed boutons that were DOR-ir. DPDPE was microinjected (10 mM, 60 nl, unilateral) into regions of respiratory field activity in the VRG of anaesthetised, vagotomised rats, and the effects on phrenic nerve discharge and mean arterial pressure were recorded. DPDPE depressed phrenic nerve amplitude, with little effect on phrenic nerve frequency in the B?tzinger complex, pre-B?tzinger complex and rVRG, the greatest effects occurring in the B?tzinger complex. The results indicate that the DOR is located on afferent inputs to respiratory neurons in the VRG. Activation of the DOR in the VRG is likely to inhibit the release of neurotransmitters from afferent inputs that modulate the pattern of activity of VRG neurons.     

Oxytocin neurones are recruited into co-ordinated fluctuations of firing before bursting in the rat

Hypothalamic oxytocin neurones have dual physiological functions with associated characteristic activity patterns: a homeostatic osmoregulatory role involving continuous low frequency firing at a relatively constant rate, and roles associated with reproduction involving periodic, brief, synchronised, high frequency bursts of spikes. Apparently the same neurones maintain both roles during reproduction, when both activity patterns occur simultaneously, although sometimes factors linked to the homeostatic response predominate and prevent bursting. With the object of understanding how oxytocin neuronal networks manage both roles during lactation, we analysed basal activity between bursts in simultaneously recorded neurones to reveal potentially adaptive changes in network behaviour. Negative autocorrelation on a time scale of 0.5-2 s occurs in basal activity between bursts but also in non-bursting oxytocin neurones, and can therefore be associated with the system's homeostatic role. Although the system responds to the pups suckling by the induction of bursting, there are also increasing fluctuations in firing that are positively correlated in some simultaneously recorded neurones during basal activity between bursts. A few seconds before bursts, cross-correlation strengthens, irregularity of firing increases, and serial correlation (autocorrelation) weakens, all substantially. After pharmacological treatments known to facilitate bursting, cross-correlation and irregularity of firing increase and autocorrelation weakens, and the reverse occurs in conditions that delay bursting (hyperosmotic stress and pharmacological interventions). Our analyses suggest heterogeneity in the population of oxytocin neurones during lactation; the range including 'leader neurones' that readily display co-ordinated fluctuations in firing in response to suckling and escape from negative autocorrelation just before bursts, and 'follower neurones' that fire at a relatively constant rate in no apparent relationship to others, except when recruited late to bursting, probably in response to massive stimulation from already bursting neurones. The steep increases in correlation a few seconds before bursts reflect an accelerating process of recruitment of follower neurones to co-ordinated fluctuations, leading to the phase transition that constitutes the critical stage of burst generation.     

Effect of testosterone and the oestrous cycle on neuronal refractory periods and firing rates of stria terminalis neurones in the female rat

Summary Absolute refractory periods of a subpopulation of corticomedial amygdala (CMA) neurones which project to the medial preoptic/ anterior hypothalamic junction (MPH) via the stria terminalis were recorded in the female rat. Previous experiments have shown that this sub-population of CMA neurones is testosterone-sensitive in the male rat. In the ovariectomised female testosterone propionate (TP, 200 g/day for 18–22 days) significantly reduced the mean absolute refractory period of these CMA neurones compared to oil treated controls (from 1.34 ms to 0.87 ms). In a second experiment the absolute refractory periods of these CMA neurones were measured during the pro-oestrus and di-oestrus stages of the oestrous cycle as well as in ovariectomised controls. The mean absolute refractory period of these neurones was significantly shortened at pro-oestrus (0.99 ms) compared both to animals in di-oestrus 1 (1.45 ms) and ovariectomised controls (1.42 ms). The median firing rate of these CMA neurones was also significantly increased at pro-oestrus (1.24 Hz) compared to di-oestrus 1 (0.13 Hz) and ovariectomised controls (0.09 Hz). No firing rate increase was observed after TP treatment of ovariectomised animals in the first experiment.Results show that the testosterone-sensitive CMA neurones originally discovered in the male rat have a similar sensitivity in the female rat. They also show that the refractory periods of these neurones are shortened at pro-oestrus. Further, these same neurones also show firing rate increases at this time, although such an increase has not been observed in gonadally intact male rats when compared to castrated ones. Results are interpreted in terms of a possible functional sexual dimorphism in the output of these CMA neurones.Supported by the Medical Research Council     

Are there serotonergic projections from raphe and retrotrapezoid nuclei to the ventral respiratory group in the rat?

The relationship of serotonergic neurons in raphe and ventrolateral medullary regions to neurons projecting to the rostral ventral respiratory group in the rat were investigated using combined immunohistochemical and retrograde labeling techniques. Serotonergic and non-serotonergic neurons retrogradely labeled with rhodamine beads were found intermingled in the larger population of serotonin-immunoreactive neurons in raphe pallidus, obscurus and magnus. In addition, a cell group analogous to the retrotrapezoid nucleus in the cat was identified in the rat ventrolateral medulla. Retrotrapezoid neurons, which exhibited exclusively ipsilateral projections to the ventral respiratory group, were located lateral to clusters of serotonergic cells near the ventral surface, but were not serotonin immunoreactive.     

Flufenamic acid blocks depolarizing afterpotentials and phasic firing in rat supraoptic neurones

Depolarizing afterpotentials (DAPs) that follow action potentials in magnocellular neurosecretory cells (MNCs) are thought to underlie the generation of phasic firing, a pattern that optimizes vasopressin release from the neurohypophysis. Previous work has suggested that the DAP may result from the Ca²⁺-dependent reduction of a resting K⁺ conductance. Here we examined the effects of flufenamic acid (FFA), a blocker of Ca²⁺-dependent non-selective cation (CAN) channels, on DAPs and phasic firing using intracellular recordings from supraoptic MNCs in superfused explants of rat hypothalamus. Application of FFA, but not solvent (0.1 % DMSO), reversibly inhibited (IC₅₀+ 13.8 μ m ;   R + 0.97  ) DAPs and phasic firing with a similar time course, but had no significant effects (   P > 0.05  ) on membrane potential, spike threshold and input resistance, nor on the frequency and amplitude of spontaneous synaptic potentials. Moreover, FFA did not affect (   P > 0.05  ) the amplitude, duration, undershoot, or frequency-dependent broadening of action potentials elicited during the spike trains used to evoke DAPs. These findings suggest that FFA inhibits the DAP by directly blocking the channels responsible for its production, rather than by interfering with Ca²⁺ influx. They also support a role for DAPs in the generation of phasic firing in MNCs. Finally, the absence of a depolarization and increased membrane resistance upon application of FFA suggests that the DAP in MNCs may not be due to the inhibition of resting K⁺ current, but to the activation of CAN channels.     

Behavioural activation induced in the rat by substance P infusion into ventral tegmental area: implication of dopaminergic A10 neurones

The functional role of the putative transmitter substance P (SP) was studied using a behavioural approach. SP infusion into the ventral tegmental area in awake rats elicited an increase in locomotor activity which could be blocked by either infusion of a neuroleptic into the nucleus accumbens septi (NAS) or by 6-hydroxydopamine (6-OHDA) lesions of the ascending dopaminergic A10 neurones. Our results suggest that SP induces its behavioural effects through activation of dopaminergic A10 neurones, and imply that endogenous SP may have an important modulatory role.     

Increased phasic activity of dopaminergic neurones in the rat ventral tegmental area following pharmacologically elevated levels of endogenous kynurenic acid

Kynurenic acid (KYNA) is an antagonist of ionotropic glutamate receptors, preferentially blocking the glycine-site of the N-methyl-D-aspartate (NMDA) receptor. In the present electrophysiological study, the firing pattern of dopamine (DA) neurones of rat ventral tegmental area (VTA) was investigated following pharmacologically elevated endogenous levels of KYNA by means of an inhibitor of kynurenine 3-hydroxylase (PNU 156561A). Pre-treatment with PNU 156561A (40 mg kg-1, i.v., 5-9 h) caused a threefold increase in endogenous KYNA in whole brain levels and also evoked a significant increase in firing rate and bursting activity of VTA DA neurones. Administration of D-cycloserine (2-128 mg kg-1, i.v.), a partial agonist at the glycine-site of the NMDA-receptor, was found to reverse the increase in firing rate and bursting activity as induced by elevated concentrations of KYNA. The electrophysiological effects of elevated KYNA levels were in all essential mimicked by administration of the NMDA-receptor antagonist MK 801 (0.05-1.6 mg kg-1, i.v.). Thus, the effects of elevated endogenous brain KYNA observed in the present study are likely to be carried out by NMDA receptor antagonism. In conclusion, this study shows that an increase in endogenous KYNA levels produces significant actions on the tonic afferent control of the firing pattern of VTA DA neurones. Given the psychotomimetic effects of NMDA-receptor antagonists, e.g. phencyclidine and ketamine, the state of hyperactivity of mesocorticolimbic DA system induced by elevated levels of KYNA may represent a pathophysiological condition analogous to that seen in schizophrenic patients.     

The effects of neural stalk stimulation upon firing patterns in rat supraoptic neurones

Summary Antidromically identified neurones were recorded from the supraoptic neucleus of urethane-anaesthetised rats. In test periods of about 10 min, each recorded action potential was followed 1 ms later by a shock to the neural stalk. In phasically firing cells, this stimulation consistently modified the observed firing pattern: burst length was shortened but intraburst activity was unchanged. The stimulation invaded most supraoptic neurones antidromically, but antidromic spikes evoked in the recorded neurone were extinguished by collision. Thus, the observed changes in firing pattern were probably the consequence of interaction between the recorded unit and other neurones projecting to the neural stalk — most probably other supraoptic neurones.The behaviour of continuously firing supraoptic neurones was studied following single shocks and following trains of shocks. The results suggest that the widely reported evidence for recurrent inhibition in the rat supraoptic nucleus has been misinterpreted, and that inhibition cannot be demonstrated in most supraoptic neurones following single shocks to the neural stalk. However, the experiments using trains of shocks produced evidence consistent with a complex interaction between magnocellular neurosecretory cells involving both excitation and inhibition.     

Complement activation in the adult respiratory distress syndrome following cardiopulmonary bypass

We investigated complement fractions in patients after extracorporeal circulation for coronary bypass operations or cardiac valve replacement, and in two cases developing an adult respiratory distress syndrome (ARDS) after this type of intervention. The patients presenting an ARDS had significantly increased levels of C3d (p less than 0.001), the small molecular breakdown product of C3, associated with decreased levels of total classic haemolytic activity (p less than 0.05) and of the complement component C1q (p less than 0.001) when compared to a group of 10 patients who had uneventful evolution after bypass. However, all patients undergoing cardiopulmonary bypass had significantly increased levels of C3d (p less than 0.005 or less) associated with significant decrease of various complement components within 24 h after bypass, when compared to a control group of 5 patients investigated after aorto-iliac bypass graft surgery. We conclude that significant complement activation can persist in patients 24 h after bypass and--at higher levels--be a pathogenic and biological marker of ARDS after extracorporeal circulation.     

Biphasic effects of substance P on respiratory activity and respiration-related neurones in ventrolateral medulla in the neonatal rat brainstem in vitro

The effects of substance P (SP) on respiratory activity in the brainstem-spinal cord preparation from neonatal rats (0-4 days old) were investigated. The respiratory activity was recorded from C4 ventral roots and intracellularly from three types of respiration-related neurones, i.e. pre-inspiratory (or biphasic E), three subtypes of inspiratory; expiratory and tonic neurones in the ventrolateral medulla (VLM). After the onset of SP bath application (10 nM-1 microM) a dose-dependent decline of burst rate (by 48%) occurred, followed by a weaker dose-dependent increase (by 17.5%) in burst rate. 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 neurones. There were no significant changes in membrane resistance in any type of neurones when SP was applied alone or when synaptic transmission was blocked with tetrodotoxin (TTX). The initial depolarization was associated with an increase in inspiratory drive potential (by 25%) as well as in bursting time (by 65%) and membrane excitability in inspiratory and pre-inspiratory neurones, which corresponded to the decrease in burst rate (C4 activity). The spiking frequency of expiratory and tonic neurones was also increased (by 36 and 48%). This activation was followed by restoration of the synaptic drive potential and bursting time in inspiratory and to a less extent in pre-inspiratory neurones, which corresponded to the increase in burst rate. The discharge frequency of expiratory and tonic neurones also decreased to control values. This phase followed the peak membrane depolarization. At the peak depolarization, SP reduced the amplitude of the action potential by 4-8% in all types of neurones. Our results suggest that SP exerts a general excitatory effect on respiration-related neurones and synaptic coupling within the respiratory network in the VLM. The transient changes in neuronal activity in the VLM may underlie the biphasic effect of SP in the brainstem respiration activity recorded in C4 roots. However, the biphasic effect of SP on inspiratory burst rate seems to be also defined by the balance in activity of other SP-sensitive systems and neurones in the respiratory network in the brainstem and spinal cord, which can modify the activity of medullary respiratory rhythm generator.