Antiserum to somatostatin prevents stress-induced inhibition of growth hormone secretion in the rat.

Plasma growth hormone levels fall and remain low for several hours after stress in the rat. When antiserums to somatostatin are administered to rats prior to stress, growth hormone secretory pulses are partially restored. The results provide evidence that circulating somatostatin plays a prominent role in stress-induced inhibition of growth hormone secretion in the rat.

Cell swelling, seizures and spreading depression: an impedance study.

The cellular processes that take place during the transition from pre-seizure state to seizure remain to be defined. In this study in awake, paralyzed rats, we used an electrical impedance measure of changes in extra-cellular intracranial volume to estimate changes in cell size in acute models of epilepsy. Animals were prepared with extradural electroencephalographic (EEG)/impedance electrodes and a venous catheter. On a subsequent day, animals were paralyzed, ventilated and treated with picrotoxin, kainic acid or fluorocitrate in doses that usually induce epileptiform discharges. We now report that increases in baseline impedance were induced by kainic acid and smaller increases by picrotoxin. We also demonstrated that epileptiform discharges were preceded by small, accelerated increases in impedance. Increases in baseline impedance were highly correlated with increases in power of non-ictal high frequency EEG activity. Seizures were accompanied by increases in impedance and all treatments induced transient, relatively large, increases in impedance often associated with unilateral reductions in low frequency EEG, likely periods of spreading depression. We conclude: cerebral cells swell in convulsant models of epilepsy, that there are pre-ictal accelerations in cell swelling, and that spreading depression-like events are frequently associated with seizures.

Automatic determination of EMG-contaminated components and validation of independent component analysis using EEG during pharmacologic paralysis.

OBJECTIVE: Validate independent component analysis (ICA) for removal of EMG contamination from EEG, and demonstrate a heuristic, based on the gradient of EEG spectra (slope of graph of log EEG power vs log frequency, 7-70 Hz) from paralysed awake humans, to automatically identify and remove components that are predominantly EMG. METHODS: We studied the gradient of EMG-free EEG spectra to quantitatively inform the choice of threshold. Then, pre-existing EEG from 3 disparate experimental groups was examined before and after applying the heuristic to validate that the heuristic preserved neurogenic activity (Berger effect, auditory odd ball, visual and auditory steady state responses). RESULTS: (1) ICA-based EMG removal diminished EMG contamination up to approximately 50 Hz, (2) residual EMG contamination using automatic selection was similar to manual selection, and (3) task-induced cortical activity remained, was enhanced, or was revealed using the ICA-based methodology. CONCLUSION: This study further validates ICA as a powerful technique for separating and removing myogenic signals from EEG. Automatic processing based on spectral gradients to exclude EMG-containing components is a conceptually simple and valid technique. SIGNIFICANCE: This study strengthens ICA as a technique to remove EMG contamination from EEG whilst preserving neurogenic activity to 50 Hz.

Gamma rhythms are not integral to EEG spindle phenomena.

OBJECTIVE: Gamma rhythms (30-100 Hz) have been shown to be associated with spindling activity induced by picrotoxin. To determine if gamma power is unique to picrotoxin spindles or is an integral part of physiological and pathological spindling activity we analysed and compared the strength and brain distribution of gamma EEG power during 4 spindling activities in the rat. METHODS: The electroencephalogram (EEG) was recorded from rats with chronically implanted electrodes during natural sleep, barbiturate anaesthesia, during naturally occurring absence epilepsy spike and wave discharges and following the systemic application of picrotoxin. Spectral analysis was applied off-line to compare the strength and brain distribution of gamma EEG power during the 4 spindling activities. RESULTS: Each spindle type contained significantly different levels of gamma power. Gamma power was significantly increased over background levels during picrotoxin spindles, slightly increased during absence spindles, slightly decreased during sleep spindles and significantly suppressed during barbiturate spindles CONCLUSIONS: Changes in the power of gamma frequencies during spindle types suggest that gamma frequencies are neither the cause of nor an integral part of a spindle. They appear to be correlated with levels of consciousness and may contribute to the process of epileptogenesis. SIGNIFICANCE: The findings are consistent with high frequency EEG activity being related to seizure-tendency.

Surface Laplacian of scalp electrical signals and independent component analysis resolve EMG contamination of electroencephalogram.

The serious impact of electromyogram (EMG) contamination of electroencephalogram (EEG) is well recognised. The objective of this research is to demonstrate that combining independent component analysis with the surface Laplacian can eliminate EMG contamination of the EEG, and to validate that this processing does not degrade expected neurogenic signals. The method involves sequential application of ICA, using a manual procedure to identify and discard EMG components, followed by the surface Laplacian. The extent of decontamination is quantified by comparing processed EEG with EMG-free data that was recorded during pharmacologically induced neuromuscular paralysis. The combination of the ICA procedure and the surface Laplacian, with a flexible spherical spline, results in a strong suppression of EMG contamination at all scalp sites and frequencies. Furthermore, the ICA and surface Laplacian procedure does not impair the detection of well-known, cerebral responses; alpha activity with eyes-closed; ERP components (N1, P2) in response to an auditory oddball task; and steady state responses to photic and auditory stimulation. Finally, more flexible spherical splines increase the suppression of EMG by the surface Laplacian. We postulate this is due to ICA enabling the removal of local muscle sources of EMG contamination and the Laplacian transform being insensitive to distant (postural) muscle EMG contamination.

Measurement of neural signals from inexpensive, wireless and dry EEG systems.

Electroencephalography (EEG) is challenged by high cost, immobility of equipment and the use of inconvenient conductive gels. We compared EEG recordings obtained from three systems that are inexpensive, wireless, and/or dry (no gel), against recordings made with a traditional, research-grade EEG system, in order to investigate the ability of these 'non-traditional' systems to produce recordings of comparable quality to a research-grade system. The systems compared were: Emotiv EPOC (inexpensive and wireless), B-Alert (wireless), g.Sahara (dry) and g.HIamp (research-grade). We compared the ability of the systems to demonstrate five well-studied neural phenomena: (1) enhanced alpha activity with eyes closed versus open; (2) visual steady-state response (VSSR); (3) mismatch negativity; (4) P300; and (5) event-related desynchronization/synchronization. All systems measured significant alpha augmentation with eye closure, and were able to measure VSSRs (although these were smaller with g.Sahara). The B-Alert and g.Sahara were able to measure the three time-locked phenomena equivalently to the g.HIamp. The Emotiv EPOC did not have suitably located electrodes for two of the tasks and synchronization considerations meant that data from the time-locked tasks were not assessed. The results show that inexpensive, wireless, or dry systems may be suitable for experimental studies using EEG, depending on the research paradigm, and within the constraints imposed by their limited electrode placement and number.

Pulsatile growth hormone and prolactin: effects of (+) butaclamol, a dopamine receptor blocking agent.

Growth hormone (rGH) and prolactin (rPRL) secretory profiles were obtained before and after treatment with a dopamine receptor blocking agent, (+) butaclamol, in 10 male rats chronically implanted with right atrial cannulae. Mean rGH plasma concentrations, determined by planimetry, were reduced (202 +/- 20 ng/ml vs. 135 +/- 20ng/ml, P less than .01), but the basic configuration and periodicity of rGH secretory bursts were unaltered. Mean rPRL plasma concentrations were elevated (11.1 +/- 2.1 ng/ml vs 65.5 +/- 8.1 ng/ml, P less than .0005), but rPRL episodic secretion was still apparent. It is concluded that dopaminergic neurons have a minor role in facilitating episodic rGH secretion. Furthermore, persisting episodic rPRL secretion in rats administered a dopamine antagonist suggests that rPRL feedback inhibition does not inactivitate the neural mechanism generating episodic rPRL secretion.

Growth hormone and prolactin secretion in genetically obese Zucker rats.

GH and PRL levels were measured by RIA in plasma samples taken from genetically obese and nonobese rats over a 6-h period at consecutive 15-min intervals. The mean GH level was 204 ng/ml for lean animals and 48 ng/ml for obese rats; the difference is significant (t = 5.8; P less than 0.01). For the group of 5 lean rats, there were 10 GH peaks that exceeded the upper limit of the assay (800 ng/ml), whereas for the group of 6 obese rats, there were only 2 peaks that exceeded the upper limit. In some of the obese rats, peaks of very small amplitude were present. No differences were seen in PRL levels. The mean plasma PRL level was 3.6 ng/ml for lean animals and 3.3 ng/ml for obese rats. Abnormalities in GH in obese rats may be related to an imbalance between hypothalamic releasing and inhibiting factors or a defect in the pituitary.

Pulsatility of immunoreactive somatomedin-C in chronically cannulated rats.

Pituitary GH secretion is pulsatile in man and the rat, but evidence of pulsatility in the GH-dependent somatomedins (insulin-like growth factors) has not been described. In this study serum immunoreactive somatomedin-C periodicity was examined in 10 chronically cannulated unstressed rats. Blood samples were taken at 15-min intervals over 6 h, and serum rat GH and somatomedin-C measured by RIA. For somatomedin-C assay samples were first extracted into acid-ethanol to dissociate protein-bound peptide. Serum GH levels indicated episodic secretion, with a frequency of 2.85 +/- 0.24 h; some secretory episodes were polyphasic. The mean frequency of all GH spikes reaching 400 ng/ml or greater was 1.99 +/- 0.87 h. Somatomedin-C levels showed fluctuations over an average 2-fold concentration range, 0.60 +/- 0.20 to 1.21 +/- 0.29 U/ml (mean, 0.86 +/- 0.18 U/ml), with peaks occurring 1-1.5 h after most GH secretory peaks. The somatomedin-C peak frequency was 1.93 +/- 0.47 h. Summed GH values from 0-5 h were significantly correlated with summed somatomedin-C values from 1-6 h (r = 0.861, P = 0.0007), suggesting a 1-h lag between GH pulses and the following rise in somatomedin-C. Somatomedin-binding protein showed no regular fluctuations. This study indicates that serum somatomedin-C levels in unstressed rats show periodicity which may be directly related to pulsatile GH secretion.

Pulsatile growth hormone release in the rat: failure to demonstrate a correlation with sleep phases.

Concurrent 5-hour profiles of growth hormone (GH) secretion and sleep phases were obtained in 7 rats chronically implanted with right atrial cannulae, and electroencephalographic electromyographic, and electroculographic electrodes. Hormone profiles confirmed pulsatile secretion of GH. Secretory GH episodes occurred every 3 to 4 hours and peaks generally exceeded 100 ng/ml, and, in 14 of the 21 troughs recorded, GH was unmeasurable (less than 1 ng/ml). The comparison of hormone profiles and concurrent sleep patterns excluded a temporal relationship between episodic GH secretion and sleep cycles, while scattergrams of hormone values plotted against preceding sleep phase durations also failed to demonstrate a relationship.

Distribution of tyrosine hydroxylase and neuropeptide Y-like immunoreactive neurons in rabbit medulla oblongata, with attention to colocalization studies, presumptive adrenaline-synthesizing perikarya, and vagal preganglionic cells.

We studied the distribution, within the rabbit medulla oblongata, of neuronal cell bodies containing either tyrosine hydroxylase or neuropeptide Y-like immunoreactivity. Both avidin-biotin and immunofluorescence procedures were used. Because the two primary antibodies were raised in different species it was possible to perform simultaneous colocalization studies with the immunofluorescence procedure. Tyrosine hydroxylase-containing neurons in the rostral medulla were demonstrated to contain a catecholamine by the colchicine-enhanced FAGLU (formaldehyde-glutaraldehyde) fluorescence histochemical procedure. These neurons are presumably adrenergic, corresponding to the C1 and C2 groups described in the rat. No C3 group was found in the rabbit. The distribution of tyrosine hydroxylase-containing neurons in the caudal medulla was in accordance with previous descriptions of the A1 and A2 groups based on the unenhanced FAGLU procedure. Neuropeptide Y-like immunoreactivity was observed in cell groups corresponding to those already described in the rat, but additional groups were discovered in the rabbit. Some neurons containing neuropeptide Y-like immunoreactivity were observed in nucleus raphe pallidus and these also contained serotonin (5-HT). In the nearby nucleus reticularis gigantocellularis there were occasional neurons that contained neuropeptide Y-like immunoreactivity without any colocalized 5-HT. Neuropeptide Y-like immunoreactivity was also observed in the dorsal motor nucleus of the vagus, rostral to the obex, and these neurons were demonstrated to be true vagal preganglionic cells by colocalization of neuropeptide Y-like immunoreactivity and Fast Blue retrogradely transported from the cervical vagus. We found that neuropeptide Y-like immunoreactivity was colocalized in approximately 75% of the tyrosine hydroxylase-containing neurons in the rostral medulla (C1 and C2 cells). A smaller proportion of the A1 cells also contained this peptide but it was absent from both the most caudal A1 cells and from the A2 cells. Some tyrosine hydroxylase-containing neurons occur in direct apposition to vagal preganglionic cells in both the dorsal motor nucleus of the vagus and the nucleus ambiguous. However, colocalization studies revealed that none of these neurons contained Fast Blue when this dye was retrogradely transported from the cervical vagus. Medullary catecholamine-synthesizing neurons apparently do not contribute axons to the vagus nerve. This finding is consistent with our own studies in the rat but is in contrast to studies in this species published by other workers.

Fos induction following systemic kainic acid: early expression in hippocampus and later widespread expression correlated with seizure.

We determined the distribution of Fos protein expression in a model of generalised epilepsy caused by excessive neuronal excitation. Fos immunoreactivity was mapped in forebrain in unrestrained rats, previously prepared with an indwelling venous catheter, after the intravenous administration of kainic acid (10 mg/kg). We determined cerebral activation following various periods of exposure to kainic acid by using intravenous administration of pentobarbitone to prevent further activation. Within a few minutes, kainic acid caused episodes of staring, sniffing, wet dog shakes, nodding and chewing. Fos induction occurred initially and simultaneously in hippocampus, subiculum, septum and entorhinal cortex as early as 9.5 min after kainate injection. After up to 40 min of staring, sniffing, wet dog shakes, nodding and chewing, Fos induction was not further increased above levels present within the first 9.5 min. After 56 +/- 6 min a motor convulsion occurred, initially affecting the jaw, head and tail and variably extending to the forelimbs, trunk or hindlimbs. Following the convulsive event, additional Fos was expressed in hippocampus, thalamus, caudate-putamen and other subcortical structures and in the cerebral cortex. Fos induction was sometimes asymmetric in entorhinal, visual, piriform, cingulum, parietal and frontal cortices and in amygdala and dorsal endopiriform area. Electroencephalographic recordings after a few minutes exposure to kainic acid revealed an increased amplitude of fast frequencies in hippocampus which appeared to correlate with Fos induction in this structure. The findings are generally consistent with the reported distribution and slow development of kainic acid-induced seizure activity using electrophysiological and deoxyglucose methods. However, the Fos distribution suggests that (i) hippocampal, possibly dentate, activation precedes significant activation elsewhere, (ii) extensive involvement of other cerebral structures and cerebral cortex occurs simultaneously and correlates with motor seizures and (iii) brain structures can be recruited asymmetrically.

Opioid receptor activation resets the hypothalamic clock generating growth hormone secretory bursts in the rat.

Plasma growth hormone (GH) concentrations were measured serially every 20 min for 6 h in unrestrained chronically-catheterised male rats to define physiological GH pulsatile secretory patterns. Bursts of GH secretion lasted 69 +/- 5 min and occurred every 177 +/- 4 min. Intravenous administration of the opioid receptor agonist morphine (200 micrograms/kg) caused an immediate GH burst of normal duration (63 +/- 3 min) in all animals. This burst of secretion occurred whatever the phase of the background GH cycle and was followed by normal trough GH levels; a second GH burst occurred 177 +/- 6 min later, an inter-burst period not different from controls. Opioid receptor blockade with naloxone (5 mg/kg) administered i.v. every 20 min during spontaneous GH bursts significantly lengthened the interburst interval from 177 +/- 4 to 200 +/- 9 min (P = 0.015). Naloxone did not affect synchronisation of the GH rhythm induced by morphine but lengthened the duration of GH secretory bursts from 69 +/- 5 to 94 +/- 9 min (P = 0.017). The findings indicate that opioid receptor activation resets the hypothalamic mechanism generating pulsatile GH secretion and that both the period of the GH rhythm and duration of the GH burst is normally shortened by opioid mechanisms.

Disappearance half-life times of exogenous and growth hormone-releasing factor-stimulated endogenous growth hormone in normal rats.

This study was performed to determine the disappearance half-life times of endogenous and exogenous rat GH in conscious normal rats and to compare these with the decay characteristics of GH at the end of spontaneous normal bursts. The endogenous half-life was determined in five rats by giving an i.v. injection of rat GH-releasing factor followed after 10 min by an i.v. injection of long-acting somatostatin analogue (octreotide) and taking blood samples for 85 min. The half-lives (mean +/- S.E.M.) were 3.4 +/- 0.4 min and 13.2 +/- 1.1 min for the first and second exponential respectively as determined by bi-exponential analysis. The exogenous GH half-life was determined in ten rats by giving i.v. octreotide followed after 10 min by i.v. rat GH and sampling for 85 min. The half-lives of exogenous GH were 3.3 +/- 0.2 min and 17.5 +/- 1.4 min by bi-exponential analysis and there was no significant difference between the half-lives of endogenous and exogenous GH. The half-life of the decline of GH levels at the end of spontaneous bursts in nine rats was 14.4 +/- 0.9 min, not different from the half-life of endogenous GH, the secretion of which was terminated by octreotide. This suggests that the end of spontaneous GH bursts is marked by sudden cessation of GH release and may provide an indication of the rapidity of change in the levels of the underlying hypothalamic hormones which control GH release.

Effects of human growth hormone on the secretion of rat growth hormone.

Human growth hormone (hGH) was administered to chronically cannulated male rats and its effect upon the physiological secretory patterns of rat growth hormone (rGH) and prolactin were observed. In comparison with injected control animals, a reduction in the size of spontaneous secretory bursts of rGH was apparent when hormone concentrations were compared 3-6 h after administration of hGH (136.27 +/- 27.31 (S.E.M.) v. 76.22 +/- 20.98 ng/ml respectively). However, the mean frequency of secretory episodes of rGH was unaltered. It is therefore postulated that endogenous rGH may modulate the amplitude but not the rhythmicity of secretory episodes of rGH.

Responses to prolactin secretagogues in oestrogen-treated rats suggest that the defect in prolactin regulation produced by oestrogen is at the level of the pituitary gland.

Prolactin responses to pharmacological agents were used to characterize the defect in prolactin regulation which occurs after administration of high doses of oestrogen to rats. Animals with chronically implanted venous cannulae were injected with 2 mg oestradiol benzoate in oil and 2-3 days later prolactin concentrations were measured after injections of saline, thyrotrophin-releasing hormone (TRH), fenfluramine, apomorphine and butaclamol. The responses were compared with those in oil-injected animals. Hyperprolactinaemia in oestrogen-treated animals was unresponsive to apomorphine, but was even more sensitive to dopamine receptor blockade than controls. These results suggest that the lactotrophs in oestrogen-treated animals are already maximally suppressed by endogenous dopamine, though ineffectively. Although there was an increased prolactin response to TRH in oestrogen-treated animals, there was an impaired response to fenfluramine, indicating suppressed serotonergic prolactin-releasing factor mechanisms. Maximal endogenous dopaminergic activity and suppressed prolactin-releasing factor mechanisms are appropriate hypothalamic responses to hyperprolactinaemia. The operation of these responses in the earliest stages of the development of pituitary hyperplasia indicates that oestrogen induces a disturbance of prolactin regulation in the lactotroph, independent of hypothalamic control.

Sustained oestrogen-induced hyperprolactinaemia results from a pituitary defect.

Pituitary enlargement and hyperprolactinaemia were induced in male rats by a single subcutaneous injection of 2 mg oestradiol benzoate in oil. Two months after treatment, when oestrogen levels were normal, serial blood samples for determination of plasma concentrations of prolactin were obtained from undisturbed animals through an indwelling right atrial cannula which had been implanted 7-10 days before. Basal concentrations of prolactin were obtained in treated and control rats, and responses of prolactin to intravenous injections of thyrotrophin releasing hormone (TRH), apomorphine, butaclamol and fenfluramine were measured. Sustained hyperprolactinaemia with pituitary hyperplasia was achieved in only 20% of animals. Responses of prolactin to TRH were the same in control, oestrogen-treated hyperprolactinaemic and non-hyperprolactinaemic rats, indicating normal pituitary responsiveness to one prolactin releasing factor. Complete suppression of prolactin concentrations by apomorphine occurred in hyperprolactinaemic animals, whereas no suppression could be demonstrated in animals with normal (low) basal prolactin levels, indicating good responsiveness of hyperplastic pituitary glands to dopamine inhibition. Dopamine receptor blockade by butaclamol resulted in a vigorous prolactin response in animals with sustained hyperprolactinaemia, indicating that dopaminergic prolactin inhibitory mechanisms remain qualitatively intact, but the response was quantitatively less than in controls, suggesting insufficient hypothalamic release of dopamine. Responses of prolactin to certain doses of fenfluramine were completely abolished in hyperprolactinaemic animals, indicating diminished sensitivity to serotoninergic prolactin releasing factor mechanisms. Prolactin releasing factor unresponsiveness and relative insufficiency of dopaminergic activity could be regarded as physiologically appropriate responses to chronic hyperprolactinaemia. Thus oestrogen-induced chronic hyperprolactinaemia appears to be entirely of pituitary origin.

Non convulsive spike-wave discharges do not induce Fos in cerebro-cortical neurons.

Immunohistochemical localisation of Fos was used as a marker of neuronal activity to demonstrate neurons active during non-convulsive spike-wave epilepsy. Fos-positive neurons in cortex and several subcortical areas were counted. In undisturbed animals. Fos counts were not related to spike-wave in any region. With the electroencephalographic (EEG) recording procedure. Fos induction occurred in all regions, even after habituation. However, in central cortex, counts were found to be inversely related to spike-wave duration. This suggests that neuronal activity is not increased during spike-wave and that the central cortex in these animals is less responsive to arousal than in non-epileptic animals.

Activation of opioid receptors in the mediobasal hypothalamus stimulates prolactin secretion in the conscious rat.

Abstract In an attempt to localize the opioid receptor(s) (mu, delta and kappa) involved in opioid-stimulated prolactin release in the conscious male rat, opioid agonists were microinjected into the mediobasal hypothalamus and prolactin levels measured before and after injection. The specific mu agonist, DAGO ((D-Ala(2), NMe-Phe(4), Gly-ol(5))-enkephalin) was the most effective in eliciting prolactin release, the smallest effective dose being 0.01 nmoles. The specific delta agonist, DPDPE ((D-Pen(2), D-Pen(5))-enkephalin) had no significant effect even at the highest dose of 10 nmoles. The specific kappa agonist, U50,488H ((trans-3,4-dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzene acetamide) was effective at the doses 1.0 and 10 nmoles. We conclude that mu and kappa opioid receptors in the mediobasal hypothalamus are involved in opioid-stimulated prolactin release and that delta receptors are not.

Activation of hypothalamic gamma-aminobutyric Acid receptors resets the pendulum of the growth hormone clock.

Abstract Plasma levels of growth hormone in male rats exhibit an ultradian rhythm having a periodicity of 3 to 4 h, bursts of growth hormone being separated by troughs when growth hormone secretion ceases. To determine if neurons in the vicinity of somatostatin neurons in the preoptic/anterior hypothalamic area participate in the generation of the rhythm, we temporarily inhibited this region with injections of the gamma-aminobutyric acid agonist, muscimol, in unanaesthetized rats previously prepared with a venous catheter and stereotaxically implanted intracerebral guide tubes. The injection resulted in an immediate burst of growth hormone release, followed by a trough period and another growth hormone burst, 3.4 +/- 0.1 h later. The induction of a trough and synchronization of growth hormone bursts was not reproduced by a burst of intravenously injected exogenous growth hormone at least as large as the first burst initiated by muscimol. These findings indicate that, in the short term, the ultradian rhythm is independent of growth hormone feedback and provide the first evidence that structures in the anterior hypothalamus receiving a gamma-aminobutyric acid input are an important component of the neural generator of the ultradian rhythm of growth hormone.