Anxiety behaviour of the male rat on the elevated plus maze: associated regional increase in c-fos mRNA expression and modulation by early maternal separation.

Stressful stimuli cause region-specific increases in c-fos expression within the rat brain. Early maternal separation (EMS) is a model of early life adversity that results in long lasting changes to stress and anxiety responses. This study examined the regional distribution of c-fos mRNA after exposure to the elevated plus-maze (EPM) and how EMS altered this pattern. On each of post-natal days 5-21 pups were separated from the dam for 6 h -- control rats remained undisturbed. At 70 days old, male offspring were either exposed to the EPM or left undisturbed in the home cage. After exposure to the EPM, c-fos mRNA expression was significantly increased in specific brain areas, including cingulate cortex, medial amygdala and hippocampus. EMS rats displayed greater anxiety behaviour on the EPM vs. controls. Although EMS caused no overall effect on basal c-fos mRNA, a significant interaction between treatment group and exposure to the EPM occurred in the dentate gyrus and piriform cortex, with lower EPM-induced mRNA levels in EMS rats. The region-specific increase in c-fos mRNA reflects activation of neural circuits associated with EPM-induced anxiety. The effect of EMS on this activation in the two regions suggests these areas may contribute to the differential response to the anxiogenic stress of the EPM.

Corticotropin-releasing factor increases in vitro firing rates of serotonergic neurons in the rat dorsal raphe nucleus: evidence for activation of a topographically organized mesolimbocortical serotonergic system.

In vivo studies suggest that the stress-related neuropeptide corticotropin-releasing factor (CRF) modulates serotonergic neurotransmission. To investigate the underlying mechanisms for this interaction, the present study examined the effects of CRF in vitro on dorsal raphe neurons that displayed electrophysiological and pharmacological properties consistent with a serotonergic phenotype. In the presence of either 1 or 2 mm Ca(2+), perfusion of ovine CRF or rat/human CRF rapidly and reversibly increased firing rates of a subpopulation (19 of 70, 27%) of serotonergic neurons predominantly located in the ventral portion of the dorsal raphe nucleus. For a given responsive neuron, the excitatory effects of CRF were reproducible, and there was no tachyphylaxis. Excitatory effects were dose-dependent (over the range of 0.1-1.6 micrometer) and were completely absent after exposure to the competitive CRF receptor antagonists alpha-helical CRF(9-41) or rat/human [d-Phe(12), Nle(21, 38), alpha-Me-Leu(37)]-CRF(12-41). Both the proportion of responsive neurons and the magnitude of excitatory responses to CRF in the ventral portion of the caudal dorsal raphe nucleus were markedly potentiated in slices prepared from animals previously exposed to isolation and daily restraint stress for 5 d. Immunohistochemical staining of the recorded slices revealed close associations between CRF-immunoreactive varicose axons and tryptophan hydroxylase-immunoreactive neurons in the area of the recordings, providing anatomical evidence for potential direct actions of CRF on serotonergic neurons. The electrophysiological properties and the distribution of responsive neurons within the dorsal raphe nucleus are consistent with the hypothesis that endogenous CRF activates a topographically organized mesolimbocortical serotonergic system.

A secondary isotope effect in the lipoxygenase reaction.

Fatty acids containing a prochiral tritium label have often been used in the study of enzymatic reactions which involve an obligatory step of hydrogen abstraction. In the lipoxygenase reaction, the primary isotope effect associated with this approach is detected as an isotopic enrichment of the substrate. Herein we characterize a previously unrecognized secondary isotope effect which changes the specific activity of both the substrate and product. The 12-lipoxygenase of human platelets removes the 10-LS hydrogen of arachidonic acid in the formation of 12-hydroperoxyeicosatetraenoic acid. We studied the specific activity changes associated with conversion of the enantiomerically labeled [10-DR-3H]arachidonic acid to 12-[10-3H]hydroxyeicosatetraenoic acid in aspirin-treated platelets. [3-14C]Arachidonic acid served as internal standard. The most pronounced change in 3H/14C ratio in the early stages of reaction was a 15-20% deficiency of tritium in the product. Later, the remaining arachidonate showed a marked increase in 3H/14C ratio. The changes in specific activity closely matched those predicted for a secondary isotope effect. Comparison of these data with the theoretical equations for a secondary isotope effect indicated the 10-DR-3H substrate reacted at about 84% of the rate of unlabeled molecules. Interestingly, this secondary isotope effect is similar in magnitude to the secondary isotope effect in autoxidation reactions, a finding compatible with a basic similarity in reaction mechanisms in enzymatic and non-enzymatic oxygenation of lipids.

Oxytocin-induced excitation of neurones in the rat central and medial amygdaloid nuclei.

Central oxytocin plays an important role in regulating emotionality. The amygdala expresses gonadal steroid-sensitive oxytocin binding sites in both the central and medial sub-nuclei, although the densities markedly differ between these nuclei. These studies examined the in vitro electrophysiological effects of oxytocin in the two amygdaloid nuclei and compared responses in female rats in different reproductive states (virgin, pregnant and lactating). Oxytocin (10(-9)-10(-6)M) caused a concentration-dependent increase in the firing rate of 20-36% of the neurones in both nuclei. Although autoradiographic studies using the oxytocin receptor antagonist [(125)I]d(CH(2))(5)[Tyr(Me)(2),Thr(4),Orn(8),Tyr-NH(2)(9)]-vasotocin showed a higher density of binding in the central nucleus of the amygdala than medial nucleus of the amygdala, neurones in the central nucleus of the amygdala had a much lower sensitivity to oxytocin: equivalent responses obtained with 10(-6)M in the central nucleus of the amygdala and 10(-8)M in the medial nucleus of the amygdala, and neurones in the central nucleus of the amygdala were insensitive to concentrations below 10(-6)M. Furthermore, repeated applications of oxytocin induced homologous desensitization in the central nucleus of the amygdala, but not medial nucleus of the amygdala-a single application of oxytocin producing long duration suppression of responses. This indicates that oxytocin has contrasting modes of action in the amygdala. Studies made across the reproductive cycle showed that lactating animals exhibited a larger proportion of oxytocin-responsive neurones in the medial nucleus of the amygdala and a smaller proportion in the central nucleus of the amygdala, compared with virgin or pregnant animals, indicating a peripartum shift in relative activation within the amygdala. However, changes in responses were not accompanied by changes in the density of oxytocin binding sites. These data show that oxytocin has a markedly different efficacy on neuronal activation in the central and medial sub-nuclei of the amygdala. The relative shift in excitatory responses between these two nuclei may underlie some of the neuroendocrine, behavioral and anxiolytic effects which have been ascribed to oxytocin in the periparturient rat.

Differential excitatory responses to oxytocin in sub-divisions of the bed nuclei of the stria terminalis.

The lateral dorsal nucleus of the bed nuclei of the stria terminalis (BST-LD) expresses dense oxytocin binding while lower binding is detected in the medial anterior BST (BST-MA) and adjacent ventrolateral septum (VLS). However, in vitro examination of neuronal responses to oxytocin showed that the BST-LD exhibited small, transient responses which desensitized upon repeated challenge. In contrast, the BST-MA and VLS exhibited significantly larger responses with no significant desensitization. This inverse relationship between oxytocin binding density and electrophysiological responsiveness is also seen in the central and medial amygdaloid nuclei, which have respective associations with the lateral and medial divisions of the BST. Thus, excitatory responses to oxytocin vary markedly between BST sub-divisions and may reflect associations within the extended amygdala.

Randomized, double-blind, placebo-controlled, short-term trial of ropinirole in restless legs syndrome.

BACKGROUND AND PURPOSE: Restless legs syndrome (RLS) is a condition characterized by an urge to move the legs, usually accompanied by lower limb paresthesias. These symptoms worsen at rest, are relieved by activity, and are worse at night. Previous studies have suggested that dopaminergic drugs such as L-dopa and dopamine agonists, as well as benzodiazepines and opioids, can treat RLS successfully. The purpose of this study was to test the clinical efficacy of ropinirole, a D2/D3 agonist, in the treatment of RLS in a double-blind, short-term, placebo-controlled clinical trial. PATIENTS AND METHODS: After undergoing successful open-label titration and dose adjustments with ropinirole for RLS symptoms over a period of 4 weeks, 22 RLS patients (mean age=50.8; mean duration of symptoms=26.1 years) were randomized to receive either placebo (n=13) or ropinirole (n=9) for 2 additional weeks. Outcome measures included assessment of periodic leg movements in sleep (PLMS) recorded with nocturnal polysomnography and RLS symptoms as assessed with the International Restless Legs Syndrome Study Group (IRLSSG) Rating Scale. Secondary outcomes included sleep macroarchitecture. RESULTS: Results indicated that relative to placebo, ropinirole, at a mean dose of 1.4mg HS significantly decreased PLMS and RLS symptoms. Sleep macroarchitecture did not change. Side effects were typical of all dopamine agonists and were dose related. The majority of patients elected to continue treatment with ropinirole upon study completion. CONCLUSIONS: Ropinirole successfully treated long-standing RLS and can be considered a viable short-term treatment for this condition.

Central oxytocin administration reduces stress-induced corticosterone release and anxiety behavior in rats.

Endocrine responses to noise stress and anxiety-related behaviors were measured in groups of ovariectomized, estradiol-treated female rats given central infusions of oxytocin. Control animals receiving isotonic saline showed a large increase in plasma corticosterone concentrations in response to 10 min of white noise. This response to noise stress was significantly and dose dependently decreased by oxytocin administered intracerebroventricularly at 10 or 100 ng/h for 5 days. Oxytocin also significantly decreased rearing behavior during this stress. When a second noise stress was given 3 days after cessation of oxytocin infusion, corticosterone responses did not differ between the control and previously oxytocin-infused animals. Administration of vasopressin had no significant effect on either the corticosterone or behavioral responses to noise stress. Anxiety-related behaviors were measured on the elevated plus-maze. No significant differences were seen in maze exploration between saline- and oxytocin-treated animals when housed and tested in the same environment. However, when animals were mildly stressed by testing in an unfamiliar environment, oxytocin-treated animals showed a higher proportion of open arm entries and spent significantly more time in the open arms of the maze. Thus, oxytocin exerts a central anxiolytic-like effect on both endocrine and behavioral systems and could play a role in moderating behavioral and physiological responses to stress.

Intracellular recordings from bovine anterior pituitary cells: modulation of spontaneous activity by regulators of prolactin secretion.

The electrophysiological properties of cultured bovine anterior pituitary cells were examined using intracellular recordings. Although all cells had high input resistances (mean 332 M omega), membrane potentials (Vm) showed a wide distribution (-32 to -80 mV). In 9 cells with low Vm (mean -38 mV) spontaneous Ca2+-dependent action potentials were observed (mean frequency 3.4 hertz). In the majority of silent cells (mean Vm -58 mV), application of TRH caused a reduction in membrane resistance and a transient hyperpolarization of Vm. This initial response was followed by a small, sustained depolarization. In 4/18 cells this depolarization led to the appearance of action potentials in previously silent cells. In 15/18 cells application of dopamine caused an increase in the membrane conductance and led to a sustained hyperpolarization of 30-35 mV. The response reversibly blocked the propagation of action potentials in spontaneously active cells. This effect could also be reversed by the dopamine receptor antagonists, sulpiride and haloperidol. In all cells, elevating the extracellular potassium concentration caused a concentration-dependent depolarization and decrease in the membrane input resistance. In some cells this effect was associated with an increased frequency of action potentials. Electrophysiological responses to TRH, dopamine, and elevated potassium were correlated with changes in the release of PRL. It is concluded that by their effects on Vm these factors modulate spontaneous electrical properties and may regulate the entry of calcium necessary for hormone secretion from lactotrophs.

The pulsatile characteristics of hypothalamo-pituitary-adrenal activity in female Lewis and Fischer 344 rats and its relationship to differential stress responses.

The dynamic patterns of basal and stimulated hypothalamo-pituitary-adrenal (HPA) activity of freely moving female Lewis and Fischer 344 rats were compared using an automated blood-sampling system. Both strains showed pulsatile corticosterone release throughout the 24 h cycle. Lewis rats showed clear circadian variation in both pulse frequency (8.4 +/- 0.4 pulses between 1700-2300 h vs. 5.3 +/- 0.8 pulses between 0500-1100 h; P < 0.05) and height (198 +/- 27 ng/ml between 1700-2300 h vs. 107 +/- 14 ng/ml between 0500-1100 h; P < 0.05). Fischer rats exhibited pulses of similar frequency and height to those in Lewis rats during the evening, but showed no circadian variation, resulting in higher mean daily corticosterone concentrations. Although both strains showed behavioral and HPA responses to white noise stress (10 min; 114 dB), Fischer rats showed much greater increases in total activity, grooming, and rearings, and two important differences in the corticosterone responses were observed. First, in Lewis rats a clear relationship existed between basal and stimulated HPA activities, in that a significant response was seen only when the stress coincided with the rising (secretory active) phase of a basal pulse. Noise stress coinciding with a falling (nonsecretory) phase elicited no significant response. In contrast, Fischer rats showed similar responses regardless of the underlying pulse phase. Second, after the peak response at 20 min (Lewis, 237 +/- 67 ng/ml; Fischer, 390 +/- 57 ng/ml), corticosterone levels fell rapidly in Lewis rats, but remained maximally elevated for 20 min in Fischer rats, resulting in a significantly greater integrated response. The corticosterone response to i.v. CRF was unaffected by pulse phase in both strains, suggesting that a suprapituitary mechanism mediates the phase-dependent response to stress in the Lewis strain. CRF-induced corticosterone levels rose more rapidly in Fischer rats, peaking at 10 min (473 +/- 95 ng/ml) compared with 30 min (390 +/- 75 ng/ml) in Lewis rats, suggesting greater pituitary sensitivity in this strain. Thus, differences in both central and pituitary control of the HPA axis contribute to the strain difference in stress responsiveness between female Lewis and Fischer rats.

Ultradian rhythm of basal corticosterone release in the female rat: dynamic interaction with the response to acute stress.

The present study investigated the dynamic regulation of the hypothalamo-pituitary-adrenal axis and its significance to acute stress responsiveness in the female rat. An automated, frequent blood-sampling technique allowed the circadian rhythm of corticosterone to be resolved into a series of pulses. These were equally distributed (mean interval, 50.9 +/- 3.7 min) throughout the 24-h cycle, but their magnitude varied significantly, being higher between 1800-2200 h (137 +/- 9 ng/ml) than between 0600-1000 h (75 +/- 17 ng/ml). This pattern of release indicates continuous, but variable, activity of the axis throughout the day. The pulsatile ultradian rhythm suggested alternate periods of secretion and inhibition, which were found to have a profound effect on the corticosterone responses to acute stress. Noise stress (10 min, 114 decibels) evoked a transient increase in corticosterone, which reached a maximum (377 +/- 87 ng/ml) 20 min after onset. However, within this group (n = 26) the response varied depending on the underlying basal activity. When stress coincided with a rising (secretory) phase of a pulse, corticosterone concentrations rose to 602 +/- 150% of mean basal concentrations (P < 0.001). In contrast, when stress coincided with a falling (nonsecretory) phase of a pulse, a significantly smaller response, no greater than a basal pulse, was evoked. Thus, the alternate periods of secretion and inhibition generating basal hypothalamo-pituitary-adrenal activity are an important determinant of responses to acute stress.

Reduced response of the hypothalamo-pituitary-adrenal axis to alpha1-agonist stimulation during lactation.

To determine whether altered noradrenergic activation of the hypothalamo-pituitary-adrenal (HPA) axis contributes to the attenuated neuroendocrine response to stress observed during lactation, the effect of intracerebroventricular injection of the alpha1-agonist methoxamine (100 microg) was compared between virgin and lactating rats. Virgin rats showed significant increases in plasma corticosterone after methoxamine, reaching 317 +/- 44 ng/ml at 10 min and remaining significantly elevated for more than 120 min, but lactating rats showed no significant increase in corticosterone levels. Furthermore, methoxamine induced an increase in paraventricular nucleus (PVN) CRF messenger RNA expression in virgin, but not lactating, animals. Both groups of rats exhibited comparable elevations in plasma PRL after methoxamine treatment. Arginine vasopressin messenger RNA expression within the parvocellular PVN was greater in the lactating animals than in the virgin controls, but methoxamine injection was without further effect. Studies performed on ovariectomized virgin rats and ovariectomized rats receiving estradiol or progesterone replacement failed to reproduce the attenuated HPA responses seen after methoxamine treatment, although methoxamine-induced PRL levels were greatly increased by estradiol, probably arising from an effect on hormone synthesis. In vitro electrophysiological recordings of PVN neurons in hypothalamic slices from proestrous virgin and lactating rats showed that 45-52% of neurons in both groups exhibited excitatory responses to 10(-4) M methoxamine, but there was a differential response to 10(-5) M methoxamine, with PVN neurons from lactating animals failing to show a response. These data show a selective down-regulation of alpha1-mediated activation of the HPA axis in lactating animals. This may contribute to the attenuated stress-induced activation of the HPA axis during lactation.

Lack of effect of protein deprivation-induced intrauterine growth retardation on behavior and corticosterone and growth hormone secretion in adult male rats: a long-term follow-up study.

To further define the neuroendocrine consequences of intrauterine growth retardation (IUGR), we have used a rat model of maternal protein restriction throughout pregnancy to examine the pattern of corticosterone and GH secretion under basal conditions and in response to psychological stress in male offspring at 4, 9, and 18 months of age. The findings were correlated with studies of behavioral activity. Despite a consistent reduction in birth weight and failure of catch-up growth, there were no significant differences in GH secretory profiles between IUGR and control rats at any age. We were unable to demonstrate a difference in the number, amplitude, length, or area of corticosterone secretory pulses between control and IUGR animals; although again, there was a significant decrease with age. The mean peak plasma concentration of corticosterone in response to a noise stress also declined with age but was unaffected by IUGR. There were no consistent, statistically significant differences in behavioral responses between normal control and IUGR animals or between groups of animals at different ages. These results do not, therefore, support the presence of major functional abnormalities in either GH or corticosterone secretory responses in adult male rats subjected to IUGR.

Acute and chronic effects of corticosterone on 5-HT1A receptor-mediated autoinhibition in the rat dorsal raphe nucleus.

Corticosteroid modulation of 5-HT(1A) receptor function may contribute to the aetiology of affective disorders. To examine this modulation, the effects of acute and chronic corticosterone administration on 5-HT(1A) autoreceptor function were investigated using in vitro electrophysiology in the rat dorsal raphe nucleus (DRN). The magnitude and time course of the inhibitory response to a submaximal dose of 5-HT was not affected by acute application of either corticosterone (30-200 nM) or dexamethasone (100 nM) in vitro, when tested either in slices from control rats or rats adrenalectomised two weeks prior to recording. For chronic treatment, rats were supplied with drinking water containing corticosterone (50 microg/ml) or ethanol vehicle (0.5%) for 25-31 days. The autoinhibitory response to 5-HT was significantly attenuated in the corticosterone-treated group; vehicle EC(50)=48+/-8 microM vs. corticosterone EC(50)=121+/-20 microM. Furthermore a subpopulation of 5-HT neurones from corticosterone-treated animals exhibited marked insensitivity to 5-HT. In situ hybridisation histochemistry showed that corticosterone did not affect the expression of mRNA encoding the 5-HT(1A) receptor or either the type 1 and type 3 subunits of the G-protein linked inwardly rectifying K+ (GIRK) channel. However, GIRK2 subunit mRNA expression was significantly reduced. Thus, 5-HT(1A) autoreceptor function in the DRN is attenuated following chronic, but not acute, exposure to elevated corticosterone levels, and this effect may involve changes to the receptor-effector coupling mechanism.

5-HT1A receptor-mediated autoinhibition does not function at physiological firing rates: evidence from in vitro electrophysiological studies in the rat dorsal raphe nucleus.

5-HT(1A)-mediated autoinhibition of neurones in the dorsal raphe nucleus (DRN) is considered to be the principal inhibitory regulator of 5-HT neuronal activity. The activation of this receptor by endogenous 5-HT was investigated using electrophysiological recordings from the rat DRN in vitro. At a concentration which blocked the inhibitory effect of exogenous 5-HT, the 5-HT(1A) antagonist WAY 100635 did not alter basal firing rate or modulate the excitatory response to the alpha(1)-agonist phenylephrine. Blockade of 5-HT reuptake by a concentration of fluoxetine, which enhanced the inhibitory effect of exogenous 5-HT, lowered phenylephrine-induced basal firing presumably due to potentiation of the effect of endogenous 5-HT. However, this effect was not firing rate dependent and neither the proportional increase nor the time-course of the response to a higher concentration of phenylephrine were altered in the presence of fluoxetine. These data suggest that the inhibitory 5-HT(1A) receptor on raphe neurones is neither tonically activated nor plays any role in modulating the response to excitatory transmitters. Thus, at physiological firing rates this receptor does not appear to function as an autoreceptor of serotonergic neurones of the DRN.

Chronic treatment with oestradiol does not alter in vitro LTP in subfield CA1 of the female rat hippocampus.

Population excitatory post-synaptic potentials (pEPSPs) were recorded in vitro from subfield CA1 of the hippocampus of female rats which had been ovariectomized and treated for 14 days with either oil or 17beta-oestradiol (10 microg/day). The currents applied to the Schaffer collateral-commissural input necessary to induce threshold, maximum and 50% maximum pEPSP responses did not differ between groups. Application of trains of pulses (0.1-1 s; 100 Hz) evoked post-tetanic and long-term (> 60 min) potentiation of pEPSP responses, the magnitude of which was related to stimulus duration in both groups. However, the degree of potentiation induced by near-threshold (0.1, 0.15 and 0.2 s) and saturating (1 s) stimuli did not differ between groups. Thus, despite reports that oestradiol can modulate synaptic spine density and glutamatergic and GABAergic components of the inputs to CA1, these data suggest that chronic oestradiol treatment has no effect on either the excitability or induction of LTP in the Schaffer collateral-commissural-CA1 pathway.

Oxytocin-containing pathway to the bed nuclei of the stria terminalis of the lactating rat brain: immunocytochemical and in vitro electrophysiological evidence.

Immunocytochemical staining within the forebrain of lactating rats revealed oxytocin-immunoreactive perikarya in a continuum running from the anterior parvocellular hypothalamic paraventricular nucleus through the anterior commissural nucleus and perifornical region. Beaded axons could be seen arising from these perikarya to enter the bed nuclei of the stria terminalis. In sections cut at a 45 degree angle to the parasagittal plane, much of this pathway could be maintained intact, and in vitro tissue slices prepared in this orientation were used for electrophysiological studies of oxytocinergic innervation of the bed nuclei. By extracellular recording, neurons of the bed nuclei of the stria terminalis were tested for their response to exogenous oxytocin and to stimulation of the paraventricular hypothalamus. Both short latency (3-40 ms) orthodromic excitation (26/78 neurons) and longer latency (greater than 100 ms) excitation (12/78 neurons) were observed following paraventricular hypothalamic stimulation, possibly representing mono- and polysynaptic inputs, respectively. Removal of extracellular Ca2+ blocked these orthodromic responses (n = 6). Antidromic invasion was seen in a further 11/78 neurons with characteristics of constant latency (mean = 5.9 +/- 0.7 ms), high frequency following (40-80 Hz) and persistence in Ca(2+)-free medium. When tested for the effect of oxytocin (10(-7) M), none (0/11) of the antidromically activated neurons were excited, but nine of 34 of the orthodromically excited neurons (both short and long latency) responded with a marked increase in activity. In three of eight cases, the orthodromic synaptic excitation following hypothalamic stimulation could be reversibly attenuated by the receptor antagonist [d(CH2)5,D-Tyr(OEt)2,Val4,Cit8]-vasopressin (0.5 or 2.5 x 10(-6) M), further substantiating the involvement of oxytocin. These data provide anatomical and electrophysiological evidence for an oxytocinergic innervation of the bed nuclei of the stria terminalis. This pathway is discussed in terms of possible involvement in mediating the facilitatory effect of oxytocin on the milk-ejection reflex of lactating rats which has been suggested to act through this part of the limbic system.

Voltage-activated currents through calcium channels in normal bovine lactotrophs.

The properties of whole cell Ba2+ currents were studied in immunocytochemically identified, normal bovine lactotrophs using the patch clamp technique. In the current clamp mode, current-induced and spontaneous Ba2+ action potentials were recorded. These were of longer duration and showed less inactivation with stimulation frequency when compared with Na+ action potentials. Under voltage clamp, isolated Ba2+ currents had an activation threshold of about -35 mV and peak value at -15 mV to +20 mV. Inactivation of the current to a potential-dependent, non-zero steady-state level indicated the presence of one rapidly and one slowly inactivating component to the current. These two components were also distinguished by: (1) the voltage dependence of the inactivation time constant of the current, (2) the differential frequency-dependent inactivation of the peak and steady-state currents, and (3) the presence of two half-inactivation potentials for the current. Analysis of the ensemble current variance of the non-inactivating component gave a single-channel amplitude of 0.19 pA at 0 mV and a slope conductance of 3 pS. Fluctuation analysis of the voltage-activated Ba2+ current noise revealed two time constants, one which was voltage dependent and the other was independent of potential. The contribution of these two currents to Ca2+-dependent hormone secretion remains to be clarified.

Circadian rhythm of neuronal activity in suprachiasmatic nucleus slices from the vasopressin-deficient Brattleboro rat.

In vitro extracellular recordings were made from tissue slices of suprachiasmatic nucleus from homozygous Brattleboro rats which are deficient in vasopressin. A high proportion (56%) of neurons were excited by application of exogenous vasopressin, indicating that the V1 receptors expressed by these neurons were functional. Basal activity of these vasopressin-sensitive neurons showed a marked circadian variation (higher during the subjective light phase) while vasopressin-insensitive neurons showed no significant variation, suggesting the presence of the V1 receptor identifies a population of highly circadian neurons. Suprachiasmatic neurons from both homozygous rats and their heterozygous (vasopressin-containing) litter mates displayed a circadian rhythm of spontaneous (basal) activity, with firing rates declining during the subjective dark phase, indicating that the endogenous pacemaker driving the circadian rhythm was not dependent upon the presence of vasopressin. However, the peak of spontaneous activity displayed during the subjective light phase was significantly lower in the vasopressin-deficient animals. These data show that the presence of endogenous vasopressin within the suprachiasmatic nucleus is not necessary for the generation of the circadian pattern of activity. However, vasopressin does function to amplify the rhythm by its excitatory effect during the light phase.

Oxytocin release evoked by electrical stimulation of the medial forebrain in the rat: analysis of stimulus parameters and supraoptic neuronal activity.

The role of the medial forebrain area (vertical limb of the diagonal band, medial septum and medial nucleus accumbens) in the control of oxytocin secretion in lactating rats was investigated. Electrical stimulation of the medial forebrain evoked a reproducible rise in intramammary pressure, equivalent to that caused by i.v. injection of 1 mU oxytocin. No pressor effect accompanied this response. Radioimmunoassay of plasma samples showed that stimulation caused a significant rise in the concentration of circulating oxytocin. The effects of changing the parameters of stimulation to the medial forebrain were compared with those evoked by stimulation of the neural stalk. The optimal frequency for stimulation of the forebrain was found to be four-fold lower (10-20 Hz) than that for stimulation of the neural stalk (50 Hz). During continuous prolonged stimulation of the forebrain (20 Hz; 2 min) only a single transient response was obtained, whereas a protracted response was obtained as a result of prolonged stimulation of the stalk. Recordings were made from antidromically identified neurosecretory cells in the supraoptic nucleus. Electrophysiological responses to electrical stimulation of the medial forebrain were characterized by two main features. (1) Single-pulse stimulation produced only a small excitation (one or two action potentials), while high-frequency trains produced a profound facilitation of this response, with each pulse evoking short-duration 'bursting' behaviour in the supraoptic neurons. (2) During long trains of stimulation this frequency-dependent facilitation declined and could only be renewed after a period of rest.(ABSTRACT TRUNCATED AT 250 WORDS)

Changing pattern of oxytocin-induced excitation of neurons in the bed nuclei of the stria terminalis and ventrolateral septum in the peripartum period.

Oxytocin acts within the limbic system (bed nuclei of the stria terminalis and ventrolateral septum) to induce maternal behaviour and to facilitate neuroendocrine activity at specific times during the peripartum period. Studies were undertaken to determine whether the timing of these effects arises from modulation of the oxytocin-induced excitation of limbic neurons. Extracellular activity of single units was recorded on urethane-anaesthetized rats and neurons were tested for responses to intracerebroventricular injection of 1.1 ng oxytocin. In the first part, animals were recorded on days 19 and 22 of pregnancy and on days 3 and 5 of lactation. No significant differences in the basal firing rates or in the proportion of oxytocin-responsive neurons were detected, but responses by neurons on day 22 of pregnancy occurred after a significant delay (10.7 +/- 2.0 min), resulting in a smaller overall response compared to the other groups. These differences in the pattern of response were not due to changes in density of oxytocin binding in the limbic areas studied, since autoradiographic detection of oxytocin binding sites using the iodinated antagonist [125I]d(CH2)5[Tyr(Me)2, Thr4, Orn3, Tyr-NH2(9)]-vasotocin showed no differences between the pregnant and postpartum animals. In the second part, parturient animals (day 22 of pregnancy) received intravenous injection of the long-acting opioid antagonist naltrexone, or unilateral knife-cut lesions to the stria terminalis, a source of inhibitory inputs (including enkephalinergic) to the bed nuclei of the stria terminalis and ventrolateral septum. Both treatments abolished the characteristic delay of oxytocin-induced excitation in non-treated animals on day 22 of pregnancy, and increased the overall excitatory response. Thus, during the peripartum period, a population of limbic neurons sensitive to oxytocin display a dynamically changing pattern of excitatory responses, apparently modulated by an endogenous opioid cone and independent of changes in oxytocin receptor expression. The attenuated neuronal response to central oxytocin seen on the day of parturition could account for the absence of a facilitatory effect of oxytocin on neuroendocrine activity at this time.