Magnocellular axons in passage through the median eminence release vasopressin

Vasopressin (arginine vasopressin, AVP) is present in two types of nerve fibres in the median eminence (ME). First, it is found in nerve terminals that originate in the parvicellular neurones of the hypothalamic paraventricular nucleus (PVN) and abut on the pericapillary space surrounding the fenestrated capillaries of the primary pituitary portal plexus in the external zone (EZ) of the ME. These neurones also synthesize corticotropin-releasing factor (CRF), which acts synergetically with vasopressin to stimulate release of adrenocorticotropin (ACTH) from the pituitary gland (see ref. 7). Second, vasopressinergic axons of the magnocellular neurosecretory system pass through the internal zone (IZ) of the ME to terminate in the neurohaemal contact zone of the neurohypophysis. The involvement of vasopressinergic magnocellular neurones in the control of ACTH secretion is much debated. Of particular interest in this context is the origin of the vasopressin found in pituitary portal blood. Although it has been demonstrated that vasopressin and CRF are present in the same neurosecretory granules of EZ fibres, parallel determinations of vasopressin and CRF in pituitary portal blood have shown alterations of the concentration of vasopressin without a concomitant change in that of CRF. Such a dissociation suggests that either differential release of vasopressin and CRF can occur from a single population of nerve endings, or there are fibres in the pituitary-stalk ME which release vasopressin but not CRF. Here we present evidence for the latter. Our results indicate that stimuli causing depolarization of the axonal membrane in vitro elicit release of vasopressin from nerve fibres in the external and internal zones of the ME.     

Co-localization of corticotropin-releasing factor and vasopressin in median eminence neurosecretory vesicles

Vasopressin (VP) potentiates the effect of corticotropin-releasing factor (CRF) on the secretion of adrenocorticotropic hormone (ACTH) from anterior pituitary cells in vitro, and both CRF and VP have been found in portal blood. These data support the hypothesis that VP acts synergistically with CRF to cause the secretion of ACTH in vivo but the origin of the CRF and VP, and the physiology of their release, have not been precisely defined. Parvocellular cell bodies in the paraventricular nucleus (PVN) which project to the external zone of the median eminence can be stained for both CRF and VP after adrenalectomy, and there is light microscopic immunocytochemical evidence that neurophysin (NP) may be located within some of the CRF-containing axons. Electron microscopic immunocytochemical studies have demonstrated the presence of CRF, VP and its 'carrier' protein, VP-associated neurophysin (NP-VP) in 100-nm neurosecretory vesicles (NSVs) in axons terminating near the portal capillary plexus in the external zone of the median eminence. If these peptides are extensively co-localized in the same NSVs in the median eminence, then coordinate secretion of CRF and VP in vivo is obligatory, at least in some physiological circumstances. We demonstrate in this report, using post-embedding electron microscopic immunocytochemistry on serial ultrathin sections, that CRF, VP and NP-VP are contained not only in the same axons and terminals, but in the same 100-nm NSVs in the median eminence of both normal and adrenalectomized rats. In addition, in the normal rat median eminence 44% of the CRF-positive axons and terminals stained strongly for VP and NP-VP, whereas in the adrenalectomized rat virtually all the CRF-positive structures in the median eminence showed strong staining for VP and NP-VP, indicating a transformation of one subpopulation of CRF-positive axons and terminals by adrenalectomy.     

Increase of corticotropin-releasing factor staining in rat paraventricular nucleus neurones by depletion of hypothalamic adrenaline

In response to stress, adrenocorticotropic hormone (ACTH) is released by corticotrophs in the anterior pituitary under the control of several central and peripheral factors including corticotropin-releasing factor (CRF), which was recently isolated from the brain and sequenced. Immunocytochemical studies have shown that most of the CRF-containing cell bodies that project to the median eminence are present in the hypothalamic paraventricular nucleus (PVN). A dense PNMT(phenylethanolamine-N-methyltransferase)-containing fibre network was also observed in the same region--PNMT is the final enzyme in the biosynthesis of adrenaline and has been demonstrated in the brain. In the present study we found an association of adrenergic nerve fibres and CRF neurones by immunohistochemistry using antisera to PNMT and CRF. To examine the functional significance of the adrenergic projection to the PVN, we blocked the synthesis of adrenaline using a specific inhibitor of PNMT. The depletion of adrenaline resulted in an increase in CRF immunoreactivity. The present results suggest that, as well as catecholamines which regulate ACTH release at the anterior pituitary level via a beta 2-adrenergic receptor mechanism, central catecholamines (mainly adrenaline) also affect ACTH release through their action on CRF cells. Peripheral catecholamines seem to have a direct stimulatory effect on the pituitary corticotroph cells, whereas the present findings suggest that central adrenaline-containing neurones have an inhibitory role in the physiological response to stress.     

Reciprocal changes in corticotropin-releasing factor (CRF)-like immunoreactivity and CRF receptors in cerebral cortex of Alzheimer's disease

Alzheimer's disease is a progressive degenerative disease of the nervous system characterized neuropathologically by the presence of senile plaques and neurofibrillary tangles in amygdala, hippocampus and neocortex. Dysfunction and death of basal forebrain cholinergic neurones projecting to forebrain targets are associated with marked decreases in cholinergic markers, including the activity of choline acetyltransferase (ChAT). Although cortical levels of somatostatin and somatostatin receptors are reduced in Alzheimer's, no consistent changes have been reported in other neuropeptide systems. We have now examined in control and Alzheimer's brain tissues pre- and postsynaptic markers of corticotropin-releasing factor (CRF), a hypothalamic peptide regulating pituitary-adrenocortical secretion which also seems to act as a neurotransmitter in the central nervous system (CNS). We have found that in Alzheimer's, the concentrations of CRF-like immunoreactivity (CRF-IR) are reduced and that there are reciprocal increases in CRF receptor binding in affected cortical areas. These changes are significantly correlated with decrements in ChAT activity. Our results strongly support a neurotransmitter role for CRF in brain and demonstrate, for the first time, a modulation of CNS CRF receptors associated with altered CRF content. These observations further suggest a possible role for CRF in the pathophysiology of the dementia. Future therapies directed at increasing CRF levels in brain may prove useful for treatment.     

Oxytocin induces morphological plasticity in the adult hypothalamo-neurohypophysial system

The hypothalamo-neurohypophysial system offers a unique example in the adult mammalian central nervous system (CNS) of a functional and structural plasticity related to a physiological state. During lactation, oxytocin neurones evolve a synchronized electrical activation which permits pulsatile hormone release at milk ejection. At the same time, in the supraoptic (SON) and paraventricular nuclei, glial coverage of neurones diminishes, so that large portions of their surface membrane become directly juxtaposed; synaptic remodelling also associates pairs of neurones through the formation of common presynaptic terminals. These structural changes, reversible after weaning, affect exclusively oxytocinergic neurones and could facilitate their synchronized electrical activity. As several observations suggest that oxytocin itself is released centrally, we have examined the effect of prolonged intracerebroventricular infusions of oxytocin on the structure of the SON of non-lactating animals. We report here that the peptide indeed engenders the structural reorganization characteristic of the oxytocin system when it is physiologically activated. Similar infusion of vasopressin has no effect. Our observations thus demonstrate that a central neuropeptide can induce anatomical changes in the adult CNS, and suggest that oxytocin can regulate its own release by contributing to the dramatic restructuring of the nuclei containing the neurones responsible for its secretion.     

Corticotropin releasing activity of the new CRF is potentiated several times by vasopressin

Initially the hypothalamic factor responsible for the release of corticotropin (CRF), was thought to be a simple peptide. More recent work has led to the conclusion that CRF is a multifactorial complex. In 1979 we proposed that vasopressin, much disputed as a CRF candidate, was a major constituent of the complex, interacting with a potentiating the CRF activity of the other component(s). The recent characterization of a 41 residue ovine hypothalamic peptide capable of releasing adrenocorticotropic hormone (ACTH) in a dose-related manner has allowed us to compare its CRF bioactivity with that of vasopressin and simple extracts of the hypothalamus, and to investigate any interaction it may have with vasopressin and other hypothalamic factors in the release of ACTH. We report here that the new CRF is more potent than vasopressin in releasing ACTH. When given simultaneously with vasopressin a fourfold potentiation of CRF activity with steep dose-response characteristics were observed. It also potentiated vasopressin-free hypothalamic extracts, suggesting that a new CRF does not account for all the nonvasopressin portion of the CRF complex.     

Reduction in number of immunostained GABAergic neurones in deprived-eye dominance columns of monkey area 17

The primary visual cortex (area 17) of the Old World monkey is divided into alternating right- and left-eye dominance columns that are highly modifiable by visual experience during a critical period in development but display little morphological or physiological plasticity during adult life. However, changes in immunocytochemical staining for a calcium/calmodulin-dependent protein kinase occur in visual cortical neurones of adult monkeys after brief monocular deprivation and concentrations of putative neurotransmitters or their related enzymes can be altered with changes in neuronal activity in other systems. We therefore examined the effects of monocular deprivation on the immunocytochemical staining for gamma-aminobutyric acid (GABA) and its synthetic enzyme, glutamic acid decarboxylase (GAD), in adult monkey area 17. The staining for GABA and GAD in neuronal somata and terminals was markedly reduced within ocular dominance columns associated with a removed or a visually deprived eye, suggesting that the GABA concentration in cortical neurones may depend on their levels of activity. Thus area 17 of adult monkeys may retain a greater degree of plasticity than previously recognized and sensory experience can profoundly affect transmitter levels, in the cortex, apparently by regulating levels of a synthetic enzyme.     

Kindling stimulation induces c-fos protein(s) in granule cells of the rat dentate gyrus

Alterations in neuronal gene expression have been proposed to account for permanent changes in brain function such as learning and memory. In particular, it has been suggested that protooncogenes such as c-fos may be rapidly induced in conditions that lead to neuronal plasticity and evoke permanent changes in the expression of effector genes. Concentrations of the c-fos proto-oncogene increase rapidly following depolarization-induced calcium influx in non-dividing neuronally differentiated PC 12 cells. Recently, the presence and induction of c-fos in the adult brain and spinal cord has been observed. Here we report that electrically-induced seizure activity, which leads to a permanent increase in the response of the brain to future seizures (kindling), rapidly and transiently increases c-fos protein-like immunoreactivity in the nuclei of granule cells in the rat dentate gyrus. These results suggest that c-fos protein is present within the nuclei of adult mammalian neurons, and could be involved in plastic changes in the nervous system associated with seizure activity.     

Modulation of stress-induced ACTH release by corticotropin-releasing factor, catecholamines and vasopressin

The stress-induced release of ACTH is believed to involve the activation of several humoral and neural pathways, including corticotropin-releasing factor (CRF), catecholamines and vasopressin. The essential role of CRF was supported by our observation that immunoneutralization of this releasing factor significantly lowers plasma ACTH levels of ether-stressed rats. However, the presence of a small but measurable residual ACTH secretion suggested the possible involvement of factors other than CRF in the stress response. We report here that pretreatment with a vasopressin antagonist decreases the plasma ACTH levels of ether-stressed rats in later (10-20 min), but not earlier (0-10 min), phases of ether stress. The ganglionic blocker chlorisondamine, inhibits ACTH release during both phases of the response to ether by 40-60% when used alone, and by 100% when administered with anti-CRF antibody. These results support a role of CRF, catecholamines and vasopressin in mediating ACTH release by ether stress.     

Cycloheximide-sensitive synthesis of substance P by isolated dorsal root ganglia

Substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2) may be used as a neurotransmitter by certain primary afferent neurones, particularly those carrying pain impulses. Substance P-like immunoreactivity has been localised to the cell bodies of one population of dorsal root ganglion neurones by immunocytochemistry. It is contained in vesicles in the central terminals of these neurones, and has also been demonstrated in the peripheral terminals. As axons and terminals have very little capacity for peptide biosynthesis, it is possible that substance P is synthesised and packaged in the perikaryon and transported to the terminals by an axoplasmic transport process. Consistent with this is the finding that substance P accumulates proximal to a ligature placed on the dorsal root. There has, however, been no direct demonstration of the biosynthesis of substance P in the nervous system. We report here that rat dorsal root ganglia incorporate 35S-methionine into substance P, characterised as authentic by immunoprecipitation followed by HPLC. There is a delay of 1-2 h between addition of label and its incorporation into substance P. Synthesis is blocked by cycloheximde suggesting that, in dorsal root ganglia, substance P is synthesised by a conventional ribosomal process. Synthesis of substance P is reduced by some 90% in ganglia from rats treated neonatally with capsaicin, a drug which is thought to destroy a population of primary afferent neurones.     

Increased affiliative response to vasopressin in mice expressing the V1a receptor from a monogamous vole.

Arginine vasopressin influences male reproductive and social behaviours in several vertebrate taxa through its actions at the V1a receptor in the brain. The neuroanatomical distribution of vasopressin V1a receptors varies greatly between species with different forms of social organization. Here we show that centrally administered arginine vasopressin increases affiliative behaviour in the highly social, monogamous prairie vole, but not in the relatively asocial, promiscuous montane vole. Molecular analyses indicate that gene duplication and/or changes in promoter structure of the prairie vole receptor gene may contribute to the species differences in vasopressin-receptor expression. We further show that mice that are transgenic for the prairie vole receptor gene have a neuroanatomical pattern of receptor binding that is similar to that of the prairie vole, and exhibit increased affiliative behaviour after injection with arginine vasopressin. These data indicate that the pattern of V1a-receptor gene expression in the brain may be functionally associated with species-typical social behaviours in male vertebrates.     

Single cocaine exposure in vivo induces long-term potentiation in dopamine neurons

How do drugs of abuse modify neural circuitry and thereby lead to addictive behaviour? As for many forms of experience-dependent plasticity, modifications in glutamatergic synaptic transmission have been suggested to be particularly important. Evidence of such changes in response to in vivo administration of drugs of abuse is lacking, however. Here we show that a single in vivo exposure to cocaine induces long-term potentiation of AMPA (alpha-amino-3-hydroxy-5-methyl-isoxazole propionic acid)-receptor-mediated currents at excitatory synapses onto dopamine cells in the ventral tegmental area. Potentiation is still observed 5 but not 10 days after cocaine exposure and is blocked when an NMDA (N-methyl-d-aspartate) receptor antagonist is administered with cocaine. Furthermore, long-term potentiation at these synapses is occluded and long-term depression is enhanced by in vivo cocaine exposure. These results show that a prominent form of synaptic plasticity can be elicited by a single in vivo exposure to cocaine and therefore may be involved in the early stages of the development of drug addiction.     

Two types of cholinergic innervation in cortex, one co-localized with vasoactive intestinal polypeptide

The existence of cholinergic neuronal cell bodies in mammalian cerebral cortex was long the subject of much controversy (see ref. 1 for review). Recently, however, a specific cholinergic marker, the acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT, E.C.2.3.1.6), was demonstrated by immunohistochemical methods to be present in bipolar neurones in rat cortex. Here we show that at least 80% of these intrinsic cholinergic neurones also contain immunoreactivity for vasoactive intestinal polypeptide (VIP), a neuroactive peptide found to be present in a subpopulation of cortical neurones. On the other hand, we find that the ChAT-positive cells in the basal forebrain, which are another major source of cholinergic innervation of the cortex, contain no detectable VIP-immunoreactivity. In addition, we have observed by both light and electron microscopy that some VIP- and some ChAT-positive structures in cortex are closely associated with blood vessels.     

Evidence for coexistence of dopamine and CCK in meso-limbic neurones

Vanderhaeghen et al. reported the occurrence of gastrin-like immunoreactivity in the mammalian brain. Subsequent studies have revealed that this immunoreactivity corresponded mainly to the COOH-terminal octapeptide of cholecystokinin (CCK-8), which has a COOH-terminal pentapeptide identical to gastrin. Also, two peptides resembling the NH- and the COOH-terminal tetrapeptide fragments of CCK-8 are present in the central nervous system (CNS). Using COOH-terminal-specific antisera raised to gastrin and/or CCK, the distribution of CCK neurones has been described with immunohistochemical techniques. Although high numbers of cells and nerve terminals are found in cortical areas, the CCK systems are also present in most other parts of the brain and spinal cord. In the CNS, true gastrin molecules, gastrin-17 and gastrin-34 have been located only in the neurohypophysis, hypothalamus and occasionally in the medulla oblongata (unpublished results). We describe here the occurrence of peptides in meso-limbic dopamine neurones in the rat brain. Evidence has also been obtained that mesencephalic dopamine neurones in the human brain contain similar peptides.     

Latent synaptic pathways revealed after tetanic stimulation in the hippocampus

Synaptic plasticity may result from changes at existing synapses or from alterations in the number of functional synaptic connections. In the hippocampus excitatory synaptic strength is persistently enhanced after tetanic stimulation. Here we report that latent synaptic pathways may also become functional. Simultaneous intracellular recordings were made from pairs of CA3 pyramidal cells in slices from guinea pig hippocampus. After stimulating afferent fibres repetitively, polysynaptic excitatory pathways between previously unconnected cells became apparent. The efficacy of recurrent inhibitory circuits was also reduced. The loss of inhibitory control is of interest because latent excitatory pathways are revealed after pharmacological suppression of inhibition. This plasticity in local synaptic circuits leads to the emergence of synchronous firing in groups of CA3 cells. The formation of groups of associated cells and the ability of some cells to initiate synchronous firing in a larger cell group through recurrent pathways is reminiscent of several models of information storage and recall in the cortex.     

Enkephalin-, VIP- and substance P-like immunoreactivity in the carotid body

The carotid body type I cell contains amines and has features, both morphological and cytochemical, which indicate that it may also produce a peptide. Many regulatory peptides are now known to be present in both central and peripheral tissues. In the periphery these neuropeptides occur in both classical endocrine (APUD) cells and the neurones of the autonomic nervous system. We have now investigated the possible presence of neuropeptides in the cat carotid body using both immunocytochemistry and radioimmunoassay. Met- and Leu-enkephalin-like material occurred in considerable quantities in carotid body extracts and enkephalin-like immunoreactivity was localised in type I cells. Both vasoactive intestinal polypeptide (VIP)- and substance P-like immunoreactivity was also present but was localised in nerve fibres distributed throughout the organ. These active neuropeptides are widely distributed in mammalian tissues, forming a diffuse regulatory system which now seems to include the carotid body.     

Inhibitory neurones of a motor pattern generator in Xenopus revealed by antibodies to glycine

Glycine and gamma-aminobutyric acid (GABA) are inhibitory transmitters of major importance. Whereas neurones using GABA as the transmitter can be visualized by immunocytochemical methods for glutamate decarboxylase (GAD) or GABA, no comparable techniques have been available for the selective visualization of glycinergic neurones. We have now produced polyclonal antibodies which specifically recognize glycine in glutaraldehyde-fixed tissue. We used these antibodies to investigate the distribution of glycine in the simple central nervous system (CNS) of the Xenopus embryo, which contains an anatomically and physiologically defined class of reciprocal inhibitory interneurones, the commissural interneurones. These interneurones have an important role in the generation of the swimming motor pattern and are thought to be glycinergic. The glycine antibodies specifically stain these interneurones, revealing their distribution and number in the embryo CNS. This is the first demonstration of the selective localization of glycine-like immunoreactivity in a putative glycinergic class of neurone that has been characterized physiologically, pharmacologically and anatomically.     

Neuropeptides modulate the beta-adrenergic response of purified astrocytes in vitro

Neuropeptides may have functions in the central nervous system (CNS) other than altering neuronal excitability. For example, they may act as regulators of brain metabolism by affecting glycogenolysis. Since it has been suggested that glial cells might provide metabolic support for neuronal activity, they may well be one of the targets for neuropeptide regulation of metabolism. Consistent with this view are reports that peptide-containing nerve terminals have been seen apposed to astrocytes, but it is also quite possible that peptides could act at sites lacking morphological specialization. Primary cultures containing CNS glial cells have been shown to respond to beta-adrenergic agonists with an increase in cyclic AMP and, as a result, with an increase in glycogenolysis and have also been shown to respond to a variety of peptides with changes in cyclic AMP. In the study reported here, we have examined the effects of several peptides on relatively pure cultures of rat astrocytes. We demonstrate that the increase in intracellular cyclic AMP induced by noradrenaline is markedly enhanced by somatostatin and substance P and is inhibited by enkephalin, even though these peptides on their own have little or no effect on the basal levels of cyclic AMP. Vasoactive intestinal peptide (VIP) on the other hand increases cyclic AMP in the absence of noradrenaline. These results suggest that neuropeptides influence glial cells as well as neurones in the CNS and, in the case of somatostatin and substance P, provide further examples of neuropeptides modulating the response to another chemical signal without having a detectable action on their own.     

Selective collateral elimination in early postnatal development restricts cortical distribution of rat pyramidal tract neurones

The pyramidal tract, comprising those axons which pass from the neocortex to the medulla and spinal cord, is among the most thoroughly studied projections of the mammalian cortex. Recent studies using anterograde axon tracing techniques have provided information concerning the time course of the growth of pyramidal tract fibres, yet much remains to be learned about its development. We have now begun to study the distribution of the neurones of origin of the pyramidal tract during the postnatal development of the rat neocortex using the recently introduced retrogradely transported fluorescent marker, True blue. During the first postnatal week, injections of True blue into the pyramidal decussation result inthe labelling of pyramidal tract neurones which are distributed virtually throughout the tangential extent of layer V of the neocortex, whereas after comparable injections during the fourth postnatal week the distribution of such cells is much more restricted and remains restricted into adult life. This developmental restriction is most dramatic in the occipital cortex: during the first postnatal week many pyramidal tract neurones are found throughout the visual cortex while none is seen in this area of the adult. When True blue is injected into the pyramidal decussation during the first postnatal week and the animals are allowed to survive until the fourth postnatal week, the distribution of pyramidal tract neurones is as widespread as in the immediate postnatal period and includes the entire visual cortex. This implies that many of the neurones in the occipital cortex initially send a collateral into the pyramidal tract which is later eliminated, although the neurones themselves persist. These findings, together with similar recent observations on the development of the callosal connections, indicate that the elimination of axon collaterals may be a general feature of the development of cortical projection systems, and that such transitory collaterals may traverse considerable distances.