Cells Of Supraoptic Nucleus Research Articles

A rat model for pituitary stalk electric lesion-induced central diabetes insipidus: application of 3D printing and further outcome assessments.

A stable and reproducible rat injury model is not currently available to study central diabetes insipidus (CDI) and the neurohypophyseal system. In addition, a system is needed to assess the severity of CDI and measure the accompanying neurobiological alterations. In the present study, a 3D-printed lesion knife with a curved head was designed to fit into the stereotaxic instrument. The neuro-anatomical features of the brain injury were determined by in vivo magnetic resonance imaging (MRI) and arginine vasopressin (AVP) immunostaining on brain sections. Rats that underwent pituitary stalk electrical lesion (PEL) exhibited a tri-phasic pattern of CDI. MRI revealed that the hyperintenseT1-weighted signal of the pituitary stalk was interrupted, and the brain sections showed an enlarged end proximal to the injury site after PEL. In addition, the number of AVP-positive cells in supraoptic nucleus (SON) and paraventricular nucleus (PVN) decreased after PEL, which confirmed the success of the CDI model. Unlike hand-made tools, the 3D-printed lesion knives were stable and reproducible. Next, we used an ordinal clustering method for staging and the k-means’ clustering method to construct a CDI index to evaluate the severity and recovery of CDI that could be used in other multiple animals, even in clinical research. In conclusion, we established a standard PEL model with a 3D-printed knife tool and proposed a CDI index that will greatly facilitate further research on CDI.

Effect of p53 Inhibition by Pifithrin-Alpha on Functional Activity of Vasopressin Neurons in Rat Hypothalamus

In the present work we investigated the effects of p53 inhibition by pifithrin-alpha (PFT) in vitro and in vivo on functioning vasopressinergic magnocellular neurones of rat hypothalamus. In vivo treatments with PFT were done by intra-hypothalamic microinjections or by intra peritoneal injections. In in vitro experiments hypothalamic slices containing supraoptic nuclei and intact pituitary were incubated with or without PFT. In all experiments we observed accumulation of vasopressin (VP) in the cell perikarya after PFT injections, however expression of VP mRNA was not changed. Analysis of VP content in the posterior pituitary demonstrated that amount of VP was significantly decreased after PFT treatments. Additionally, long-term inhibition of p53 in experiments with intra-hypothalamic injections of PFT resulted in an increased diuresis rate. The obtained results demonstrated that in all experiments PFT treatments inhibited VP anterograde transport from the cells of supraoptic nuclei. Moreover, analysis of MEK/ERK activities revealed that phosphorylation levels of MEK1/2 and ERK1/2 were decreased after PFT treatments. Our findings provide new evidences that p53 could be involved in the control of VP secretion from hypothalamo-hypophyseal system and that this action probably can be mediated by ERK signalling pathway.

Nav1.9 expression in magnocellular neurosecretory cells of supraoptic nucleus

Osmoregulation in mammals is tightly controlled by the release of vasopressin and oxytocin from magnocellular neurosecretory cells (MSC) of the supraoptic nucleus (SON). The release of vasopressin and oxytocin in the neurohypophysis by axons of MSC is regulated by bursting activity of these neurons, which is influenced by multiple sources, including intrinsic membrane properties, paracrine contributions of glial cells, and extrinsic synaptic inputs. Previous work has shown that bursting activity of MSC is tetrodotoxin (TTX)-sensitive, and that TTX-S sodium channels Nav1.2, Nav1.6 and Nav1.7 are expressed by MSC and upregulated in response to osmotic challenge in rats. The TTX-resistant sodium channels, NaV1.8 and Nav1.9, are preferentially expressed, at relatively high levels, in peripheral neurons, where their properties are linked to repetitive firing and subthreshold electrogenesis, respectively, and are often referred to as “peripheral” sodium channels. Both sodium channels have been implicated in pain pathways, and are under study as potential therapeutic targets for pain medications which might be expected to have minimal CNS side effects. We show here, however, that Nav1.9 is expressed by vasopressin- and oxytocin-producing MSC of the rat supraoptic nucleus (SON). We also show that cultured MSC exhibit sodium currents that have characteristics of Nav1.9 channels. In contrast, Nav1.8 is not detectable in the SON. These results suggest that Nav1.9 may contribute to the firing pattern of MSC of the SON, and that careful assessment of hypothalamic function be performed as NaV1.9 blocking agents are studied as potential pain therapies.

MORPHOFUNCTIONAL CHANGES OF PITUITARY-NEUROHYPOPHYSIS SYSTEM IN IMMATURE RATS WITH STREPTOZOTOCIN DIABETES MELLITUS

The research is devoted to the study of morphofunctional organization of the pituitary-neurohypophysis system of diabetes type 1. It was found that on the 14 th day after the streptozotocin diabetes simulation in 1-month old animals with hyperglycemia the increasing of functional activity of neurosecretory cells of supraoptic nuclei and increasing of bulk density in their neurosecretory granules are observed. Because of such conditions in the nervous lobe of neurohypophysis increase of bulk density of neurosecretory granules by the diffusing and the residual types are observed. On the 42 th day of the experiment constant high hyperglycemia leads to the stage of depletion of neurosecretory cells of supraoptic and paraventricular nuclei and development of dystrophic-destructive changes in them. In the neurohypophysis the reducing of bulk density of young and mature neurosecretory granules is observed. In the pituicytes of type-2 a large number of different sizes and shapes of lipid drops are observed.

Transient Receptor Potential Channel M4 and M5 in Magnocellular Cells in Rat Supraoptic and Paraventricular Nuclei

The neurohypophysial hormones, vasopressin (VP) and oxytocin (OT), are synthesised by magnocellular cells in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN) of the hypothalamus. The release of VP into the general circulation from the neurohypophysis increases during hyperosmolality, hypotension and hypovolaemia. VP neurones increase hormone release by increasing their firing rate as a result of adopting a phasic bursting. Depolarising after potentials (DAPs) following a series of action potentials are considered to be involved in the generation of the phasic bursts by summating to plateau potentials. We recently discovered a fast DAP (fDAP) in addition to the slower DAP characterised previously. Almost all VP neurones expressed the fDAP, whereas only 16% of OT neurones had this property, which implicates the involvement of fDAP in the generation of the firing patterns in VP neurones. Our findings obtained from electrophysiological experiments suggested that the ionic current underlying the fDAP is mediated by those of two closely-related Ca(2+) -activated cation channels: the melastatin-related subfamily of transient receptor potential channels, TRPM4 and TRPM5. In the present study, double/triple immunofluorescence microscopy and reverse transcriptase-polymerase chain reaction techniques were employed to evaluate whether TRPM4 and TRPM5 are specifically located in VP neurones. Using specific antibodies against these channels, TRPM5 immunoreactivity was found almost exclusively in VP neurones, but not in OT neurones in both the SON and PVN. The most prominent TRPM5 immunoreactivity was in the dendrites of VP neurones. By contrast, most TRPM4 immunoreactivity occurred in cell bodies of both VP and OT neurones. TRPM4 and TRPM5 mRNA were both found in a cDNA library derived from SON punches. These results indictate the possible involvement of TRPM5 in the generation of the fDAP, and these channels may play an important role in determining the distinct firing properties of VP neurones in the SON.

Specific changes in TRPV4 expression in the vasopressinergic cells in supraoptic nucleus of the rat brain following bile duct ligation (BDL) induced cirrhosis

Inappropriate vasopressin (AVP) results in hyponatremia, a risk factor in pathophysiological states such as cirrhosis. This study tested the hypothesis that changes in the expression of TRPV4, a transient receptor potential channel that has been shown to be activated by osmotic stimulation, in vasopressin neurons contributes to inappropriate vasopressin release in BDL rats. Four weeks after surgery, BDL rats demonstrated significantly increased plasma vasopressin and plasma renin activity (PRA), hypervolemia, and decreased plasma osmolality compared to sham ligated controls. Western Blot analysis revealed that TRPV4 expression was significantly increased in brain punches from BDL rats containing the supraoptic nucleus (SON) (100% ± 11 to 157% ± 4.8), but not the paraventricular nucleus (PVN) of the hypothalamus. Immunohistochemistry analysis demonstrated that TRPV4 is co‐localized in AVP neurons in both PVN and SON. The percentage of AVP neurons expressing TRPV4 was significantly increased (36 % ± 3 to 55.3% ± 4.3) only in the SON of BDL rats. It could be that increased expression of TRPV4 in AVP neurons in the SON resets their osmosensitivity allowing them to continue to release AVP in the face of hypoosmolality and hyponatremia contributing to the pathogenesis of inappropriate AVP release. NIH R01 HL55692

Effects of selective Bcl-2 inhibitor HA14-1 treatments on functional activity of magnocellular vasopressinergic neurons of rat hypothalamus

In this study we examined whether in vivo treatments with Bcl-2 inhibitor HA14-1 can affect the function of vasopressinergic system of rat. HA14-1 is a novel organic compound that has micromolar affinity for Bcl-2 and Bcl-xL and acts as a mimetic of BH3-only proteins by antagonizing the anti-apoptotic Bcl-2 proteins and triggering Bax-dependent apoptosis. We found that intrahypothalamic injections of HA14-1 did not induce apoptosis of vasopressin (VP) cells of supraoptic nucleus, but led to activation of VP synthesis and release, resulting in decreased diuresis. Our data has also demonstrated that injections of HA14-1 increased phospho-MEK1/2, phospho-CREB and phospho-Elk-1 levels in magnocellular neurons. Thus we propose that injections of HA14-1 into the hypothalamus do not lead to neuronal death, but change the functional activity of VP neurons of hypothalamus centres.

Impaired ability to increase water excretion in mice lacking the taurine transporter gene TAUT

Cellular taurine uptake or release counteracts alterations of cell volume. Na+-coupled taurine transporter TAUT mediates concentrative cellular uptake of taurine. Inhibition of vasopressin secretion by hypotonicity may involve taurine release from glial cells of supraoptic nucleus. We compared renal function of mice lacking TAUT (taut-/-) and wild-type littermates (taut+/+). We observed renal taurine loss and subsequent hypotaurinemia in taut-/- mice. With free access to water, plasma and urine osmolality, urinary flow rate as well as urinary excretion and plasma concentrations of Na+ and K+ were similar in taut-/- and taut+/+ mice, whereas plasma concentrations of urea were enhanced in taut-/- mice. An oral water load (1 ml/16 g body weight) induced a similar diuresis in both genotypes. Repeating the oral water load immediately after normalization of urine flow rate, however, resulted in delayed diuresis and higher urinary vasopressin/creatinine ratios in taut-/- mice. In comparison, the repeated diuretic response to vasopressin V2 receptor blockade was not different between genotypes. Water deprivation for 36 h led to similar antidiuresis and increases of urinary osmolality in both genotypes. Upon free access to water after deprivation, taut-/- mice continued to concentrate urine up to 6 days, while taut+/+ mice rapidly returned to normal urinary osmolality. Urinary vasopressin/creatinine ratios and plasma aldosterone concentrations were not different under basal conditions but were significantly higher in taut-/- mice than in taut+/+ mice at 6 days after water deprivation. In conclusion, taut-/- mice suffer from renal taurine loss and impaired ability to lower urine osmolality and to increase urinary water excretion. The latter defect could reside extrarenally and result from a role of taurine in the suppression of vasopressin release which may be attenuated in taut-/- mice.

Morphofunctional Analysis of Effect of Short-Term Sleep Deprivation on the Wakefulness–Sleep Cycle in Young and Adult Rats

The work presents comparative data on changes of neurophysiological, time characteristics of the wakefulness–sleep cycle (WSC) and morphofunctional state of neurosecretory cells in supraoptic nucleus of the hypothalamus, which develop under influence of a 6-h long sleep deprivation in adult and one-month old rats. It is shown that the rebound of sleep develops in adult animals with a delay, after the 3rd hour and is characterized by a moderate increase of portions of slow-wave (SSP) and fast-wave (FSP) sleep phases in WSC and by a decrease of the wakefulness portion. Morphological analysis of the hypothalamus nonapeptidergic system has revealed a rise of content of neurosecretory material in fibers of supraoptic nucleus cells an in area of supraoptic-pituitary tract, as well as marked hyperemia that indicates activation of processes of secretion of neurohormones into the general blood flow; these reactions are similar to reactions of this system to stress. In rat pups the sleep rebound develops in 0.5 h after the end of the deprivation procedure and is characterized by more pronounced, statistically significant changes in WSC. Individual WSC become very short and almost all of them are completed with episodes of FSP. A statistically significant rise of power of the δ-wave band in electrogram spectra of hippocampus and somatosensory cortex in SSP, whereas peak of the activity in FSP is shifted to α-waves. Ratios of SSP and FSP to wakefulness in individual WSC in mature animals increase after the deprivation 1.53 and 1.85 times, while they are elevated in one-month old animals 5.25 and 6.75 times, respectively. The obtained morphofunctional data allow believing that deprivation is the stress factor of low intensity for adult animals, whereas it may be considered as the stress action of intermediate and even high intensity for rat pups, which changes essentially the interrelations in WSC. Participation of central mechanisms of regulation of sleep and vigilance, which provide processes of compensation of damaging action of deprivation on WSC in the maturing animals, is discussed.

Effects of the endogenous opioid peptide, endomorphin 1, on supraoptic nucleus oxytocin and vasopressin neurones in vivo and in vitro.

We investigated the actions of the endogenous opioid tetra-peptide endomorphin 1, a selective mu-opioid receptor agonist, on oxytocin and vasopressin cell activity in vivo and in vitro. The activity of antidromically-identified supraoptic nucleus cells were recorded from urethane-anaesthetized female rats. The firing rates of both oxytocin and vasopressin cells were reduced by intracerebroventricular endomorphin 1 (5 - 100 pmol); this inhibition was prevented by intravenous naloxone (5 mg kg(-1)). A second group of rats was infused intracerebroventricularly with endomorphin 1 (27 pmol min(-1)) over 5 days. The firing rates of oxytocin and vasopressin cells in endomorphin 1 pre-treated rats were similar to those of endomorphin 1 naïve rats, indicating tolerance to the inhibitory effects of endomorphin 1. Intravenous naloxone induced similar modest and transient increases in the firing rate of oxytocin cells in endomorphin 1 pre-treated rats and endomorphin 1 naïve rats, indicating that endomorphin 1, unlike the mu-opioid alkaloid agonist, morphine, does not induce mu-opioid dependence in these cells. In vitro, whole-cell current clamp recordings were made from supraoptic nucleus cells in superfused coronal hypothalamic slices from young female rats. Endomorphin 1 (100 nM) inhibited the firing rate of oxytocin cells but had no significant effect on vasopressin cells at up to 10 microM. Inhibition of oxytocin cells was reversed by naloxone, and remained when synaptic transmission was blocked by superfusion with low Ca(2+)/Co(2+)-containing medium. Thus, endomorphin 1 directly inhibits oxytocin cells but inhibits vasopressin cells by indirect actions. Chronic endomorphin 1 administration induces mu-opioid tolerance in oxytocin and vasopressin cells but not mu-opioid dependence in oxytocin cells.

Central actions of the nonpeptide growth hormone secretagogue GHS-25.

Growth hormone secretagogues (GHSs) increase the activity of hypothalamic arcuate nucleus neurons thought to be involved in controlling the release of growth hormone (GH). The GHS receptor is also found in hypothalamic regions not associated with the release of GH, suggesting that GHSs may influence other hypothalamic systems. This study utilized double-labeling immunocytochemical techniques to examine the hypothalamic actions of a novel nonpeptide GHS, GHS-25. In common with other GHSs, GHS-25 induced significant amounts of Fos immunoreactivity in the arcuate nucleus of conscious male rats. However, unlike other GHSs, GHS-25 also induced Fos immunoreactivity in the supraoptic nucleus. Double labeling revealed that approx 66% of supraoptic nucleus cells that were Fos positive after the administration of GHS-25 were also immunoreactive for oxytocin. Thus, in addition to its actions on the GH axis, GHS-25 may influence the release of neurohypophyseal hormone.

Direct pathways to the supraoptic nucleus from the brainstem and the main olfactory bulb are activated at parturition in the rat

Sensory input from female reproductive structures is paramount for the co-ordination of neuroendocrine changes at parturition. Using a retrograde tracer (fluorescent latex microspheres) in combination with Fos (as an indicator of neuronal activation) and tyrosine hydroxylase (to identify catecholaminergic neurons) immunocytochemistry we identified cells within the brainstem and main olfactory bulb that project to the supraoptic nucleus, and which become significantly activated at parturition (compared to virgin rats and rats on the day of expected parturition). Within the A2/C2 region in the nucleus tractus solitarii, 60% of the projecting activated cells were catecholaminergic, as were 59% of such cells in the A1/C1 region of the ventrolateral medulla. This suggests that oxytocin and vasopressin neurons within the supraoptic nucleus are stimulated at parturition via afferent inputs from the brainstem, but the input is not exclusively noradrenergic. Within the mitral layer of the main olfactory bulb, cells that projected to the supraoptic nucleus were significantly activated, suggesting that the olfactory system may regulate supraoptic nucleus cell firing at parturition. The preoptic area, organum vasculosum of the lamina terminalis and medial amygdala contained cells that projected to the supraoptic nucleus but these projections were not significantly activated at parturition, although non-projecting cells in these regions were. On the expected day of parturition, but before birth, projections from the organum vasculosum of the lamina terminalis to the supraoptic nucleus became significantly activated. These findings provide evidence of direct afferent pathways to the supraoptic nucleus from the brain stem and olfactory bulbs that are activated at parturition.

Local Morphine Withdrawal Increases c-fosGene, Fos Protein, and Oxytocin Gene Expression in Hypothalamic Magnocellular Neurosecretory Cells

We measured stimulation of c-fos and oxytocin gene expression during excitation of oxytocin cells induced by systemic or local morphine withdrawal. Female rats were made morphine-dependent by intracerebroventricular morphine infusion over 5 d. Morphine withdrawal, induced by systemic injection of the opioid antagonist naloxone (5 mg/kg) in conscious or anesthetized rats, increased the density of c-fos messenger RNA and of oxytocin heterogeneous nuclear RNA in supraoptic nucleus cells compared with those of nonwithdrawn rats; c-fos messenger RNA was also increased in the magnocellular and parvocellular paraventricular nuclei of withdrawn rats. Morphine withdrawal increased the number of Fos-immunoreactive cells in the supraoptic and magnocellular paraventricular nuclei of conscious or pentobarbitone-anesthetized rats. Morphine withdrawal also increased Fos-immunoreactive cell numbers in the parvocellular paraventricular nucleus of conscious but not anesthetized rats. Central administration of the alpha(1)-adrenoreceptor antagonist benoxathian (5 microg/min) did not prevent morphine withdrawal-induced increases in the numbers of Fos-immunoreactive neurons in the supraoptic or magnocellular paraventricular nucleus. Unilateral microdialysis administration of naloxone (10(-5) M) into the supraoptic nucleus of anesthetized morphine-dependent rats increased Fos-immunoreactive cell numbers compared with the contralateral nucleus. Finally, we investigated whether dependence could be induced by chronic unilateral infusion of morphine into a supraoptic nucleus; systemic naloxone (5 mg/kg) increased Fos-immunoreactive cell numbers in the morphine-infused nucleus compared with the contralateral nucleus. Thus, morphine withdrawal excitation increases c-fos and oxytocin gene expression in supraoptic nucleus neurons. This occurs independently from excitation of their ascending noradrenergic inputs, and both dependence and withdrawal can be induced within the supraoptic nucleus.

Kappa-opioid receptor activation inhibits post-spike depolarizing after-potentials in rat supraoptic nucleus neurones in vitro.

Endogenous agonists acting at kappa-opioid receptors modulate the discharge activity of hypothalamic supraoptic nucleus vasopressin cells in vivo. Phasic activity in vasopressin cells is known to depend critically on intrinsic mechanisms involving post-spike depolarizing after-potentials and we hypothesized that inhibition of phasic bursting by an endogenous kappa-agonist may result from reducing the magnitude of depolarizing after-potentials. To investigate this possibility, intracellular sharp electrode recordings were obtained from supraoptic nucleus cells impaled in superfused explants of rat hypothalamus. Bath application of the selective kappa-agonist, U50,488H (0.1-1 microM), decreased the spontaneous firing rate of magnocellular neurosecretory cells (by 94. 0+/-4.5% at 1 microM, mean+/-SEM; P = 0.02, n = 4). U50,488H did not alter membrane potential (0.9+/-0.8 mV hyperpolarization at 1 microM, P = 0.17, n = 8) or input resistance (11.0+/-4.5% increase at 1 microM, P = 0.09, n = 5). U50,488H (0.1 and 1 microM, both n = 5) reduced depolarizing after-potential amplitude (by 29.9+/-9.3 and 78.0+/-10. 6%, respectively, P<0.001) in eight cells in which the baseline membrane potential was kept constant by dc-current injection and in which a depolarizing after-potential was evoked every 25-40 s by a brief (40-80 ms) train of 3-6 action potentials (the number of spikes in the trains was kept constant for each cell). Thus, kappa-opioid receptor activation reduces depolarizing after-potential amplitude in supraoptic nucleus cells and this may underlie the reduction in burst duration of vasopressin cells caused by an endogenous kappa-agonist in vivo.

Excitation of oxytocin cells in the hypothalamic supraoptic nucleus by electrical stimulation of the dorsal penile nerve and tactile stimulation of the penis in the rat

In urethane-anaesthetized male rats, electrical stimulation of the dorsal penile nerve (DPN) excited 29 of 48 (60%) oxytocin cells in the contralateral supraoptic nucleus, whereas only 5 of 28 (18%) vasopressin cells were excited by the stimulation. The stimulus applied to the ipsilateral DPN to the recorded neurone also excited a similar proportion of oxytocin cells (25 of 43; 58%). Tactile stimulation of the glans penis excited 7 of 12 (58%) oxytocin cells, whereas the same stimulation excited only 3 of 16 (19%) vasopressin cells. The results suggest that sensory information arising from the penis preferentially excites oxytocin cells in the supraoptic nucleus.

Stress-induced activation of CRF and c- fos mRNAs in the paraventricular nucleus are not affected by serotonin depletion

The role of serotonin in regulating the stress response is controversial. We have investigated the effects of serotonin depletion by p-chlorophenylalanine (PCPA) on corticotrophin-releasing factor (CRF) mRNA and c- fos mRNA responses in the paraventricular nucleus (PVN) together with circulating levels of ACTH and corticosterone to both physical and psychological stressors in the rat. PCPA pretreatment, which resulted in a 95% depletion in hypothalamic serotonin, had no effect on basal levels of ACTH or the increase in response to the physical stress of hypertonic saline. Plasma ACTH concentrations were also not affected by serotonin depletion in response to the predominantly psychological stress of restraint. Both basal and restraint stress-induced circulating corticosterone levels were however further stimulated in the PCPA-pretreated rats suggesting a possible inhibitory serotoninergic tone at the adrenal level. C- fos mRNA was undetectable in control animals. Activation of c- fos mRNA in response to stress was unaffected by serotonin depletion and the activation of magnocellular PVN and supraoptic nucleus cells was demonstrated to be stressor dependent. Basal and stress-induced levels of CRF mRNA were unaffected by PCPA pretreatment. It appears therefore that under these experimental conditions there is little if any involvement of serotonin in either basal levels or the stress-induced activation of the hypothalamo-pituitary-adrenal axis in vivo.

Depolarizing action of cholecystokinin on rat supraoptic neurones in vitro.

1. Cholecystokinin is co-localized within the oxytocin- and, to a lesser extent, vasopressin-synthesizing magnocellular neurones in the hypothalamic supraoptic and paraventricular nuclei. These nuclei are also prominent binding sites for cholecystokinin. In the present study we used intracellular current- and voltage-clamp recordings from fifty-seven supraoptic nucleus cells, maintained in superfused explants of rat hypothalamus, to assess their membrane responses to exogenous cholecystokinin and define the nature of their cholecystokinin receptors. 2. In a majority of the fifty-seven cells tested, bolus infusions into the superfusion media of cholecystokinin fragments (maximum concentrations estimated at 0.3-15 microM) were followed within 1-5 s by a transient and reversible membrane depolarization. Active peptides included sulphated cholecystokinin octapeptide (26-33) (28 of 33 cells responded), non-sulphated cholecystokinin octapeptide (26-33) (21 of 25 cells responded), cholecystokinin tetrapeptide (30-33) (20 of 24 cells responded and caerulein (4 of 4 cells responded). None of five cells responded to cholecystokinin (26-28). Depolarizing responses to cholecystokinin analogues persisted in the presence of tetrodotoxin (0.2-0.4 microM), and in Ca(2+)-free solutions containing MnCl2 (2.5 mM). 3. Under voltage clamp, cholecystokinin fragments evoked an inward current accompanied by an increase in membrane conductance. The amplitude of the inward current varied linearly as a function of membrane voltage, with an extrapolated reversal potential of approximately -15 mV. Reversal potentials were not altered by chloride injection. These features suggest that cholecystokinin activates a non-selective cationic conductance. 4. Active cholecystokinin analogues were approximately equipotent in their depolarizing actions, a feature that supports the activation of cholecystokinin-B type receptors. Moreover bath application of 200 nM L-365,260, an antagonist with a high affinity for cholecystokinin-B receptors, reversibly attenuated the cholecystokinin-induced responses in four of six cells tested. 5. These observations indicate that cholecystokinin can directly influence the excitability of rat supraoptic nucleus neurones and provide evidence for an additional site where this peptide may act within the hypothalamo-neurohypophysial axis.

Lesions of the tissue surrounding the preoptic recess (AV3V region) affect neurosecretory cells in the paraventricular nuclei in the rat

Lesions of the tissue surrounding the preoptic recess (AV3V region) have severe effects on body fluid homeostasis; these include acute adipsia and failure of the antidiuretic response. Because neurosecretory cells in supraoptic nuclei comprise the major source of antidiuretic hormone (ADH) in this species, we have previously observed the fine structure of supraoptic nuclei in rats with AV3V lesions. Paraventricular nuclei are the other major source of ADH in rats. Therefore, in this investigation we compared the fine structure of paraventricular nuclei in rats which had received AV3V lesions 3 days earlier with that of control rats which had received sham lesions and either had drinking water available or had water withheld for 3 days. Degenerating axons and axon terminals were present in paraventricular nuclei of lesioned rats. The degenerating terminals were in axodendritic and less often in axosomatic synapses. Morphometric evaluation revealed that neurosecretory cells did respond to the dehydrated state of the adipsic-lesioned animals, but the response was significantly attenuated compared to that which occurred in sham-lesioned rats deprived of water for 3 days. It appears that AV3V lesions damage afferent connections and impair the response of neurosecretory cells to dehydration in paraventricular as well as supraoptic nuclei. However, in paraventricular nuclei the response is not completely prevented by AV3V lesions during the adipsic period as was observed in supraoptic nuclei. The presence of a response in paraventricular nuclei may be at least partially stimulated by reduced body fluid volume. Information from volume receptors would be carried from the medulla to paraventricular nuclei by ascending pathways which are not affected by AV3V lesions.

Calcium-dependent action potentials in rat supraoptic neurosecretory neurones recorded in vitro.

Intracellular recordings obtained from thirty-nine supraoptic nucleus neurones in perfused hypothalamic explants displayed a mean resting membrane potential of -69 mV and spike amplitude of 79 mV. Voltage-current plots were linear in the hyperpolarizing direction and revealed a mean slope resistance of 197 M omega. After Na+ channel blockade with tetrodotoxin (TTX; 0.3-16 microM), the voltage-current relationship did not change significantly for hyperpolarizing pulses. An increase in spike threshold in TTX permitted visualization of a reduction in slope resistance to depolarizing current pulses. This rectification was reduced by the addition of the Ca2+ channel blockers Cd2+, Co2+ or Mn2+. High threshold TTX-resistant spikes with amplitudes ranging between 25 and 64 mV were evoked in an all-or-none manner by brief intracellular current pulses applied above 1.0 Hz. Current pulses presented at lower frequencies (less than or equal to 0.5 Hz) evoked graded responses. In seventeen of nineteen cells, prolonged depolarizing current pulses elicited repetitive firing of TTX-resistant spikes with a progressive increase in their amplitude, rise and fall times and after-hyperpolarizations. TTX-resistant spikes were reversibly abolished when CaCl2 was replaced by equimolar amounts of EGTA or by the addition of either Cd2+, Co2+ or Mn2+ to the perfusion medium. In artificial medium containing EGTA, both the shoulder on the repolarization phase of the spike and the subsequent after-hyperpolarization were reversibly abolished. Tetraethylammonium (TEA; 2-5 mM) induced prolongation of mean action potential durations from 1.9 to 12.3 ms (nineteen cells); in TTX, TEA also prolonged the duration and increased the over-all peak amplitude of the TTX-resistant (Ca2+) spike. While TEA also enhanced the amplitude of the Na+ spike, no comparable prolongation in spike duration was observed. These data indicate that somatic action potentials of supraoptic nucleus cells arise from the co-activation of a low threshold Na+ conductance and a higher threshold Ca2+ conductance; the latter is expressed as a shoulder on the repolarization phase of the action potential.

The effect of lipofuscin on cellular function

The neurons of the supraoptic nucleus of male C57BL/Icrfa 1 mice at 6 or 28 months of age were examined from normally hydrated, osmotically loaded and osmotically loaded-rehydrated animals. Using quantitative morphological techniques, a reduction in the concentration of lipofuscin in the neurons was observed in osmotically loaded mice at both ages, and these levels were restored to control values during rehydration. In addition, there was a significant difference in the pattern of response of lipofuscin levels between the two age groups during the experiment. The concentration of hormone containing neurosecretory granules in the neurons of the supraoptic nucleus did not differ significantly between the two age groups during the experiment. However, the surface area of rough endoplasmic reticulum per unit volume of the supraoptic nucleus cell did differ significantly between the two age groups over the course of the experiment. It is concluded that increasing concentrations of lipofuscin do not affect the ability of the cell to control the concentration of neurosecretory granules or rough endoplasmic reticulum. The simplistic view that lipofuscin accumulates with age to the detriment of cell function must be revised.