Relaxin-3/RXFP3 signalling in mouse hypothalamus: no effect of RXFP3 activation on corticosterone, despite reduced presynaptic excitatory input onto paraventricular CRH neurons in vitro.

Relaxin-3/RXFP3 signalling is proposed to be involved in the neuromodulatory control of arousal- and stress-related neural circuits. Furthermore, previous studies in rats have led to the proposal that relaxin-3/RXFP3 signalling is associated with activation of the hypothalamic-pituitary-adrenal axis, but direct evidence for RXFP3-related actions on the activity of hypothalamic corticotropin-releasing hormone (CRH) neurons is lacking. In this study, we investigated characteristics of the relaxin-3/RXFP3 system in mouse hypothalamus. Administration of an RXFP3 agonist (RXFP3-A2) intra-cerebroventricularly or directly into the paraventricular nucleus of hypothalamus (PVN) of C57BL/6J mice did not alter corticosterone levels. Similarly, there were no differences between serum corticosterone levels in Rxfp3 knockout (C57BL/6JRXFP3TM1) and wild-type mice at baseline and after stress, despite detection of the predicted stress-induced increases in serum corticosterone. We examined the nature of the relaxin-3 innervation of PVN in wild-type mice and in Crh-IRES-Cre;Ai14 mice that co-express the tdTomato fluorophore in CRH neurons, identifying abundant relaxin-3 fibres in the peri-PVN region, but only sparse fibres associated with densely packed CRH neurons. In whole-cell voltage-clamp recordings of tdTomato-positive CRH neurons in these mice, we observed a reduction in sEPSC frequency following local application of RXFP3-A2, consistent with an activation of RXFP3 on presynaptic glutamatergic afferents in the PVN region. These studies clarify the relationship between relaxin-3/RXFP3 inputs and CRH neurons in mouse PVN, with implications for the interpretation of current and previous in vivo studies and future investigations of this stress-related signalling network in normal and transgenic mice, under normal and pathological conditions.

Embedded synaptic feedback in the neuroendocrine stress axis.

Neural regulation of blood glucocorticoid levels is critical for defence of homeostasis during physiological or psychoemotional challenges. In mammals, this function is carried out by the neuroendocrine stress axis, coordinated by parvocellular neuroendocrine cells (PNCs) of the paraventricular hypothalamic nucleus. Feedback regulation of PNCs by glucocorticoids provides complex experience-dependent shaping of neuroendocrine responses. We review recent evidence for metaplastic actions of glucocorticoids as 'circuit breakers' at synapses directly regulating PNC excitability and explore how such mechanisms may serve as substrates for stress adaptation.

Physiological regulation of magnocellular neurosecretory cell activity: integration of intrinsic, local and afferent mechanisms.

The hypothalamic supraoptic and paraventricular nuclei contain magnocellular neurosecretory cells (MNCs) that project to the posterior pituitary gland where they secrete either oxytocin or vasopressin (the antidiuretic hormone) into the circulation. Oxytocin is important for delivery at birth and is essential for milk ejection during suckling. Vasopressin primarily promotes water reabsorption in the kidney to maintain body fluid balance, but also increases vasoconstriction. The profile of oxytocin and vasopressin secretion is principally determined by the pattern of action potentials initiated at the cell bodies. Although it has long been known that the activity of MNCs depends upon afferent inputs that relay information on reproductive, osmotic and cardiovascular status, it has recently become clear that activity depends critically on local regulation by glial cells, as well as intrinsic regulation by the MNCs themselves. Here, we provide an overview of recent advances in our understanding of how intrinsic and local extrinsic mechanisms integrate with afferent inputs to generate appropriate physiological regulation of oxytocin and vasopressin MNC activity.

Glial regulation of neuronal function: from synapse to systems physiology.

Classically, glia have been regarded as non-excitable cells that provide nourishment and physical scaffolding for neurones. However, it is now generally accepted that glia are active participants in brain function that can modulate neuronal communication via several mechanisms. Investigations of anatomical plasticity in the magnocellular neuroendocrine system of the hypothalamic paraventricular and supraoptic nuclei led the way in the development of much of our understanding of glial regulation of neuronal activity. In this review, we provide an overview of glial regulation of magnocellular neurone activity from a historical perspective of the development of our knowledge of the morphological changes that are evident in the paraventricular and supraoptic nuclei. We also focus on recent data from the authors' laboratories presented at the 9th World Congress on Neurohypophysial Hormones that have contributed to our understanding of the multiple mechanisms by which glia modulate the activity of neurones, including: gliotransmitter modulation of synaptic transmission; trans-synaptic modulation by glial neurotransmitter transporter regulation of neurotransmitter spillover; and glial neurotransmitter transporter modulation of excitability by regulation of ambient neurotransmitter levels and their action on extrasynaptic receptors. The magnocellular neuroendocrine system secretes oxytocin and vasopressin from the posterior pituitary gland to control birth, lactation and body fluid balance, and we finally speculate as to whether glial regulation of individual magnocellular neurones might co-ordinate population activity to respond appropriately to altered physiological circumstances.

The intricate link between glucocorticoids and endocannabinoids at stress-relevant synapses in the hypothalamus.

The relationship between glucocorticoids and endocannabinoids at hypothalamic synapses in the presence of stress is particularly complex. Under conditions of acute stress, glucocorticoids trigger the synthesis of endocannabinoids, which through activation of type I cannabinoid receptors (CB1Rs), inhibit stress-relevant neurons in the paraventricular nucleus of the hypothalamus (PVN). Through this signaling mechanism, endocannabinoids constrain the activity of the hypothalamic-pituitary-adrenal axis. However, following chronic or repeated stress, the ability of endocannabinoids to modulate synaptic activity is compromised because of a functional down-regulation in CB1Rs. Here we examine recent findings that highlight important aspects of endocannabinoid signaling in response to stress in the PVN and the dorsomedial hypothalamus (DMH), two hypothalamic nuclei that play integral roles in regulating the neuroendocrine and autonomic responses to stress.

Metabotropic glutamate receptors: gatekeepers of homeostasis.

The capacity to appropriately respond to physiological challenges or perturbations in homeostasis is a requisite for survival. It is becoming increasingly clear that long-lasting alterations in synaptic efficacy are a fundamental mechanism for modifying neuroendocrine and autonomic output. We review recent advances in our understanding of plasticity at glutamate synapses onto magnocellular neurones (MNCs) in the paraventricular and supraoptic nuclei of the hypothalamus, with a focus on the contributions of metabotropic glutamate receptors (mGluRs) to long-lasting modifications in synaptic efficacy. Special attention is paid to the role of presynaptic mGluRs as gatekeepers for metaplasticity and regulation of body fluid homeostasis. The work highlighted here provides insight into the synaptic mechanisms that couple MNC activity to physiological states.

Isolation of various forms of sterol beta-D-glucoside from the seed of Cycas circinalis: neurotoxicity and implications for ALS-parkinsonism dementia complex.

The factors responsible for ALS-parkinsonism dementia complex (ALS-PDC), the unique neurological disorder of Guam, remain unresolved, but identification of causal factors could lead to clues for related neurodegenerative disorders elsewhere. Earlier studies focused on the consumption and toxicity of the seed of Cycas circinalis, a traditional staple of the indigenous diet, but found no convincing evidence for toxin-linked neurodegeneration. We have reassessed the issue in a series of in vitro bioassays designed to isolate non-water soluble compounds from washed cycad flour and have identified three sterol beta-d-glucosides as potential neurotoxins. These compounds give depolarizing field potentials in cortical slices, induce alterations in the activity of specific protein kinases, and cause release of glutamate. They are also highly toxic, leading to release of lactate dehydrogenase (LDH). Theaglycone form, however, is non-toxic. NMDA receptor antagonists block the actions of the sterol glucosides, but do not compete for binding to the NMDA receptor. The most probable mechanism leading to cell death may involve glutamate neuro/excitotoxicity. Mice fed cycad seed flour containing the isolated sterol glucosides show behavioral and neuropathological outcomes, including increased TdT-mediated biotin-dUTP nick-end labelling (TUNEL) positivity in various CNS regions. Astrocytes in culture showed increased caspase-3 labeling after exposure to sterol glucosides. The present results support the hypothesis that cycad consumption may be an important factor in the etiology of ALS-PDC and further suggest that some sterol glucosides may be involved in other neurodegenerative disorders.

Statistical model relating CA3 burst probability to recovery from burst-induced depression at recurrent collateral synapses.

When neuronal excitability is increased in area CA3 of the hippocampus in vitro, the pyramidal cells generate periodic bursts of action potentials that are synchronized across the network. We have previously provided evidence that synaptic depression at the excitatory recurrent collateral synapses in the CA3 network terminates each population burst so that the next burst cannot begin until these synapses have recovered. These findings raise the possibility that burst timing can be described in terms of the probability of recovery of this population of synapses. Here we demonstrate that when neuronal excitability is changed in the CA3 network, the mean and variance of the interburst interval change in a manner that is consistent with a timing mechanism comprised of a pool of exponentially relaxing pacemakers. The relaxation time constant of these pacemakers is the same as the time constant describing the recovery from activity-dependent depression of recurrent collateral synapses. Recovery was estimated from the rate of spontaneous transmitter release versus time elapsed since the last CA3 burst. Pharmacological and long-term alterations of synaptic strength and network excitability affected CA3 burst timing as predicted by the cumulative binomial distribution if the burst pace-maker consists of a pool of recovering recurrent synapses. These findings indicate that the recovery of a pool of synapses from burst-induced depression is a sufficient explanation for burst timing in the in vitro CA3 neuronal network. These findings also demonstrate how information regarding the nature of a pacemaker can be derived from the temporal pattern of synchronous network activity. This information could also be extracted from less accessible networks such as those generating interictal epileptiform discharges in vivo.

Slowly inactivating potassium conductance (I(D)): a potential target for stroke therapy.

BACKGROUND AND PURPOSE: Excessive accumulation of extracellular glutamate results in the death of most, but not all, neurons in the central nervous system. Understanding the unique properties of cells that can withstand this excitotoxic challenge may identify specific targets for novel stroke therapies. METHODS: A combination of in vivo methods for analysis of excitotoxic cell death after activation of N-methyl-D-aspartate (NMDA) receptors and in vitro patch-clamp analysis of specific conductances in hypothalamic slices and dissociated cells has been used to assess the roles of specific potassium conductances in delayed cell death after NMDA receptor activation. RESULTS: We report that a specific D-type potassium conductance (I(D)), necessary for the rapid repolarization of the membrane after a strong depolarization, serves such a protective purpose in magnocellular neurons of the paraventricular nucleus. Manipulations that inhibit this current (4-aminopyridine or angiotensin II) increase neuronal excitability and augment cell death after NMDA receptor activation. In addition, this protection is not observed in magnocellular neurons of spontaneously hypertensive rats, and intriguingly it can be reestablished by blocking angiotensin II receptors in these animals. CONCLUSIONS: These observations provide a persuasive experimental explanation for the unexpected finding that therapeutic treatments for hypertension that block central as well as peripheral angiotensin type 1 receptors reduce the severity and occurrence of stroke.

Methionine sulfoximine shows excitotoxic actions in rat cortical slices.

Methionine sulfoximine (MSO) is a rare amino acid. It occurs in nature or as a by-product of some forms of food processing. A notable example of the latter was a former method for bleaching wheat flour, using nitrogen trichloride, the "agene process," in use for most of the first 50 years of this century. "Agenized" flour was found to be responsible for various neurological disorders in animals, and MSO was identified as the toxic factor. The agene process was subsequently discontinued in the United States and the United Kingdom circa 1950. MSO inhibits the synthesis of both glutathione and glutamine, and it is possible that its actions on the nervous system arise from alterations in the amount or distribution of these molecules. Structurally, MSO resembles glutamate, an observation that has also raised the possibility that it might have more direct glutamate-like actions on neurons. In the present investigation, we report excitatory and toxic actions of MSO in an in vitro preparation of adult rat cortex. Field potential recordings in this preparation show that MSO application evokes a sustained depolarization, which can be blocked by the N-methyl-D-aspartate (NMDA) antagonist L-(+)-2-amino-5-phosphonovalerate (AP5). However, competition assays using MSO on [3H]CGP-39653 (DL-(E)-2-amino-4-propyl-1-phosphono-3-pentenoate) binding in rat cortical homogenates show only 20% displacement of total binding, suggesting that MSO is acting indirectly, perhaps by releasing glutamate. To investigate this possibility, we measured glutamate release during MSO application. Time course and dose-response experiments with MSO showed significant [3H]glutamate release, which was partially attenuated by AP5. To assess cellular toxicity, we measured lactate dehydrogenase (LDH) release from cortical sections exposed to MSO. MSO treatment led to a rapid increase in LDH activity, which could be blocked by AP5. These data suggest that MSO acts by increasing glutamate release, which then activates NMDA receptors, leading to excitotoxic cell death. These data suggest the possibility that MSO in processed flour had excitotoxic actions that may have been contributing factors to some human neuronal disorders.

Glutathione and signal transduction in the mammalian CNS.

The tripeptide glutathione (GSH) has been thoroughly investigated in relation to its role as antioxidant and free radical scavenger. In recent years, novel actions of GSH in the nervous system have also been described, suggesting that GSH may serve additionally both as a neuromodulator and as a neurotransmitter. In the present article, we describe our studies to explore further a potential role of GSH as neuromodulator/neurotransmitter. These studies have used a combination of methods, including radioligand binding, synaptic release and uptake assays, and electrophysiological recording. We report here the characteristics of GSH binding sites, the interrelationship of GSH with the NMDA receptor, and the effects of GSH on neural activity. Our results demonstrate that GSH binds via its gamma-glutamyl moiety to ionotropic glutamate receptors. At micromolar concentrations GSH displaces excitatory agonists, acting to halt their physiological actions on target neurons. At millimolar concentrations, GSH, acting through its free cysteinyl thiol group, modulates the redox site of NMDA receptors. As such modulation has been shown to increase NMDA receptor channel currents, this action may play a significant role in normal and abnormal synaptic activity. In addition, GSH in the nanomolar to micromolar range binds to at least two populations of binding sites that appear to be distinct from all known excitatory amino acid receptor subtypes. GSH bound to these sites is not displaceable by glutamatergic agonists or antagonists. These binding sites, which we believe to be distinct receptor populations, appear to recognize the cysteinyl moiety of the GSH molecule. Like NMDA receptors, the GSH binding sites possess a coagonist site(s) for allosteric modulation. Furthermore, they appear to be linked to sodium ionophores, an interpretation supported by field potential recordings in rat cerebral cortex that reveal a dose-dependent depolarization to applied GSH that is blocked by the absence of sodium but not by lowering calcium or by NMDA or (S)-2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate antagonists. The present data support a reevaluation of the role of GSH in the nervous system in which GSH may be involved both directly and indirectly in synaptic transmission. A full accounting of the actions of GSH may lead to more comprehensive understanding of synaptic function in normal and disease states.

Reciprocal interactions between CA3 network activity and strength of recurrent collateral synapses.

In hippocampal slices, synchronous CA3 network activity induced persistent strengthening of active positive-feedback synapses. This altered network operation by increasing probability of future synchronous network activation. Long-term depression of synaptic strength induced by partial blockade of NMDA receptors during synchronous network activity reversed changes in probability of spontaneous network activation. These results suggest that specific network activity patterns selectively alter strength of active synapses. Stable, reversible alterations in network activity can also be effected by corresponding alterations in synaptic strength. These findings confirm the Hebb memory model at the neural-network level and suggest new therapies for pathological patterns of network activity in epilepsy.

Activation of N-methyl-D-aspartate receptors evokes calcium spikes in the dendrites of rat hypothalamic paraventricular nucleus neurons.

Activation of dendritic voltage-dependent calcium (Ca2+) conductances in neuroendocrine cells of the hypothalamus may underlie previously documented Ca2+ spikes in these cells. The present study, in which whole-cell recordings were obtained from paraventricular nucleus neurons in a hypothalamic slice preparation, addresses this issue by directly activating dendritic N-methyl-D-aspartate receptors in the presence of tetrodotoxin. Application of tetrodotoxin abolished spontaneous action potentials in all paraventricular nucleus neurons tested (n = 27). Following tetrodotoxin, spikes were evoked by depolarizing current pulses, in an all-or-none fashion in the majority of cells (n = 20). Removal of extracellular Ca2+ (n = 6) or addition of 500 microM CdCl2 (n = 4) abolished the spikes in response to pulses. Repetitive spiking activity (in tetrodotoxin) was also observed following N-methyl-D-aspartate agonist application in 75% of the cells tested (n = 15). The spikes, underscored by a slow membrane depolarization, were abolished by the administration of CdCl2 (n = 4). N-Methyl-D-aspartate agonist elicited a slow inward current in cells voltage-clamped at -60 mV (n = 5). Additionally, larger amplitude, transient inward currents were observed near the onset of the response. The activation threshold to elicit spikes following N-methyl-D-aspartate agonist application was significantly more negative (-54.6+/-3.6 mV) than the potential at which spikes were initiated as a result of depolarizing current injection (-32.3+/-1.8 mV; Student's t-test: P < 0.0001). In contrast to this, Na+ spikes in control solution had an invariable threshold (-49.6+/-0.7 mV vs -51.5+/-1.2 mV; P > 0.05), regardless of the stimulus used to initiate the spikes. These observations suggest that direct activation of N-methyl-D-aspartate receptors located on the dendrites of paraventricular nucleus neurons triggers Ca2+ spikes. Although the precise function of these spikes is unclear, previous data reporting dendritic neuropeptide release in the paraventricular nucleus raise the possibility that dendritically initiated spikes may serve as a local signal to trigger such release.

Hyperpolarizing after-potentials regulate generation of long-duration plateau depolarizations in rat paraventricular nucleus neurons.

Activation of N-methyl-D-aspartate (NMDA) receptors in a population of neurons of the paraventricular nucleus (PVN) results in long-duration plateau depolarizations during which the membrane rapidly depolarizes, reaching a stable plateau near -20 mV. These responses were observed in 29% of the Type II PVN neurons tested with 1 microM NMDA agonist (n = 84). The stable plateau phase is characterized by an increase in ionic conductance, from 1.19+/-0.11 nS to 5.24+/-2.17 nS (n = 5). Bath application of tetrodotoxin (n = 4) or alternatively inclusion of QX-314 in the pipette solution (n = 3) prevented the generation of these events. The remaining cells tested (n = 56) also depolarized in response to NMDA agonist, but long duration plateau depolarizations were not observed. Previous evidence from hypothalamic cultures has demonstrated synaptically driven plateau potentials following the blockade of repolarizing conductances. Pharmacological blockade of the post-spike hyperpolarizing afterpotential with 4-aminopyridine (200 microM), in cells that did not generate plateaux, resulted in the observance of long duration plateau depolarizations in response to a subsequent application of NMDA agonist (n = 4). Our results demonstrate that this 4-aminopyridine-sensitive ionic conductance plays a critical role in determining whether a cell will depolarize for a prolonged duration in response to NMDA receptor activation. As a prolonged depolarization of the postsynaptic membrane and accompanying membrane permeability changes are essential for neurotoxicity, these findings provide evidence for a potential protective mechanism that depends solely on the ability of the cell, through its ionic conductances, to control imposed changes in membrane potential.

Effect of butylated hydroxyanisole on catalase activity and malondialdehyde content in aging Zaprionus paravittiger (diptera).

Catalase activity and malondialdehyde (MDA) content were measured in whole body and mitochondrial homogenates of the banana fruit fly, Zaprionus paravittiger, fed on control and butylated hydroxyanisole (BHA, 10 mM) mixed diets. Catalase activity increased during the reproductive period and decreased thereafter with age. However, the MDA content increased with advancing age in both sexes. In general, females exhibited higher catalase activity and lower MDA content as compared to their male counterparts. BHA feeding increased catalase activity significantly during all age intervals in both sexes. Mitochondrial fractions had lower catalase activity and lower MDA content than whole body homogenates. However, the pattern of changes was similar in both homogenates with age as well as on antioxidant feeding. These results suggest that BHA strengthens the defense mechanism of the insects by increasing catalase activity and reducing MDA content which may be responsible for increased longevity of insects.

Leptin depolarizes rat hypothalamic paraventricular nucleus neurons.

Leptin, the protein product of the ob/ob gene, is thought to have a central site of action, presumably within the hypothalamus, through which it regulates feeding behavior. THe paraventricular nucleus (PVN) is one structure that has been implicated in regulating feeding behavior. Using patch-clamp recording techniques, this study examines the direct membrane effects of leptin on neurons in a coronal PVN slice. Bath application of the physiologically active leptin fragment (amino acids 22-56) elicited dose-related depolarizations in 82% of the type I cells tested (n = 17) and 67% of the type II cells tested (n = 9). By contrast, the physiologically inactive leptin fragment (amino acids 57-92) had no discernible effect on membrane potential (n = 7). The effects of this peptide were unaffected following synaptic isolation of the cells by bath application of the sodium channel blocker tetrodotoxin (n = 5). Voltage clamp recordings in six cells demonstrated that leptin increased a nonspecific cation conductance with a reversal potential near -30 mV. These findings suggest that neurons in PVN may play an important role in the central neuronal circuitry involved in the physiological response to leptin.

Did consumption of flour bleached by the agene process contribute to the incidence of neurological disease?

The present report proposes the hypothesis that increased levels of neurodegenerative disorders in humans may have arisen due to inclusion in the diet of methionine sulfoximine (MSO), a byproduct of the bleaching of flour by nitrogen trichloride. This method of bleaching, the 'agene process' was in use from early in the century and continued until at least 1949/1950. Estimates indicate that, at least in the UK, as much as 80% of all flour during this period was produced by this process. MSO acts directly to inhibit the production of two crucial molecules, glutathione (GSH) and glutamine. Decreases in GSH, a key antioxidant and free radical scavenger, diminish the body's antioxidant defenses and may lead to increased oxidative stress. Decreases in glutamine synthesis may act to increase free glutamate and give rise to increased levels of ammonia. Cells in the nervous system are particularly sensitive to a decline in either GSH or glutamine. The combined effects of decreases in these molecules, particularly with long-term exposure to MSO in bleached flour, may have had quite drastic effects on neuronal health and survival. The present hypothesis may provide clues to the etiology of neurological disorders such as Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS), suggesting that such disorders may arise in part due to toxic actions of some compounds in processed human foods.

Presynaptic modulation of CA3 network activity.

The simultaneous discharge of hippocampal CA3 pyramidal cells is a widely studied in vitro model of physiological and pathological network synchronization. This network is rapidly activated because of extensive positive feedback mediated by recurrent axon collaterals. Here we show that population-burst duration is limited by depletion of the releasable glutamate pool at these recurrent synapses. Postsynaptic inhibitory conductances further limit burst duration but are not necessary for burst termination. The interval between bursts in vitro depends on the rate of replenishment of releasable glutamate vesicles and the probability of release of those vesicles at recurrent synapses. Therefore presynaptic factors controlling glutamate release at recurrent synapses regulate the probability and duration of synchronous discharges of the CA3 network.

Long duration pressor responses following activation of subfornical organ neurons in rats are the result of increased circulating vasopressin.

Electrical stimulation in the subfornical organ (SFO) of male Sprague-Dawley rats resulted in biphasic increases in blood pressure (BP) without a change in heart rate. The initial short duration (0-10 s) increase in BP lasted throughout the 10 s stimulation period (area under the curve (AUC) = 104.3+/-15.26 mmHg/s, (mean+/-SEM) P < 0.001). Upon termination of the electrical stimulus, the BP remained elevated for approximately 55 s (long duration response, AUC = 327.5+/-48.22 mmHg/s, P < 0.001). This long duration BP response was determined to be the result of an increase in circulating vasopressin (VP) as administration of a V1 receptor antagonist abolished this response (AUC = -210.7 +/- 42.38 mmHg/s, P < 0.01). The results of the present study demonstrate that the long duration component of the biphasic increase in BP observed on response to electrical stimulation of the SFO is the result of increased concentrations of circulating VP.

Reduced NMDA receptor sensitivity may underlie the resistance of subpopulations of PVN neurons to excitotoxicity.

Magnocellular neurons in the paraventricular nucleus are resistant to excitotoxic cell damage. We tested the hypothesis that a modified post-synaptic response following NMDA receptor activation may underlie this resistance. Whole-cell recordings from hypothalamic slices showed that NMDA receptor activation caused dose-dependent depolarizations in both Type I (putative magnocellular) and Type II (putative parvocellular) neurons. Type II cells, however, were an order of magnitude more sensitive (10 nM) than Type I neurons (100 nM). The depolarizations recorded in Type II cells were also significantly greater (> 35% resulting in sodium channel inactivation) than those recorded in Type I neurons. This differential sensitivity of neurons to NMDA receptor activation may explain the selective resistance of magnocellular PVN neurons to excitatory neurotoxins.