Loss of beta-III spectrin leads to Purkinje cell dysfunction recapitulating the behavior and neuropathology of spinocerebellar ataxia type 5 in humans.

Mutations in SPTBN2, the gene encoding beta-III spectrin, cause spinocerebellar ataxia type 5 in humans (SCA5), a neurodegenerative disorder resulting in loss of motor coordination. How these mutations give rise to progressive ataxia and what the precise role beta-III spectrin plays in normal cerebellar physiology are unknown. We developed a mouse lacking full-length beta-III spectrin and found that homozygous mice reproduced features of SCA5 including gait abnormalities, tremor, deteriorating motor coordination, Purkinje cell loss, and cerebellar atrophy (molecular layer thinning). In vivo analysis reveals an age-related reduction in simple spike firing rate in surviving beta-III(-/-) Purkinje cells, whereas in vitro studies show these neurons to have reduced spontaneous firing, smaller sodium currents, and dysregulation of glutamatergic neurotransmission. Our data suggest an early loss of EAAT4- (protein interactor of beta-III spectrin) and a subsequent loss of GLAST-mediated uptake may play a role in neuronal pathology. These findings implicate a loss of beta-III spectrin function in SCA5 pathogenesis and indicate that there are at least two physiological effects of beta-III spectrin loss that underpin a progressive loss of inhibitory cerebellar output, namely an intrinsic Purkinje cell membrane defect due to reduced sodium currents and alterations in glutamate signaling.

Responses to cholecystokinin in the ventromedial nucleus of the rat hypothalamus in vivo.

The peptide cholecystokinin (CCK) is a short-term satiety signal released from the gastrointestinal tract during food intake. From the periphery, CCK signalling travels via the vagus nerve to reach the brainstem from which it is relayed higher into the brain. The hypothalamus is a key integrator of appetite-related stimuli and the ventromedial nucleus of the hypothalamus (VMN) is thought to have an important role in the regulation of satiety. We investigated the effect of intravenous injections of CCK on the spontaneous firing activity of single VMN neurons in urethane-anaesthetised rats in vivo. We found that the predominant effect of CCK on the electrical activity in the VMN is inhibitory. We analysed the responses to CCK according to electrophysiologically distinct subpopulations of VMN neurons and found that four of these VMN subpopulations were inhibited by CCK, while five were not significantly affected. Finally, CCK-induced inhibitory response in VMN neurons was not altered by pre-administration of intravenous leptin.

Activation of the rat hypothalamic supramammillary nucleus by food anticipation, food restriction or ghrelin administration.

The circulating orexigenic hormone ghrelin targets many brain areas involved in feeding control and signals via a dedicated receptor, the growth hormone secretagogue receptor 1A. One unexplored target area for ghrelin is the supramammillary nucleus (SuM), a hypothalamic area involved in motivation and reinforcement and also recently linked to metabolic control. Given that ghrelin binds to the SuM, we explored whether SuM cells respond to ghrelin and/or are activated when endogenous ghrelin levels are elevated. We found that peripheral ghrelin injection activates SuM cells in rats, reflected by an increase in the number of cells expressing c-Fos protein in this area, as welll as by the predominantly excitatory response of single SuM cells recorded in in vivo electrophysiological studies. Further c-Fos mapping studies reveal that this area is also activated in rats in situations when circulating ghrelin levels are known to be elevated: in food-restricted rats anticipating the consumption of food and in fed rats anticipating the consumption of an energy-dense food. We also show that intra-SuM injection of ghrelin induces a feeding response in rats suggesting that, if peripheral ghrelin is able to access the SuM, it may have direct effects on this brain region. Collectively, our data demonstrate that the SuM is activated when peripheral ghrelin levels are high, further supporting the emerging role for this brain area in metabolic and feeding control.

Spontaneous discharge characteristic of neurons in the ventromedial nucleus of the rat hypothalamus in vivo.

The ventromedial nucleus of the hypothalamus (VMN) is one of the main central regulators of two vital behaviours in rat: feeding behaviour and sexual behaviour. To better understand how these behaviours are regulated in the brain requires assessing how physiological stimuli are encoded by the electrical activity of populations of neurons, but there is still little known about the electrical activity of neurons in the VMN, in particular how it is regulated in vivo. Here, we recorded spontaneous firing activity from single VMN neurons in urethane-anaesthetized rats in vivo, and characterized their electrophysiological identities. For each of 271 cells, we constructed hazard functions from interspike interval histograms to show how the excitability of the cell changes with time after a spike. We completed the statistical characterization of each cell by analysis of its mean firing rate and coefficient of variation, and features of its interspike interval distribution, including kurtosis and skew (around the mean and around the mode). We thereby identified nine subpopulations of neurons in the VMN, which we named according to the main features of their firing pattern. One of the subpopulations fires very regularly, another almost randomly and another in intermittent clusters of two-three spikes, but perhaps the most interesting subpopulation are 'oscillatory cells' whose activity seems to be governed by an extrinsic 3-Hz rhythm. Whether these electrophysiologically distinct populations are also functionally and neurochemically distinct has now to be tested.

Population dynamics in vasopressin cells.

Most neurons sense and code change, and when presented with a constant stimulus they adapt, so as to be able to detect a fresh change. However, for some things it is important to know their absolute level; to encode such information, neurons must sustain their response to an unchanging stimulus while remaining able to respond to a change in that stimulus. One system that encodes the absolute level of a stimulus is the vasopressin system, which generates a hormonal signal that is proportional to plasma osmolality. Vasopressin cells sense plasma osmolality and secrete appropriate levels of vasopressin from the neurohypophysis as needed to control water excretion; this requires sustained secretion under basal conditions and the ability to increase (or decrease) secretion should plasma osmolality change. Here we explore the mechanisms that enable vasopressin cells to fulfill this function, and consider how coordination between the cells might distribute the secretory load across the population of vasopressin cells.

Oxytocin - The Sweet Hormone?

Mammalian neurons that produce oxytocin and vasopressin apparently evolved from an ancient cell type with both sensory and neurosecretory properties that probably linked reproductive functions to energy status and feeding behavior. Oxytocin in modern mammals is an autocrine/paracrine regulator of cell function, a systemic hormone, a neuromodulator released from axon terminals within the brain, and a 'neurohormone' that acts at receptors distant from its site of release. In the periphery oxytocin is involved in electrolyte homeostasis, gastric motility, glucose homeostasis, adipogenesis, and osteogenesis, and within the brain it is involved in food reward, food choice, and satiety. Oxytocin preferentially suppresses intake of sweet-tasting carbohydrates while improving glucose tolerance and supporting bone remodeling, making it an enticing translational target.

High-Sugar, but Not High-Fat, Food Activates Supraoptic Nucleus Neurons in the Male Rat.

Oxytocin is a potent anorexigen and is believed to have a role in satiety signaling. We developed rat models to study the activity of oxytocin neurons in response to voluntary consumption or oral gavage of foods using c-Fos immunohistochemistry and in vivo electrophysiology. Using c-Fos expression as an indirect marker of neural activation, we showed that the percentage of magnocellular oxytocin neurons expressing c-Fos increased with voluntary consumption of sweetened condensed milk (SCM). To model the effect of food in the stomach, we gavaged anesthetized rats with SCM. The percentage of supraoptic nucleus and paraventricular nucleus magnocellular oxytocin-immunoreactive neurons expressing c-Fos increased with SCM gavage but not with gastric distention. To further examine the activity of the supraoptic nucleus, we made in vivo electrophysiological recordings from SON neurons, where anesthetized rats were gavaged with SCM or single cream. Pharmacologically identified oxytocin neurons responded to SCM gavage with a linear, proportional, and sustained increase in firing rate, but cream gavage resulted in a transient reduction in firing rate. Blood glucose increased after SCM gavage but not cream gavage. Plasma osmolarity and plasma sodium were unchanged throughout. We show that in response to high-sugar, but not high-fat, food in the stomach, there is an increase in the activity of oxytocin neurons. This does not appear to be a consequence of stomach distention or changes in osmotic pressure. Our data suggest that the presence of specific foods with different macronutrient profiles in the stomach differentially regulates the activity of oxytocin neurons.

Alpha-melanocyte-stimulating hormone stimulates oxytocin release from the dendrites of hypothalamic neurons while inhibiting oxytocin release from their terminals in the neurohypophysis.

The peptides alpha-melanocyte stimulating hormone (alpha-MSH) and oxytocin, when administered centrally, produce similar behavioral effects. alpha-MSH induces Fos expression in supraoptic oxytocin neurons, and alpha-MSH melanocortin-4 receptors (MC4Rs) are highly expressed in the supraoptic nucleus, suggesting that alpha-MSH and oxytocin actions are not independent. Here we investigated the effects of alpha-MSH on the activity of supraoptic neurons. We confirmed that alpha-MSH induces Fos expression in the supraoptic nucleus when injected centrally and demonstrated that alpha-MSH also stimulates Fos expression in the nucleus when applied locally by retrodialysis. Thus alpha-MSH-induced Fos expression is not associated with electrophysiological excitation of supraoptic neurons because central injection of alpha-MSH or selective MC4 receptor agonists inhibited the electrical activity of oxytocin neurons in the supraoptic nucleus recorded in vivo. Consistent with these observations, oxytocin secretion into the bloodstream decreased after central injection of alpha-MSH. However, MC4R ligands induced substantial release of oxytocin from dendrites in isolated supraoptic nuclei. Because dendritic oxytocin release can be triggered by changes in [Ca2+]i, we measured [Ca2+]i responses in isolated supraoptic neurons and found that MC4R ligands induce a transient [Ca2+]i increase in oxytocin neurons. This response was still observed in low extracellular Ca2+ concentration and probably reflects mobilization of [Ca2+]i from intracellular stores rather than entry via voltage-gated channels. Taken together, these results show for the first time that a peptide, here alpha-MSH, can induce differential regulation of dendritic release and systemic secretion of oxytocin, accompanied by dissociation of Fos expression and electrical activity.

60 YEARS OF NEUROENDOCRINOLOGY: The posterior pituitary, from Geoffrey Harris to our present understanding.

Geoffrey Harris pioneered our understanding of the posterior pituitary, mainly with experiments that involved the electrical stimulation of the supraoptico-hypophysial tract. In the present essay, we explain how his observations included clues to the pulsatile nature of the oxytocin signal - clues that were followed up by subsequent workers, including his students and their students. These studies ultimately led to our present understanding of the milk-ejection reflex and of the role of oxytocin in parturition. Discoveries of wide significance followed, including: the recognition of the importance of pulsatile hormone secretion; the recognition of the importance of stimulus-secretion coupling mechanisms in interpreting the patterned electrical activity of neurons; the physiological importance of peptide release in the brain; the recognition that peptide release comes substantially from dendrites and can be regulated independently of nerve terminal secretion; and the importance of dynamic morphological changes to neuronal function in the hypothalamus. All of these discoveries followed from the drive to understand the milk-ejection reflex. We also reflect on Harris's observations on vasopressin secretion, on the effects of stress, and on oxytocin secretion during sexual activity.

The active role of dendrites in the regulation of magnocellular neurosecretory cell behavior.

The interactions of the dendritically released neuropeptides vasopressin and oxytocin with co-released neuroactive substances such as opioids and nitric oxide are reviewed. Endogenous opioids regulate magnocellular neurons at the level of the supraoptic nucleus and the relationship of dendritically released peptides and co-released opioids seems to be dependent on the stimulus given and the physiological state of the animal. Nitric oxide has a prominent inhibitory action on supraoptic neurons and these actions are predominantly mediated indirectly by GABA inputs. The role of these co-released neuroactive substances in differentially regulated release of neuropeptides from dendrites versus distant axon terminals has to be determined in more detail. A picture emerges in which release of vasopressin and oxytocin from different anatomical compartments of a single neuron may arise from different intracellular secretory pools and their preparation before release.

Oxytocin released from magnocellular dendrites: a potential modulator of alpha-melanocyte-stimulating hormone behavioral actions?

Alpha-melanocyte-stimulating hormone (alpha-MSH) is implicated in a variety of behavioral processes that are remarkably similar to those behaviors in which centrally acting oxytocin has been implicated. Central oxytocin derives in part from centrally projecting parvocellular neurons of the paraventricular nucleus, but large amounts of oxytocin are also released from dendrites of magnocellular oxytocin neurons in the supraoptic and paraventricular nuclei of the hypothalamus. Oxytocin release from dendrites is semi-independent of electrical activity and can be modulated by peptidergic signals independently of release from nerve terminals. Oxytocin is released from dendrites by stimuli that mobilize intracellular calcium stores, and such stimuli also prime dendritic stores of oxytocin, making them available for subsequent activity-dependent secretion. Evidence exists for efferent projections to the supraoptic nucleus from the arcuate nucleus where alpha-MSH neurons are located, and the supraoptic and paraventricular nuclei show high levels of expression of mRNA for the melanocortin receptor MC4R. These projections may be involved specifically in the regulation of dendritic oxytocin release.

Discharge patterning in rat olfactory bulb mitral cells in vivo.

Here we present a detailed statistical analysis of the discharge characteristics of mitral cells of the main olfactory bulb of urethane-anesthetized rats. Neurons were recorded from the mitral cell layer, and antidromically identified by stimuli applied to the lateral olfactory tract. All mitral cells displayed repeated, prolonged bursts of action potentials typically lasting >100 sec and separated by similarly long intervals; about half were completely silent between bursts. No such bursting was observed in nonmitral cells recorded in close proximity to mitral cells. Bursts were asynchronous among even adjacent mitral cells. The intraburst activity of most mitral cells showed strong entrainment to the spontaneous respiratory rhythm; similar entrainment was seen in some, but not all nonmitral cells. All mitral cells displayed a peak of excitability at ~25 msec after spikes, as reflected by a peak in the interspike interval distribution and in the corresponding hazard function. About half also showed a peak at about 6 msec, reflecting the common occurrence of doublet spikes. Nonmitral cells showed no such doublet spikes. Bursts typically increased in intensity over the first 20-30 sec of a burst, during which time doublets were rare or absent. After 20-30 sec (in cells that exhibited doublets), doublets occurred frequently for as long as the burst persisted, in trains of up to 10 doublets. The last doublet was followed by an extended relative refractory period the duration of which was independent of train length. In cells that were excited by application of a particular odor, responsiveness was apparently greater during silent periods between bursts than during bursts. Conversely in cells that were inhibited by a particular odor, responsiveness was only apparent when cells were active. Extensive raw (event timing) data from the cells, together with details of those analyses, are provided as supplementary material, freely available for secondary use by others.

Oxytocin, feeding, and satiety.

Oxytocin neurons have a physiological role in food intake and energy balance. Central administration of oxytocin is powerfully anorexigenic, reducing food intake and meal duration. The central mechanisms underlying this effect of oxytocin have become better understood in the past few years. Parvocellular neurons of the paraventricular nucleus project to the caudal brainstem to regulate feeding via autonomic functions including the gastrointestinal vago-vagal reflex. In contrast, magnocellular neurons of the supraoptic and paraventricular nuclei release oxytocin from their dendrites to diffuse to distant hypothalamic targets involved in satiety. The ventromedial hypothalamus, for example, expresses a high density of oxytocin receptors but does not contain detectable oxytocin nerve fibers. Magnocellular neurons represent targets for the anorexigenic neuropeptide α-melanocyte stimulating hormone. In addition to homeostatic control, oxytocin may also have a role in reward-related feeding. Evidence suggests that oxytocin can selectively suppress sugar intake and that it may have a role in limiting the intake of palatable food by inhibiting the reward pathway.

Oxytocin and appetite.

Oxytocin has potent central effects on feeding behaviour, as well as on social and sexual behaviours, and one likely substrate for its anorectic effect is the ventromedial nucleus of the hypothalamus. This nucleus expresses a high density of oxytocin receptors, but contains very few oxytocin-containing fibres, hence it is a likely target of 'neurohormonal' actions of oxytocin, including possibly oxytocin released from the dendrites of magnocellular oxytocin neurones. As oxytocin release from dendrites is regulated independent of electrical activity and of secretion from the neurohypophysis, exactly how this release is regulated by metabolic and reproduction-related signals remains to be established fully. Intriguingly though, it looks as though this central release of oxytocin from magnocellular neurons might be instrumental in a fundamental shift in motivational behaviour - switching behaviour from being driven by the need to find and consume food, to the need to reproduce.

Presynaptic actions of endocannabinoids mediate alpha-MSH-induced inhibition of oxytocin cells.

We recently showed that central injections of alpha-melanocyte-stimulating hormone (alpha-MSH) inhibits oxytocin cells and reduces peripheral release of oxytocin, but induces oxytocin release from dendrites. Dendritic oxytocin release can be triggered by agents that mobilize intracellular calcium. Oxytocin, like alpha-MSH, mobilizes intracellular calcium stores in oxytocin cells and triggers presynaptic inhibition of afferent inputs that is mediated by cannabinoids. We hypothesized that this mechanism might underlie the inhibitory effects of alpha-MSH. To test this, we recorded extracellularly from identified oxytocin and vasopressin cells in the anesthetized rat supraoptic nucleus (SON). Retrodialysis of a CB1 cannabinoid receptor antagonist to the SON blocked the inhibitory effects of intracerebroventricular injections of alpha-MSH on the spontaneous activity of oxytocin cells. We then monitored synaptically mediated responses of SON cells to stimulation of the organum vasculosum of the lamina terminalis (OVLT); this evoked a mixed response comprising an inhibitory component mediated by GABA and an excitatory component mediated by glutamate, as identified by the effects of bicuculline and 6-cyano-7-nitroquinoxaline-2,3-dione applied to the SON by retrodialysis. Application of CB1 receptor agonists to the SON attenuated the excitatory effects of OVLT stimulation in both oxytocin and vasopressin cells, whereas alpha-MSH attenuated the responses of oxytocin cells only. Thus alpha-MSH can act as a "switch"; it triggers oxytocin release centrally, but at the same time through initiating endocannabinoid production in oxytocin cells inhibits their electrical activity and hence, peripheral secretion.

Suppressed oxytocin neuron responses to immune challenge in late pregnant rats: a role for endogenous opioids.

Cytokine challenge (mimicking infection) with systemic interleukin-1beta (IL-1beta) stimulates oxytocin neurons via a noradrenergic brainstem pathway similar to that involved in parturition. As the responses of oxytocin neurons to several stimuli are reduced in late pregnancy, we have investigated whether responses to IL-1beta are also suppressed. In virgin Sprague-Dawley rats, IL-1beta (500 ng/kg i.v.) rapidly increased oxytocin secretion (3.2-fold), via a central action as the firing rate of oxytocin neurons in the supraoptic nucleus was increased. In contrast, IL-1beta had no significant effect on the electrical or secretory activity of oxytocin neurons in late pregnant rats. In pregnancy activation of a central inhibitory opioid mechanism restrains oxytocin neuron responses to various stimuli. Accordingly, we tested the effects of the opioid antagonist, naloxone, on oxytocin neuron responses to IL-1beta in pregnancy. Naloxone (5 mg/kg i.v.) did not affect the oxytocin secretory response to IL-1beta in virgin rats, whereas in late pregnant rats naloxone revealed a greater oxytocin secretory response to IL-1beta (3.5-fold) than in virgin rats. In virgin rats, naloxone decreased oxytocin neuron firing rate after IL-1beta, however, in pregnant rats naloxone increased the firing rate response to IL-1beta to the level seen in virgin rats. Thus, systemic IL-1beta acts centrally to increase oxytocin secretion. In pregnancy this response is suppressed by endogenous opioids, thus preserving neurohypophysial oxytocin stores for parturition and minimizing the risk of preterm labour. The exaggerated oxytocin secretory response to IL-1beta in pregnancy after naloxone reflects increased oxytocin stores and/or increased efficiency of excitation-secretion coupling at the posterior pituitary.

Regulation of oxytocin secretion.

A baby sucks at a mother's breast for comfort and, of course, for milk. Milk is made in specialized cells of the mammary gland, and for a baby to feed, the milk must be released into a collecting chamber from where it can be extracted by sucking. Milk "let-down" is a reflex response to the suckling and kneading of the nipple--and sometimes in response to the sight, smell, and sound of the baby--and is ultimately affected by the secretion of oxytocin. Oxytocin has many physiological roles, but its only irreplaceable role is to mediate milk let-down: oxytocin-deficient mice cannot feed their young; the pups suckle but no milk is let down, and they will die unless cross-fostered. Most other physiological roles of oxytocin, including its role in parturition, are redundant in the sense that the roles can be assumed by other mechanisms in the absence of oxytocin throughout development and adult life. Nevertheless, physiological function in these roles can be altered or impaired by acute interventions that alter oxytocin secretion or change the actions of oxytocin. Here we focus on the diverse stimuli that regulate oxytocin secretion and on the apparent diversity of the roles for oxytocin.

Regulation of activity-dependent dendritic vasopressin release from rat supraoptic neurones.

Magnocellular neurones of the hypothalamus release vasopressin and oxytocin from their dendrites and soma. Using a combination of electrophysiology, microdialysis, in vitro explants, and radioimmunoassay we assessed the involvement of intracellular Ca(2+) stores in the regulation of dendritic vasopressin release. Thapsigargin and cyclopiazonic acid, which mobilize Ca(2+) from intracellular stores of the endoplasmic reticulum, evoked vasopressin release from dendrites and somata of magnocellular neurones in the supraoptic nucleus. Thapsigargin also produced a dramatic potentiation of dendritic vasopressin release evoked by osmotic or high potassium stimulation. This effect is long lasting, time dependent, and specific to thapsigargin as caffeine and ryanodine had no effect. Furthermore, antidromic activation of electrical activity in the cell bodies released vasopressin from dendrites only after thapsigargin pretreatment. Thus, exposure to Ca(2+) mobilizers such as thapsigargin or cyclopiazonic acid primes the releasable pool of vasopressin in the dendrites, so that release can subsequently be evoked by electrical and depolarization-dependent activation. Vasopressin itself is effective in inducing dendritic vasopressin release, but it is ineffective in producing priming.

Phasic spike patterning in rat supraoptic neurones in vivo and in vitro.

In vivo, most vasopressin cells of the hypothalamic supraoptic nucleus fire action potentials in a 'phasic' pattern when the systemic osmotic pressure is elevated, while most oxytocin cells fire continuously. The phasic firing pattern is believed to arise as a consequence of intrinsic activity-dependent changes in membrane potential, and these have been extensively studied in vitro. Here we analysed the discharge patterning of supraoptic nucleus neurones in vivo, to infer the characteristics of the post-spike sequence of hyperpolarization and depolarization from the observed spike patterning. We then compared patterning in phasic cells in vivo and in vitro, and we found systematic differences in the interspike interval distributions, and in other statistical parameters that characterized activity patterns within bursts. Analysis of hazard functions (probability of spike initiation as a function of time since the preceding spike) revealed that phasic firing in vitro appears consistent with a regenerative process arising from a relatively slow, late depolarizing afterpotential that approaches or exceeds spike threshold. By contrast, in vivo activity appears to be dominated by stochastic rather than deterministic mechanisms, and appears consistent with a relatively early and fast depolarizing afterpotential that modulates the probability that random synaptic input exceeds spike threshold. Despite superficial similarities in the phasic firing patterns observed in vivo and in vitro, there are thus fundamental differences in the underlying mechanisms.

Intracellular calcium stores regulate activity-dependent neuropeptide release from dendrites.

Information in neurons flows from synapses, through the dendrites and cell body (soma), and, finally, along the axon as spikes of electrical activity that will ultimately release neurotransmitters from the nerve terminals. However, the dendrites of many neurons also have a secretory role, transmitting information back to afferent nerve terminals. In some central nervous system neurons, spikes that originate at the soma can travel along dendrites as well as axons, and may thus elicit secretion from both compartments. Here, we show that in hypothalamic oxytocin neurons, agents that mobilize intracellular Ca(2+) induce oxytocin release from dendrites without increasing the electrical activity of the cell body, and without inducing secretion from the nerve terminals. Conversely, electrical activity in the cell bodies can cause the secretion of oxytocin from nerve terminals with little or no release from the dendrites. Finally, mobilization of intracellular Ca(2+) can also prime the releasable pool of oxytocin in the dendrites. This priming action makes dendritic oxytocin available for release in response to subsequent spike activity. Priming persists for a prolonged period, changing the nature of interactions between oxytocin neurons and their neighbours.