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.

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.

Effect of gonadal steroids on the oxytocin-induced excitation of neurons in the bed nuclei of the stria terminalis at parturition in the rat.

Experiments were undertaken to examine the role of ovarian steroids in peripartum programming of oxytocin sensitivity of limbic neurons implicated in oxytocin-induced facilitation of the milk-ejection reflex. In vivo recordings of neurons in the bed nuclei of the stria terminalis and ventrolateral septum of pre-parturient rats which had undergone prior ovariectomy and hysterectomy showed that oestradiol significantly increased the excitatory responses of bed nuclei/ventrolateral septum neurons to intracerebroventricular oxytocin, compared to oil-treated controls. Oestradiol also increased the excitation of bed nuclei neurons to the selective oxytocin agonist, [Thr4,Gly7]oxytocin in brain slices from steroid pre-treated ovariectomized hysterectomized rats, so that both the proportion of responsive neurons, and the magnitude of their responses were significantly increased. Parallel autoradiographic studies showed that oxytocin binding in the medial bed nuclei and ventrolateral septum was selectively increased following oestradiol treatment. Progesterone pre-treatment had no effect on either oxytocin sensitivity of bed nuclei/ventrolateral septum neurons recorded in vivo, or on oxytocin binding in the medial bed nuclei and ventrolateral septum, compared to oil-treated controls. Mean responses to [Thr4,Gly7]oxytocin in bed nuclei neurons recorded in slices from progesterone-treated rats were larger than controls, but this effect was highly variable. These results demonstrate that oestradiol greatly enhances oxytocin receptor expression and sensitivity of bed nuclei/ventrolateral septum neurons to oxytocin over the peripartum period, consistent with involvement of this steroid in enhancing oxytocin regulation of neuroendocrine and behavioural adaptations required for lactation.

Electrophysiological effects of oxytocin within the bed nuclei of the stria terminalis: influence of reproductive stage and ovarian steroids.

The bed nuclei of the stria terminalis (BNST) is a target site for the central actions of oxytocin (OT) in promoting behavioural and neuroendocrine responses involved in female reproduction, and binding studies suggest that OT sensitivity may be modulated over the peripartum period. Electrophysiological recordings from brain slices in vitro showed that OT sensitivity of BNST neurones is relatively low in late pregnancy, but is high during lactation. In vivo studies over the immediate peri-partum period revealed that although BNST neurones can be excited by i.c.v. OT at day 22 of pregnancy, there is a 5-10 min delay in their response which is not present in lactation. This delay can be reversed by naltrexone, or lesioning the stria terminalis, and may involve an inhibitory opioid input to the BNST from the amygdala. Examination of the role of steroids in regulating OT responses of BNST neurones showed that oestradiol pre-treatment in late pregnant ovariectomized rats increased OT excitation of BNST neurones in vitro, and a similar result was observed with in vivo recordings. Progesterone also augmented OT excitation of BNST neurones in vitro, but no such effect was observed in vivo. This difference could indicate that an additional effect of progesterone is to potentiate extraneous inhibitory inputs to the BNST, or may reflect the ability of this steroid to suppress OT sensitivity by a direct membrane action. Changes in the response of BNST neurones to OT may have functional implications for the action of central OT in facilitating the neuroendocrine milk-ejection reflex (i.e. increasing milk-ejection frequency), an effect which first appears at around day 3 of lactation. Studies involving steroid treatment of late pregnant ovariectomized rats showed that this facilitatory mechanism can be induced to appear early (i.e. on day 22 of pregnancy) by oestradiol, but not progesterone treatment. Collectively, these results support this view, that the action of OT in the BNST is regulated by the changing levels of steroids towards the end of pregnancy, thereby ensuring appropriate neuroendocrine responses necessary for motherhood.

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.

Efferent connections from the anterior pretectal nucleus to the diencephalon and mesencephalon in the rat.

The anterior pretectal nucleus has been described as part of the visual pretectal complex. However, several electrophysiological and behavioural studies showed that this area is involved in somatosensory modulation, more specifically, antinociception. The efferents of the anterior pretectal nucleus have not been identified taking into account the different function of this nucleus in relation to the rest of the pretectal complex. In the study herein described, a sensitive anterograde tracer Phaseolus vulgaris leucoagglutinin was used to trace the mesencephalic and diencephalic efferents of the anterior pretectal nucleus in the rat. The majority of the connections were ipsilateral. Fibres with varicosities were observed in discrete areas of the thalamus (central lateral, posterior complex), hypothalamus (lateral, posterior and ventromedial), zona incerta, parvocellular red nucleus, intermediate and deep layers of the superior colliculus, central grey, deep mesencephalon, pontine parabrachial region, and pontine nuclei. Fibres en passant were detected in the medial lemniscus, from the level of the injection site to rostral medullary levels. Some labelled axons were seen coursing to the contralateral side through the posterior commissure and the decussation of the superior cerebellar peduncle. These results show that the anterior pretectal nucleus projects principally to areas involved in somatosensory and motor control in a manner that permits sensory modulation at higher and lower levels of the brain. These connections may explain the antinociceptive and antiaversive effects of stimulating the anterior pretectal nucleus in freely moving animals.

Anterior pretectal nucleus facilitation of superficial dorsal horn neurones and modulation of deafferentation pain in the rat.

1. Functional relationships between the anterior pretectal nucleus (APTN) and nociceptive dorsal horn neurones were investigated electrophysiologically in the anaesthetized rat. The effects of APTN lesions were assessed behaviourally in a model of deafferentation pain. 2. Cells in the dorsal and rostral parts of the APTN were excited orthodromically by electrical stimulation of the ipsilateral dorsolateral funiculus or the contralateral dorsal columns, and by noxious and innocuous cutaneous stimuli. 3. Electrical stimulation of the APTN excited nociceptive lamina I spinal neurones. These cells all projected rostrally in the contralateral dorsolateral funiculus. Identical APTN stimulation also inhibited multireceptive spinal neurones which lay deep in the dorsal horn. These particular cells were shown to project to the brain in the ventrolateral funiculus. 4. It is proposed that noxious stimuli excite spinal lamina I projection neurones which send excitatory axons to the brain, including the APTN. The APTN inhibits deep multireceptive neurones, to reduce the perception of noxious stimuli. The discharge of spinal lamina I neurones, however, will be sustained by the noxious stimulus and by facilitation from the APTN. A sustained descending inhibition of this nature would reduce responses to prolonged injury. 5. The involvement of the APTN in responses to a chronic pain state was examined by comparing the behaviour of animals with bilateral lesions of the APTN with normal controls. Lesions of the APTN strongly enhanced the autotomy behaviour triggered by sectioning of the dorsal roots. 6. These observations support the suggestion that the APTN reduces the debilitating effects of prolonged injury.

Direct projections from the anterior pretectal nucleus to the ventral medulla oblongata in rats.

The anterior pretectal nucleus has recently been implicated in the descending modulation of nociception. Electrical stimulation of the nucleus was found to reduce the nociceptive responses of deep dorsal horn neurons and to inhibit spinally integrated withdrawal reflexes. It is believed that at least part of the descending inhibitory effects of the anterior pretectal nucleus are mediated by reticulospinal cells of the ventrolateral medulla. The purpose of the present study was to trace the direct medullary projections of the anterior pretectal nucleus, to describe their topographical organization and to reveal the chemical nature of some of their putative target cells. The connections were studied using anterograde tract-tracing with Phaseolus vulgaris leucoagglutinin. Direct projections from the anterior pretectal nucleus to the ipsilateral rostral ventral medulla were found in all cases. A dense innervation of the dorsal inferior olive, the gigantocellular reticular nucleus pars ventralis and pars alpha and the ventral pontine reticular nucleus was found from all aspects of the anterior pretectal nucleus. Descending labelled terminals were also observed in the gigantocellular reticular nucleus proper and, laterally, in the lateral paragigantocellular nucleus and in the region of the A5 noradrenergic cell group. A relatively lower density of labelled terminals was noted in the medullary raphe nuclei and in the rostroventrolateral reticular nucleus. Following tract-tracer injections into five distinct subregions of the anterior pretectal nucleus, the topographical organization of the projection was examined and the relatively highest density and most widespread projection was found to originate from the caudoventral part of the anterior pretectal nucleus. A combined tract-tracing and immunolabelling study revealed that some of the descending, labelled terminals were in close proximity of tyrosine hydroxylase-immunoreactive dendrites in the C1 and A5 cell groups. Some labelled fibres were also noted among the serotonin-immunoreactive cells in the lateral extension of the B3 cell population. The existence of direct projections to the ventral medulla and pons correlates well with physiological data which showed that the descending, antinociceptive effects of the anterior pretectal nucleus are relayed via the rostral ventrolateral medulla. The data are also in keeping with pharmacological studies that suggested the role of catecholaminergic cells in the mediation of these descending effects. It is proposed that the rostral ventral medullary projections provide a path through which antinociceptive effects of the anterior pretectal nucleus are mediated to the spinal cord.

A combined immunocytochemical and retrograde tracing study of noradrenergic connections between the caudal medulla and bed nuclei of the stria terminalis.

Region-specific noradrenergic inputs to the bed nuclei of the stria terminalis (BST) from the caudal medulla were studied using combined Fast Blue injections and tyrosine hydroxylase immunoreactivity (TH-ir). Injections into the rostral, dorsal, ventral and lateral BST resulted in predominantly ipsilateral retrograde labelling restricted to the mediodorsal and ventrolateral caudal medulla. Mediodorsal projecting neurones comprised the A2 TH-ir and a second non-aminergic group medial to A2. All ventrolateral retrogradely labelled neurons showed TH-ir and corresponded to A1. Injections into the caudal BST did not label the A2 and very few A1 neurones, indicating a paucity of noradrenergic inputs from this area of the medulla.

Influence of reproductive state and ovarian steroids on facilitation of the milk-ejection reflex by central oxytocin.

In the rat, the synchronous bursting activity of oxytocin neurones associated with the milk-ejection reflex displays important changes during the peri-partum and lactational periods. The most dramatic of these changes is the appearance of a facilitatory response to centrally-administered oxytocin, involving an increase in the frequency and amplitude of bursting in the oxytocin neurones, as well as elevation of their background activity. Studies of rats at different times in the pre- and post-partum period show that this response first appears on day 3 of lactation. Ovariectomy on day 21 of gestation, or treatment with the anti-oestrogen tamoxifen on day 22, does not prevent the appearance of this response. However, ovariectomy and treatment with ovarian steroids for 3 days prior to parturition can dramatically alter the character of the facilitatory response. Oestradiol treatment causes an early (pre-partum) appearance of the facilitatory response, whereas progesterone causes the appearance of an inhibitory response (reduction in milk-ejection frequency) to central oxytocin. A major target for the central effects of oxytocin are the bed nuclei of the stria terminalis (BST) and modulation of the neuronal responses in this region may, in part, underlie the changing facilitatory effects. In vitro recordings indicate that sensitivity of BST neurones to oxytocin is increased between pregnancy and lactation, and oestradiol treatment enhances responsiveness coincident with the appearance of a facilitatory response. Progesterone pre-treatment also increases the ability of BST neurones to respond to oxytocin in vitro (although less than oestradiol), an unexpected result given the absence of oxytocin-induced facilitation of the milk-ejection reflex in late pregnancy or following progesterone treatment in vivo. In vivo recordings of BST neurones suggest that one explanation of this lack of correlation may reside in the presence of a mechanism which attenuates the excitatory response to oxytocin, perhaps serving to prevent premature expression of the facilitatory action of oxytocin. Collectively, these data show that there are dramatic reproductive state and steroid-dependent changes in the central action of oxytocin on the synchronous bursting of magnocellular oxytocin neurones. These changes, which have important consequences for the optimization of bursting in oxytocin neurones, may involve plasticity of transduction mechanisms in the oxytocin-responsive elements of the limbic system.

Mortyn Jones Memorial Lecture. Limbic regions mediating central actions of oxytocin on the milk-ejection reflex in the rat.

Central oxytocin administration has a profound facilitatory effect on the patterning of the milk-ejection reflex in the lactating rat. Lesion and microinjection studies indicate that this action is, in part, mediated via a population of limbic neurones in the bed nuclei of the stria terminalis and ventrolateral septum, which have been shown to possess oxytocin receptors and to be activated by selective oxytocin-receptor agonists in vitro. In vivo electrophysiological recordings reveal that some of these neurones display cyclical activity which is highly correlated to each milk ejection, and are rapidly activated following i.c.v. administration of oxytocin, coincident with the facilitation of milk ejection activity. A hypothetical model is proposed in which this population of limbic neurones serves to gate the activity of a pacemaker which, in turn, coordinates the bursting of hypothalamic magnocellular neurones. The oxytocin innervation of these neurones and their expression of oxytocin receptors increases in the postpartum period, and the resultant enhanced sensitivity leads to a greater facilitatory response during lactation. Inhibitory opioid and noradrenergic inputs which converge on these oxytocin-sensitive neurones may function to switch off the facilitatory circuit during periods of stress. Thus, this population of limbic neurones participates in the regulation of neuroendocrine activity during lactation by providing an appropriate degree of feedback to alter the patterning of the milk-ejection reflex.

The pontine parabrachial region mediates some of the descending inhibitory effects of stimulating the anterior pretectal nucleus.

Electrical stimulation of the anterior pretectal nucleus (APtN) elicits antinociception by inhibiting the responses of spinal multireceptive neurones to noxious stimuli. This descending inhibition is mediated, in part, by activating cells in the ventrolateral medulla. Neuronal tract tracing has previously shown that the APtN also projects directly to the pontine parabrachial region (PPR). The PPR, investigated by Katayama et al. (Brain Res., 296 (1984) 263-283), corresponds to the cholinergic cell group Ch5 of Mesulam et al. (Neuroscience, 10 (1983) 1185-1201). In this study, the pathway from APtN to PPR was investigated using urethane anaesthetised rats. Electrical stimulation (single square wave 0.2 ms pulses, 1-10 V, 5 Hz) of the APtN potently excites 40% of the cells recorded in the PPR. In the reverse experiment, stimulation of the PPR at the same parameters excited 36% of the cells recorded in the APtN. The contribution of this pathway to the spinal inhibitory effects of APtN stimulation was then examined. Unanaesthetised animals received electrical stimulation to the APtN (35 microA r.m.s., 15 s) and the increase in tail-flick latencies was measured. Bilateral electrolytic lesions of the PPR caused a 67% reduction of the antinociceptive effect of APtN stimulation. In urethane anaesthetised rats, microinjection of tetracaine into the PPR blocked the inhibition of multireceptive dorsal horn neurones caused by APtN stimulation (20 s train of 50 microA square wave 0.1 ms pulses, 100 Hz). In conclusion, these experiments strongly sugget that the PPR may be an important part of a descending antinociceptive pathway originating in the APtN.

The antinociception evoked by anterior pretectal nucleus stimulation is partially dependent upon ventrolateral medullary neurones.

Electrical stimulation (35 microA rms/15 s) of the anterior pretectal nucleus (APtN) inhibits the spinal reflex of the tail-flick (TF) to noxious heat in unanaesthetised rats. APtN stimulation also reduces the nociceptive response of spinal dorsal horn neurones in halothane-anaesthetised rats. This study determined if the antinociceptive effects of APtN stimulation depended on neurones in the ventral medulla. Bilateral electrolytic lesions of the ventrolateral medulla, but not the nucleus raphe magnus, reduced by 70% the antinociceptive effect of APtN stimulation in the TF test. In rats anaesthetised with halothane, electrical stimulation of the APtN (single square wave 0.1 msec pulses, 2-20 microA, 1 Hz) excited cells in the ventrolateral medulla. These data suggest a connection between both areas. This connection is further confirmed by neuroanatomical tract tracing studies in which the retrograde dye Fast Blue was injected into the ventrolateral medulla. Fluorescent cell bodies were found in the APtN. We therefore conclude that the ventrolateral medulla is part of a descending antinociceptive pathway from the APtN.

Antinociception elicited by electrical or chemical stimulation of the rat habenular complex and its sensitivity to systemic antagonists.

The effects of intraperitoneal administration of antagonists to morphine, norepinephrine, acetylcholine, dopamine and 5-hydroxytryptamine (5-HT) have been studied on the antinociceptive effect of electrical stimulation of the rat habenular complex (HbC). The antinociceptive effect of agonists microinjected into the HbC was also examined. A 15-s period of 53 microA rms sine-wave stimulation of the HbC significantly increased the latency of the tail-flick reflex to noxious heat for periods of up to 15 min. This effect was significantly attenuated by pretreating rats with naloxone (1 mg/kg) or phenoxybenzamine (5 mg/kg). Methysergide (5 mg/kg), haloperidol (5 mg/kg), atropine (1 mg/kg), and mecamylamine (1 mg/kg) had little effect on the antinociceptive effect of HbC stimulation. L-Glutamate (3.5 and 7.0 micrograms), morphine (1.0 and 5.0 micrograms), and carbachol (0.4 and 0.8 micrograms), but not 5-HT (5 micrograms), dopamine (5 micrograms) or norepinephrine (5 micrograms), induced a dose-dependent increase in the tail-flick latency when microinjected into the HbC. The effect of carbachol was significantly attenuated in rats previously treated with intraperitoneal administration of atropine or mecamylamine and fully depressed in rats previously treated with a combination of these two cholinergic antagonists. It is concluded that antagonists of opiate receptors and alpha-adrenoceptors, but not dopamine or cholinergic receptors, reduce the antinociceptive effects of HbC stimulation. These observations differ from the reported effects of these antagonists on the antinociception caused by stimulating the periaqueductal gray, but resemble the antinociception caused by stimulating the ventrolateral medulla and locus coeruleus.(ABSTRACT TRUNCATED AT 250 WORDS)

Antinociception induced by stimulation of the habenular complex of the rat.

The changes in the tail-flick latency to noxious heat stimulation and in the threshold for defensive/affective reactions to noxious pressure of the skin were studied following electrical stimulation of the habenular complex (HbC) and adjacent brain structures in the male rat. Single brief (15 s), low intensity (53 microA r.m.s.) stimulation of the HbC caused no significant increase in the locomotor activity or motor deficit but induced a potent and short-lasting antinociception as revealed by both algesimetric tests. Animals stimulated in the HbC also displayed poor avoidance learning in a conditioned place preference paradigm, thus suggesting that aversion is unlikely to determine antinociception. Rats daily stimulated in the HbC became tolerant to the antinociception induced by HbC stimulation or to a high systemic dose of morphine (6 mg/kg i.p.). These results indicate that stimulation of the HbC may increase the thresholds of spinally and supraspinally integrated reflexes, thus supporting the hypothesis that this nucleus may play a role in pain control, probably involving an opioid-dependent mechanism.

Nuclear magnetic relaxation of 19F as a novel assay method of superoxide dismutase.

The 19F longitudinal nuclear magnetic relaxation rate (T-1(1p)) of F- solutions is greatly enhanced by copper, zinc or manganese superoxide dismutase. The measured T-1(1p) values are at least an order of magnitude, and in most cases 2 orders of magnitude, higher than those of other metal-containing proteins and low molecular weight complexes. This property is suitable for a direct, specific, and sensitive assay of superoxide dismutase, free of interference by other molecules. For mixtures of the copper, zinc and manganese enzymes, addition of CN- permits quantification of the relative amounts of each enzyme. In the case of copper, zinc enzyme, the method proved exquisitely sensitive to the native state of the active site. Results are reported to show that the 19F relaxation method can be used to assay biological fluids and crude homogenates for copper, zinc and manganese superoxide dismutases, under conditions that offer considerable advantages in comparison to other assay methods.

The binding of copper ions to copper-free bovine superoxide dismutase. Properties of the protein recombined with increasing amounts of copper ions.

1. E.p.r. (electron-paramagnetic-resonance), proton-relaxation and u.v.-absorption parameters, and enzyme activity of samples of Cu2+-free bovine superoxide dismutase recombined with different amounts of Cu2+ up to the stoicheiometric [Cu2+]/protein] ratio were investigated after attainment of equilibrium in the recovery process. 2. The e.p.r. spectra were identical with the spectrum of the native protein at all [Cu2+]/[protein] ratios. The relaxation rate of the water protons (T1) and the u.v. absorption increase as linear functions of the added Cu2+. 3. On the other hand, in recombination experiments in the range pH 7.6-10.5 the enzyme activity shows a non-linear increase as the [Cu2+]/[protein] ratio rises. The experimental curves can be interpreted in terms of the model of co-operative binding of Cu2+ to the two sites proposed on the basis of the electrophoretic analyses of the samples, and show that the specific activity of the molecules containing only one Cu2+ ion is twice as high as that of the molecules with two Cu2+ ions. 4. These results support the hypothesis of an anti-co-operative interaction between the two sites during the activity, which allows only one Cu2+ ion to function in catalysis.