Biochemical isolation and physiological identification of the egg-laying hormone in Aplysia californica.

It has been determined that the bag cells of Aplysia californica produce two polypeptide species that comigrate on electrophoretic gels containing sodium dodecyl sulfate. By this separation procedure both species can be assigned a molecular weight of approximately 6,000. One of these molecules has an Rf of 0.65 on alkaline discontinuous electrophoresis gels, an isoelectric point at pH 4.8, a gel filtration molecular weight of approximately 12,000, and has no known biological function. The other does not enter alkaline disk gels, has an isoelectric point at approximately pH 9.3, shows a gel filtration molecular weight consistent with that determined by SDS gel electrophoresis, and is the egg-laying hormone.

Precursor and product processing in the bag cell neurons of Aplysia californica.

Posttranslational processing in the biosynthesis of the egg-laying hormone (ELH) by the bag cell neurons of Aplysia californica was studied. The precursor (pro-ELH) to ELH was found to be resistant to solubilization in denaturant-free media throughout its lifetime. Its principle products show a similar insolubility for 3 h, but two of these, ca. 6,000 daltons, subsequently become readily recoverable in the low-speed supernatant of a homogenate of the cells. The remaining product shows no change in solubility characteristics. From studies employing ultracentrifugation and examination of axoplasmic transport, the solubility shift for the lower molecular weight products is interpreted to represent the liberation of secretory vesicles into the cytoplasm from larger membranous associations. This event is accompanied by, but does appear to be dependent upon, a 15% reduction in the molecular weight of one of the products. These findings are considered in the light of the extensively studied posttranslational processing regimen for the production of insulin in the pancreatic beta cell.

Effects of ACTH(1-24) on single unit activity in the brainstem of the rat.

Central administration of corticotropin-like peptides generally blocks the analgesic action of opiate ligands, yet it is unclear whether this is due to an independent hyperalgesic action of corticotropin or to some other mechanism. Single cells in the ventromedial medulla of the anaesthetized rat were characterized as either excited by noxious stimuli or inhibited. Injections of ACTH(1-24) excited cells that were excited by noxious stimuli and inhibited cells that were inhibited by noxious stimuli. The results support an independent role for corticotropin-like peptides in pain-modulating mechanisms.

A cytochemical study of the bag cell organs of Aplysia californica.

Egg-laying hormone in Aplysia californica is synthesized and secreted by cells that seem to be homogeneous ultrastructurally and electrophysiologically. Several conventional methods have been used to demonstrate histochemical homogeneity and special staining techniques based on the known properties of the hormone show the neuroendocrine organ to be uniform in appearance. Furthermore, since stain specificity for egg-laying hormone is demonstrable using release and biochemical studies, the authors concluded that the organ consists of a population of biochemically homogeneous neurons.

A peptidergic basis for sexual behavior in mammals.

Vasopressin (VP) is a peptide neurotransmitter in the limbic system of rats. It is synthesized in the medial amygdaloid nucleus in the presence of sex steroids, transported to other limbic structures such as the hippocampus and septum and secreted there by a calcium-dependent process. In the hippocampus, VP acts on cerebral microvessels and local circuit interneurons. Its excitatory action on the inhibitory interneurons produces near-total shutdown of electrical activity of the efferent fibers of pyramidal cells, the projection neurons of the hippocampus. Stimulation of the medial amygdala and release of the endogenous VP duplicates these effects and, since they are blocked by ventricular application of a VP antagonist, the effects are almost certainly mediated by endogenous VP. Recording from the VP-containing cell bodies or of the hippocampal action of the peptide indicates that the system is selectively involved with the early stages of sexual behavior, specifically those appetitive behaviors that anticipate coitus. Stimulation of the VP cells produces alterations in sexual behavior in a manner consistent with the hypothesis that the medial amygdala organizes the appetitive phase of recognition of an appropriate partner and sexual arousal. This role for the medial amygdala complements the proposed role of nearby structures in the consummatory, reward and learned aspects of sexual behavior. Association between VP, oxytocin (OT) and homologs with sexual behavior is very widespread among vertebrates, including amphibians, reptiles, primates and humans. Humans and other primates display a phenomenon called 'concealed ovulation' that may have played a role in the evolution of social structures. The review concludes with a discussion of possible experimental strategies for evaluating the possible role of VP in concealed ovulation and other conditions in which sexual behavior occurs outside of estrus.

Peptidergic transmission in the brain. III. Hippocampal inhibition by the amygdala.

The mechanism of arginine vasopressin (AVP) action in the rat hippocampus has been determined. The peptide activates inhibitory interneurons and constricts cerebral microvessels. In the whole animal, each of these direct actions has secondary consequences for the excitability of pyramidal cells. Recent studies have shown that a peptide similar to AVP mediates endogenous neurotransmission in the hippocampus. Here we report experiments showing that the endogenous peptide activates the same mechanisms as exogenously applied AVP. The endogenous AVP-like peptide has no effect on the presynaptic fiber volley, or on pure somatic and dendritic postsynaptic potentials. These results are taken to exclude presynaptic mechanisms as explanations for the peptide's action. The endogenously released peptide inhibits individual pyramidal cells in single unit recording and excites presumed interneurons, just as AVP itself is known to act. The endogenous peptide is released only by stimuli applied to a nucleus that contains immunoreactive AVP and projects to the hippocampus.

Orthodromic activation of peptidergic cells in the medial amygdala.

Electrical recordings from vasopressin-containing cells in the medial amygdala were obtained. Electrical stimulation of one major afferent structure, the accessory olfactory bulb, invariably elicited single unit discharge in the peptidergic cells and set up a field potential indicating widespread excitation in the structure. Pheromonal stimuli, normally borne into the brain by the accessory olfactory bulb, were ineffective in activating the medial amygdala. These results in combination with preexisting research suggest that the accessory olfactory bulb is an important influence, but not the only influence, on the activity of the peptidergic cells.

Peptidergic transmission in the brain. V. Systemic correlates of central activation.

Inhibition of the hippocampus by the medial amygdala is mediated by vasopressin-like peptide. Because vasopressin has action on the periphery as well as the brain, we conducted experiments to evaluate the relationship between possible peripheral actions and the central effects of the endogenous peptide. In the acutely anesthetized rat, peptide-mediated inhibition of the hippocampus is not associated with significant changes in heart rate, blood pressure or body temperature. Peripheral injections of peptide agonist fail to evoke the central inhibition, and peripheral injections of peptide antagonist fail to block the central inhibition. Stimulation of central nuclei that contain vasopressin or a similar peptide also fail to duplicate the effect of stimulating the amygdala. We conclude that the peptidergic transmission is independent of peripheral causes or correlates.

Peptidergic transmission in the brain. IV. Sex hormone dependence in the vasopressin/oxytocin system.

The medial amygdaloid nucleus (AME) synthesizes a peptide similar to arginine vasopressin and projects peptidergic fibers to the ipsilateral hippocampus in the male rat. In previous studies, we have shown that the peptide acts as a transmitter and, using in vitro and in vivo electrophysiology, we have characterized its mechanism of action. Previous anatomical work has shown that the peptidergic fibers are more dense in male rats than females and are obliterated entirely by castration. Here we report the results of our attempt to find an electrophysiological correlate of these anatomical findings. First, we show specific mediation by a vasopressin- or oxytocin-like peptide by use of a structural vasotocin antagonist. Then we show that castration obliterates the peptidergic signal in males. However, we were unable to find any sex difference that corresponded to the male/female disparity noted in the density of the peptidergic fibers. The strength, nature and stimulus-response characteristics were the same between males and females. Apart from a very subtle difference in the duration of the signal, no physiological correlate of the sexual dimorphism could be found with our techniques. We conclude that the neurophysiology partly complements the anatomy and biochemistry of this system.

Interaction between long-term potentiation and the action of a peptide transmitter.

The interaction between long-term potentiation (LTP) and the action of a peptide transmitter was examined with evoked field potential recording in the CA1 region of the hippocampus in anesthetized male rats. The peptide transmitter transiently reversed and opposed the effects of LTP, and LTP reversed the action of the peptide transmitter. Because the peptide transmitter is released and has action particularly during sexual behaviors, the results are interpreted to mean that any memory trace encoded by potentiating mechanisms in the rat hippocampus is probably not accessible in the 20-30-min period of time surrounding reproductive activity in male rats.

Peptidergic transmission in the brain. VI. Behavioral consequences of central activation.

Having described a peptidergic transmitter system in the rat brain, we now begin to evaluate its behavioral function. We stimulated cell bodies in the medial amygdaloid nucleus (AME) with indwelling bilateral electrodes. These cell bodies contain a vasopressin-like peptide and send fibers to the hippocampus where the peptide is released upon stimulation. There the peptide inhibits hippocampal output in the awake rat just as it does in the anesthetized rat and in the rat brain slice. The stimulation reorganizes behavior with the same latency and duration as the hippocampal effect. For about 15-20 minutes after the brief stimulus, rats remain motionless with eyes wide open. This "freezing" state is punctuated by episodes of exploratory behavior. The stimulus appears to have a positive affective quality. Review of the literature in light of the present results indicates a probable role for this peptidergic system in the generation of sexual behavior in male rats.

A mechanism for vasopressin action in the hippocampus.

The action of arginine vasopressin (AVP) in the rat hippocampal slice has been extensively studied. Extracellular recording indicates that the peptide excites spontaneously active neurons in the slice, though uncertainty exists regarding the identity of this cell type. Intracellular recording from pyramidal cells also reveals an excitatory action of the peptide, but these results are confounded by simultaneous constriction of small blood vessels that surround each pyramidal cell. Here we use field potential recordings to show that AVP inhibits pyramidal cell discharge and employs a pressor-type (V1) receptor to bring about its action. The results resolve issues regarding the identity of AVP targets in the slice. Each reported result is consistent with a model that posits direct AVP excitation of inhibitory interneurons and direct AVP constriction of slice microvessels. Inhibition of pyramidal cells recorded extracellularly and excitation of pyramidal cells recorded intracellularly are respective indirect consequences of the two direct effects.

ACTH1-24 blocks opiate - induced analgesia in the rat.

Samples of 1 microliter containing 15 microgram of morphine sulfate with or without 1 microgram of synthetic ACTH were injected into the fourth ventricle of rats. The inclusion of ACTH eliminated the analgesic effect of morphine as evaluated by the tail-flick test, both in restrained and in unrestrained, lightly sedated animals. The same result was obtained when beta-endorphin was used to bring about analgesia. Since the effect of the peptides was shown to be mediated by central actions alone, the results are discussed in light of the brain ACTH/beta-endorphin system.

Blood-borne factors regulating microvascular constriction in the rat hippocampal slice.

Previous work shows that blood serum contains a factor that elicits constriction of large cerebral arteries and may be responsible for cerebral vasospasm in subarachnoid hemorrhage patients. However, few studies have considered the action of potential spasmogens on intraparenchymal resistance vessels. We used a common preparation for neurophysiology, the rat hippocampal slice, to study penetrating arterioles and their response to a variety of potential vasoconstrictors. Dilute serum from clotted rat blood evoked profound microvascular constriction when applied to the slice, but plasma incubated with heparin to prevent coagulation was unable to do so. A variety of potential vasoconstrictor substances were tested to see if they could duplicate the effects of serum. Norepinephrine (10 microM), serotonin (one microM) and prostaglandin E2 (one nM) were ineffective in this regard. However, when a stable eicosanoid, thromboxane B2, was applied at a concentration achieved in the cerebrospinal fluid of subarachnoid hemorrhage patients, it duplicated a portion of the vasoconstriction produced by serum. It is concluded that thromboxane B2 may account for part of the spasmogenic property of serum and that unidentified molecules in the coagulation pathway may also play a role.

Vasoconstriction and neural excitation in response to transient hypoxia in the rat hippocampal slice.

The vascular and neural responses to transient hypoxia in the rat hippocampal slice were studied. Neural hyperexcitability produced by tissue hypoxia was associated with localized decreases in the diameter of precapillary arterioles. Vasoconstriction occurred periodically along the length of vessels observed. The mean percent decrease in vessel diameter in these narrowed regions was 10.25%. Population spikes recorded in the cell body layer of the dentate gyrus showed a mean increase in amplitude of 71.3%. The mean latency to peak response was similar for both the vessels and neurons. The results suggest mechanisms by which autoregulatory influences on microvessel caliber may be counteracted in conditions of hypoxia and hypotension in the whole animal.

Peptidergic transmission in the brain. II. Mediation by a vasopressin-like peptide.

The medial amygdaloid nucleus (AME) sends a projection of fibers containing a peptide similar to arginine vasopressin (AVP) to the rat hippocampus. Electrical stimulation of the AME has an impact on evoked hippocampal field potentials that is identical to the effect obtained by applied AVP in vitro or in vivo. Here we show that the AVP-like peptide released by AME stimulation has action that is blocked by a structural AVP analog. The antagonist specificity suggests that the native transmitter is a peptide similar, but not identical, to AVP. We conclude that the AME projection to the hippocampus satisfies all the criteria for recognition as a central peptidergic system important in the generation of behavior.

Microvascular spasm is mediated by vasopressin fibers in the rat hippocampal slice.

Microvessels in the rat hippocampal slice can be used as an in vitro model for the study of cerebral vasospasm. Serum from coagulated blood causes prompt and long-lasting microvascular constriction that is neurogenic in nature. Here we show that a candidate spasmogen, thromboxane B2, has excitatory action on neural elements in the slice. However, spasmogenic serum lacks this excitatory effect. Instead, it is inhibitory for a major population of slice neurons. Thus, neurogenic microvascular spasm is produced by a subpopulation of slice neurons or projection fibers, not all neurons acting in concert. Arginine vasopressin (AVP) is contained in fibers that project to the hippocampus, and hippocampal microvessels contain pressor (V1) receptors for the peptide. AVP causes vasoconstriction in the slice, and a specific V1 antagonist for the peptide blocks the microvascular spasm induced by blood serum. The results are interpreted to mean that neurogenic microvascular spasm is mediated by locally released AVP.

Neurogenic mediation of serum-induced microvascular constriction.

We have used the rat hippocampal slice preparation as a model system for the study of microvascular vasospasm. Penetrating cerebral microvessels in the slice constrict in response to a variety of stimuli, including serum from coagulated blood. Microvascular responses to many stimuli are associated with changes in the activity of nearby neurons, and in some cases can be shown to be neurogenically mediated. Here we use the selective neurotoxin, tetrodotoxin (TTX), to show that serum-induced constriction is also neurogenically mediated. This neural regulation of microvessel caliber may participate in pathological vasoconstriction mechanisms in the whole animal.

Peptidergic transmission in the brain. I. Vasopressin-like signal in the hippocampus.

Studies of the action of arginine vasopressin (AVP) in the rat hippocampal slice have produced a model of the peptide's neural action. AVP excites local circuit inhibitory interneurons and causes consequent inhibition of pyramidal cells that is apparent as a reduction in the amplitude of the evoked population spike in field potential recording. Here we show that applied AVP does the same thing to the evoked population spike in the whole animal. Then we show that stimulation of the source of hippocampampal AVP, the medial amygdaloid nucleus, also inhibits the evoked population spike. Analysis of the synaptic potential indicates that the same mechanisms are employed by exogenously applied and endogenously released peptide. The inhibition can only be obtained by stimulating those brain structures known to project vasopressin fibers to the hippocampus. The stimulus-response characteristics and kinetics of the endogenous signal correspond to the properties of peptidergic signals in simple systems. The results are taken to support a transmitter role for AVP in the rat hippocampus.

Antidromic activation of a peptidergic pathway in the limbic system of the male rat.

Stimulation of the medial amygdaloid nucleus (AME) produces a long-latency and long-lasting inhibition of pyramidal cells in both the dorsal and the ventral hippocampus. The inhibition is blocked by a specific antagonist to vasopressin, which is a candidate neurotransmitter in the system. Antidromic activation of the AME from the hippocampus occurs with a latency suggestive of the conduction velocity of small diameter unmyelinated fibers. Immunocytochemistry for vasopressin reveals small diameter, unmyelinated immunoreactive fibers in the vicinity of the stimulating electrode in the hippocampus, and immunoreactive cell bodies in the vicinity of the recording electrode in the AME.