[THE EFFECT OF NEUROPEPTIDE Y ON SPIKE ACTIVITY OF NEURONS IN THE SUPRACHIASMATIC NUCLEUS OF RAT IN VITRO].

In the experiments on sagittal hypothalamic slices of male Wistar rats, the effect of 10 nM neuropeptide Y on the electrical activity of the suprachiasmatic nucleus neurons and spike information coding parameters were studied. Applications of neuropeptide Y induced a reduction of action potential frequency in 35 of 81 tested neurons, in 8 cells an increase in the parameter was observed; the remaining 38 neurons did not change their spike activity level. The decrease in spike frequency generation by neurons of the suprachiasmatic nucleus accompanied by an increase in both entropy of interspike interval distribution and mutual information between adjacent interspike intervals. These suggest an increased irregularity of the later as well as an increased patterning of spike information induced by neuropeptide Y. The obtained data indicate the ability of neuropeptide Y to modulate a level of activity and influence a spike code in a relatively numerous population of neurons in circadian oscillator of the suprachiasmatic nucleus.

Local GABAergic modulation of the activity of serotoninergic neurons in the nucleus raphe magnus.

Experiments on rat brainstem sections in membrane potential clamping conditions addressed the effects of serotonin and GABA on serotoninergic neurons in the nucleus raphe magnus. Local application of serotonin stimulated inhibitory postsynaptic currents (IPSC) in 45% of the serotoninergic neurons studied. This response was not seen in the presence of the fast sodium channel blocker tetrodotoxin. The GABAA receptor antagonist gabazine blocked IPSC in both serotonin-sensitive and serotonin-insensitive neurons. Application of GABA evoked generation of a membrane current (IGABA), which was completely blocked by gabazine. These results indicate self-regulation of the activity of serotoninergic neurons in the nucleus raphe magnus via a negative feedback circuit involving local GABAergic interneurons.

Mechanisms of the respiratory activity of leptin at the level of the solitary tract nucleus.

Acute experiments on anesthetized laboratory rats were performed to study the effects microinjections of 10(-4) M leptin into the solitary tract nucleus on the extent of the Hering-Breuer inflation reflex in ventilatory reactions to hypercapnia. Local administration of leptin into this area led to inhibition of the Hering-Breuer reflex. The extent of ventilatory responses to hypercapnia, conversely, increased, which may provide evidence that leptin has a modulatory influence on central chemoreceptors. These physiological mechanisms of action probably play a leading role in mediating the stimulatory respiratory effects of leptin at the level of the solitary tract nucleus.

Secretory cells of the supraoptic nucleus have central as well as neurohypophysial projections.

Conventional neuroanatomical methods may fail to demonstrate the presence of axons that are finer than 1 microm in diameter because such processes are near or below the limit of resolution of the light microscope. The presence of such axons can, however, be readily demonstrated by recording. The most easily interpreted type of recording for this purpose is the demonstration of antidromic activation of the cell body following stimulation of the region through which the axon passes. We have exploited this technique in the hypothalamus and have demonstrated the presence of double axonal projections or axons branching very near the cell bodies of the secretory cells of the neurohypophysial system in the rat supraoptic nucleus. We found that a small proportion of supraoptic magnocellular cells could be antidromically activated both from the neural stalk and from elsewhere in the hypothalamus, including the suprachiasmatic nucleus (8 cells of a total of 182) and the antero-ventral third ventricular region (AV3V; 4 of 182 cells) near the organum vasculosum of the lamina terminalis (OVLT). Collision of antidromic and orthodromic spikes showed that the cells were clearly antidromically (rather than synaptically, or orthodromically) activated from both sites. A stimulus applied to one of the axons prevented propagation of a spike evoked by a pulse delivered to the other axon until sufficient time had elapsed after the first stimulus for the resultant spike to have propagated from the first stimulus site along one cell process (towards the cell body or branch point), and from this point along the other axonal branch to the second stimulus site (there was also a short additional delay period during which the axon at the site of the second stimulus recovered from its absolute refractory period). If the interval between the stimuli was progressively reduced, there came a point where the second spike failed. Such a clear demonstration of dual projections in a system where the cells were previously thought to have only a single axon raises the possibility that many nerve cells in the CNS have previously unsuspected projections.

Leptin modulates spike coding in the rat suprachiasmatic nucleus.

The mammalian circadian pacemaker, the suprachiasmatic nucleus (SCN), contains receptors to the adipose tissue hormone leptin. In the present study, the effects of leptin on the electrophysiological activity of the SCN cells were characterised in vitro in rat brain slices. During extracellular recording, application of 20 nm leptin (n = 36) decreased mean spike frequency (Wilcoxon signed rank test, z = -3.390, P < 0.001) and increased the irregularity of firing measured by the entropy of the log interspike interval distribution (Student's paired t-test, t = 2.377, P = 0.023), but had no consistent effect on spike patterning as measured by the mutual information between adjacent log interspike intervals (z = 0.745, P = 0.456). Intracellular current-clamp recordings (n = 25) revealed a hyperpolarising effect of 20 nm leptin on SCN neurones (z = -2.290, P = 0.022). The hyperpolarisation largely resulted from the effect of leptin on the subgroup of cells (n = 13) that generated 'rebound' spikes upon termination of a hyperpolarising current pulse (z = -2.697, P = 0.007). Leptin application also increased the group mean duration of the afterhyperpolarisation (n = 25, t = 2.512, P = 0.023). The effects of leptin on extracellularly recorded spike activity were consistent with the changes in membrane potential and spike shape. They suggest that leptin can directly modulate the electrical properties of SCN neurones and, in this way, contribute to the mechanism by which metabolic processes influence the circadian clock.

Respiratory responses to microinjections of leptin into the solitary tract nucleus.

The regulatory peptide leptin has a respiratory stimulating effect along with its well known hypothalamic effects. The present study, performed on anesthetized rats, addressed respiratory responses to microinjections of 10(-10)-10(-4) M leptin into the solitary tract nucleus, which contains a high concentration of leptin receptors. Injections of 10(-8)-10(-4) M leptin led to stimulation of respiration, inducing a dose-dependent increase in the level of pulmonary ventilation and an increase in respiratory volume, accompanied by an increase in bioelectrical activity in the inspiratory muscles; 10(-6) M leptin also induced a transient increase in respiratory rate due to shortening of inhalation and exhalation. A characteristic feature of the response was the appearance of "sighs" - deep, prolonged inhalations accompanied by increased volley activity on the electromyograms of the inspiratory muscles and lengthening of the subsequent intervolley interval. These leptin effects, along with data on the high concentrations of specific leptin receptors (ObRb) in the solitary tract nucleus, suggested that endogenous leptin has a role in controlling respiration at the level of the dorsal segment of the respiratory center.

Effects of leucine-enkephalin on potassium currents in neurons in the rat respiratory center in vitro.

Experiments to identify the neuronal mechanisms underlying the respiratory activity of the opioid peptide leucine-enkephalin were performed on transverse slices of the rat brainstem in voltage-clamped conditions; studies addressed the effects of this peptide (10 nM-1 microM) on the potassium A current and the inward potassium current of neurons in two areas of the respiratory center: the ventrolateral area of the solitary tract nucleus and the pre-Bötzinger complex. The parameters of the A current assessed in all respiratory center neurons studied showed no change in the presence of leucine-enkephalin. At the same time, leucine-enkephalin produced reversible increases in the amplitude of the inward potassium current. These results provide evidence that the inhibitory effect of leucine-enkephalin at the level of respiratory center neurons is at least in part explained by its stimulatory action on the inward potassium current but is not associated with modulation of the potassium A current.

Melatonin modulates spike coding in the rat suprachiasmatic nucleus.

The effects of the application of melatonin in vitro on the electrophysiological activity of suprachiasmatic neurones were characterised using novel measures of coding based on the analysis of interspike intervals. Perfusion of 1 nM melatonin in vitro (n = 53) had no consistent effect on mean spike frequency (Wilcoxon's sign rank, z = -0.01, P = 0.989), but increased the irregularity of firing (Student's paired t-test, t = -3.02, P = 0.004), as measured by the log interval entropy, and spike patterning (z = -3.43, P < 0.001), as measured by the mutual information between adjacent log intervals. Intracellular recordings in vitro in current clamp mode showed that 1 nM melatonin significantly hyperpolarised (n = 11, z = -2.35, P = 0.019) those cells that showed 'rebound' spikes upon termination of a hyperpolarising current pulse. Grouping all cells together (n = 27), melatonin application decreased the duration of the afterhyperpolarisation (z = -2.49, P = 0.013) and increased the amplitude of the depolarising afterpotential (z = -2.71, P = 0.007). The effects of melatonin seen in vitro from extracellular recordings on interspike interval coding were consistent with the changes in spike shape seen from intracellular recordings. A melatonin-induced increase in the size of the depolarising afterpotential of suprachiasmatic cells might underlie the increased irregularity of spike firing seen during the subjective night time. The method of analysis demonstrated a difference in spike firing that is not revealed by frequency alone and is consistent with the presence of a melatonin-induced depolarising current.

The effects of leucine-enkephalin on the membrane potential and activity of rat respiratory center neurons in vitro.

Studies of transverse slices of Wistar rat brainstem using a patch clamp technique addressed the effects of the opioid peptide leucine-enkephalin (10 nM-1 microM) on the membrane potential and pattern of spontaneous activity of neurons in two parts of the respiratory center: the ventrolateral area of the solitary tract nucleus and the pre-Bötzinger complex. Leucine-enkephalin induced membrane hyperpolarization of respiratory center neurons and decreased the level of spike activity in spontaneously active cells. In pre-Bötzinger complex neurons showing a burst pattern of activity, leucine-enkephalin decreased the burst frequency, and two cells showed a transition from burst activity to tonic activity. These results provide evidence that the mechanism of the central respiratory activity of leucine-enkephalin results from its direct action on the membranes of respiratory center neurons.

Respiratory reactions to microinjection of bombesin into the solitary tract nucleus and their mechanisms.

Acute experiments were performed on urethane-anesthetized adult laboratory rats to investigate the effects of microinjections of 10(-13)-10(-4) M bombesin into the solitary tract nucleus on measures of respiration. Bombesin microinjections were found to stimulate respiration, inducing significant increases in the level of pulmonary ventilation, increases in respiratory volume, and increases in the bioelectrical activity of the inspiratory muscles. The most marked respiratory reactions were seen after intermediate peptide doses (10(-10)-10(-7) M). These respiratory effects of bombesin were found to result from its ability to suppress the inspiration-inhibiting Hering-Breuer reflex at the level of the solitary tract nucleus. The fact that ultralow doses of bombesin were active, along with the distribution of endogenous bombesin and its specific receptors in the solitary tract nucleus, and the ability of this peptide to modulate the Hering-Breuer reflex all provide evidence that bombesin is involved in controlling respiration at the level of the dorsal structures of the respiratory center.

Spike coding during osmotic stimulation of the rat supraoptic nucleus.

Novel measures of coding based on interspike intervals were used to characterize the responses of supraoptic cells to osmotic stimulation. Infusion of hypertonic NaCl in vivo increased the firing rate of continuous (putative oxytocin) cells (Wilcoxon z= 3.84, P= 0.001) and phasic (putative vasopressin) cells (z= 2.14, P= 0.032). The irregularity of activity, quantified by the log interval entropy, was decreased for continuous (Student's t= 3.06, P= 0.003) but not phasic cells (t= 1.34, P= 0.181). For continuous cells, the increase in frequency and decrease in entropy was significantly greater (t= 2.61, P= 0.036 and t= 3.06, P= 0.007, respectively) than for phasic cells. Spike patterning, quantified using the mutual information between intervals, was decreased for phasic (z=-2.64, P= 0.008) but not continuous cells (z=-1.14, P= 0.256). Although continuous cells showed similar osmotic responses to mannitol infusion, phasic cells showed differences: spike frequency decreased (z=-3.70, P < 0.001) and entropy increased (t=-3.41, P < 0.001). Considering both cell types together, osmotic stimulation in vitro using 40 mm NaCl had little effect on firing rate (z=-0.319, P= 0.750), but increased both entropy (t= 2.75, P= 0.010) and mutual information (z=-2.73, P= 0.006) in contrast to the decreases (t= 2.92, P= 0.004 and z=-2.40, P= 0.017) seen in vivo. Responses to less severe osmotic stimulation with NaCl or mannitol were not significant. Potassium-induced depolarization in vitro increased firing rate (r= 0.195, P= 0.034), but the correlation with decreased entropy was not significant (r=-0.097, P= 0.412). Intracellular recordings showed a small depolarization and decrease in input resistance during osmotic stimulation with NaCl or mannitol, and membrane depolarization following addition of potassium. Differences in responses of oxytocin and vasopressin cells in vivo, suggest differences in the balance between the synaptic and membrane properties involved in coding their osmotic responses. The osmotic responses in vivo constrasted with those seen in vitro, which suggests that, in vivo, they depend on extrinsic circuitry. Differences in responses to osmolality and direct depolarization in vitro indicate that the mechanism of osmoresponsiveness within a physiological range is unlikely to be fully explained by depolarization.

Thyroliberin blocks the potassium A-current in neurons in the respiratory center of adult rats in vitro.

Thyroliberin is a neuropeptide with marked respiratory activity. The neuronal mechanisms underlying this activity were addressed in experiments on transverse slices of brainstem from adult rats in conditions of membrane potential clamping to study effect effects of thyroliberin (10 nM) on the potassium A-current in neurons of two areas of the respiratory center--the ventrolateral areas of the solitary tract nucleus and the pre-Betzinger complex. The A-current, seen in all study neurons in the respiratory center, was partially and reversibly blocked by thyroliberin. A significant reduction in the amplitude of the current was accompanied by an increase in the inactivation constant. The effect of thyroliberin on the amplitude of the A-current was analogous to that of 5 mM 4-aminopyridine. These results show that the stimulatory effects of thyroliberin at the level of respiratory center neurons is at least partly explained by its ability to block the potassium A-current.

Rhythmic changes in spike coding in the rat suprachiasmatic nucleus.

The suprachiasmatic nucleus is regarded as the main mammalian circadian pacemaker but evidence for rhythmic firing of single units in vivo has been obtained only recently. The present study was undertaken to determine if rhythms could be seen using measures of activity in addition to the mean spike frequency. We investigated whether there were changes in the irregularity of cell activity measured by the disorder of the interspike interval distribution for neurones recorded in vivo and in vitro. By plotting the entropy of the log interval histogram that quantifies the coding capacity for each action potential against the respective zeitgeber time, we describe oscillations of spike activity in vivo. Entropy measures have the advantage over variances in that they quantify aspects of the shape of the distribution and not just the dispersion. One hundred and sixty-six cell recordings from the suprachiasmatic nucleus showed a significant rhythm in entropy with an oscillatory trend in the data (P < 0.001) showing a trough towards the end of the light period and a peak in the mid-dark period. There was a similar rhythm for the cells recorded from the peripheral zone (n = 209, P = 0.037). In separate experiments in vitro, to investigate the relationship between mean spike frequency and entropy, potassium-induced depolarization of cells recorded during the subjective night was correlated with a significant increase in mean spike frequency (r = 0.259, P = 0.011) and a decrease in entropy (r = -0.296, P = 0.004). The negative correlation between the entropy and mean spike frequency of cells recorded in vitro was significantly different from that seen in vivo (F = 15.5, P < 0.001), which may reflect differences in the balance between deterministic and stochastic influences on spike occurrence. The study shows that while there is a rhythm of mean spike frequency, parameters based on the variability of interspike interval distributions also display rhythmic changes over the day-night cycle.

Effects of thyroliberin on membrane potential and the pattern of spontaneous activity of neurons in the respiratory center in in vitro studies in rats.

Patch-clamp experiments on transverse brainstem slices from rats were performed to study the effects of thyroliberin (10(-8) M) on the membrane potential and spontaneous activity of neurons in two areas of the respiratory center: the ventrolateral area of the solitary tract nucleus and the pre-Botzinger complex. Thyroliberin induced membrane depolarization of neurons in the respiratory center and increased their spike activity. The pattern of activity of neurons in the pre-Botzinger complex showed decreases in the time intervals between the beginnings of bursts in response to thyroliberin. In some cases, thyroliberin led to the appearance of spike activity in initially "silent" neurons; "silent" neurons in the solitary tract nucleus became tonically active, while those in the pre-Botzinger complex showed burst activity. These results provide evidence for the existence of an indirect regulatory influence for thyroliberin on respiratory center neurons, operating at the membrane level.

Assessment of spike activity in the supraoptic nucleus.

Novel approaches to the characterization of coding carried by spike trains are discussed. Measuring firing frequency alone may only partially reflect spike patterning, and can only quantify changes of the most obvious kind. We have devised a method that combines probabilistic and information approaches to quantify the variability of the interspike intervals in a way that is independent of spike frequency. To illustrate the technique, the firing of an oxytocin cell and a vasopressin cell were compared before and after osmotic stimulation. A bimodal lognormal function was fitted to the interspike interval histograms. The entropy of the log interval histogram was used to measure the variability of intervals and to reflect the coding capacity of the cell per spike. A perfect metronome shows no variability in interval and thus has no greater coding capacity than is conveyed by its frequency, whereas the variability of intervals of magnocellular neurones means that their irregular activity has greater potential for coding. While the mean spike frequency increased in both the oxytocin and vasopressin cells in response to osmotic stimulation, the changes in their irregularity showed differences. Osmotic stimulation reduced the entropy of the oxytocin cell, reflecting an increase in the regularity of its spike activity. Conversely, osmotic stimulation had little effect on the entropy of the vasopressin cell. Such differences are not evident from a simple inspection of ratemeter activity. The comparison highlights the limitations of mean spike frequency as a measure of spike coding. Parameters based on the interspike intervals constitute informative measures of spike activity that allow objective comparisons to be made between the activity under different physiological conditions.

Comparative characteristics of respiratory pattern responses to microinjection of kainic acid into different parts of the nucleus ambiguus.

Experiments on anesthetized rats were performed using local chemical exclusion of neurons with kainic acid to study the relative roles of the rostral, intermediate, and caudal parts of the nucleus ambiguus in the mechanisms controlling respiration. The characteristics of the respiratory rhythm and pattern responses to chemical exclusion of different parts of this nucleus were observed. In particular, sequential exclusion of the left and right rostral parts of the nucleus ambiguus reproducibly induced significant decreases in the respiration rate and respiratory volume in the first minutes; in 83% of experiments, there was also irreversible respiratory arrest. Exclusion of symmetrical intermediate parts of the nucleus ambiguus was followed by bradypnea and decreases in pulmonary ventilation, the greatest respiratory effects being noted only after injection of kainic acid into the second symmetrical area, irreversible respiratory arrest being seen in 50% of cases. Exclusion of symmetrical caudal areas of the nucleus ambiguus resulted only in small decreases in respiratory frequency without significant changes in respiratory volume and gave rise to the smallest incidence of respiratory arrest, i.e., 33%.

Respiratory effects of sensorimotor cortex and their mechanisms in rats.

Acute experiments on anesthetized rats showed differential effect of various areas of the sensorimotor cortex on activity of the respiratory center. It is hypothesized that GABAergic structures of the solitary tract nucleus play an important role in the mechanisms of respiratory effects of the sensorimotor cortex.

Amygdalofugal modulation of Hering-Breuer inspiration-inhibiting reflex.

Acute experiments on rats showed that the central nucleus of the amygdaloid complex modulates realization of the Hering-Breuer inspiration-inhibiting reflex. It is hypothesized that amygdalobulbar interrelations are realized via modulation of respiratory reflexes by the amygdala. This interaction is mediated by the GABAergic system of the amygdala.

The pre-Bötzinger complex participates in generating the respiratory effects of thyroliberin.

Experiments on anesthetized rats were performed to study the effects of microinjection of thyroliberin (10 fM-100 microM) into the area of the pre-Bötzinger complex on respiratory and circulatory parameters. Thyroliberin dose-dependently increased respiration frequency, with shortening of inspiration and expiration. Tidal volume and the amplitude of the integrated EMG recorded from the inspiratory muscles decreased after administration of concentrated solutions. Using this dosage method, thyroliberin had weak effects on systemic hemodynamics. The data suggest that structures located in the area of the pre-Bötzinger complex take part in generating the respiratory effects of thyroliberin.