Supraoptic Magnocellular Neurons Research Articles

2307 Simultaneous measurements with Ca2+ imaging and patch-clamp techniques in rat supraoptic magnocellular neurons

2307 Simultaneous measurements with Ca2+ imaging and patch-clamp techniques in rat supraoptic magnocellular neurons

Differential distribution of a potassium current in immunocytochemically identified supraoptic magnocellular neurones of the rat.

Magnocellular hypothalamo-neurohypophysial neurones display characteristic firing patterns, related to the hormone they release. To identify the membrane currents that may underlie these firing patterns, we performed whole-cell recording of freshly dissociated magnocellular neurones from the supraoptic nucleus. After recording, cells were immunocytochemically identified by using highly selective monoclonal antibodies raised respectively against vasopressin (AVP) and oxytocin (OT) neurophysin. In 64 out of 131 neurones (48.8%), we detected the presence of a transient potassium current whose kinetic properties were characteristic of an A-current. The A-current was activated by depolarisation over -40 mV, and inactivated rapidly with a monoexponential decay (tau = 28 +/- 2.7 ms; n = 33 at 0 mV). Using conditioning prepulses of 50 ms, the voltage dependence of the inactivation was determined, and the data were adequately fit with a Boltzman equation (half-maximal inactivation: -42.5 mV). The steady-state time-dependent inactivation curve was determined using a prepulse potential at -40 mV, and data were best described with a mono-exponential equation (tau = 89.7 ms). The sensitivity to 1 mM 4-amino-pyridine (63 +/- 9% inhibition, n = 6), and a reversal potential close to the theoretical Nernst equilibrium for potassium (-56.3 +/- 1 mV, n = 6, vs. -58 mV) confirmed that the transient current studied was indeed an A-type potassium current. Immunocytochemical identification revealed that the A-current was selectively expressed in OT-neurophysin-positive cells. As previous work in hypothalamic slice preparations suggests that the A-current is expressed by both AVP cells and OT cells, the present data suggest that whereas the A-current is expressed in the soma of OT cells, it may be expressed only on the dendritic tree of AVP cells, which is truncated in the dispersed cell preparation used here. This distribution may play a role in the specific firing characteristics of magnocellular neurones.

Simultaneous measurements with Ca imaging and patch-clamp techniques in rat supraoptic magnocellular neurons

Simultaneous measurements with Ca imaging and patch-clamp techniques in rat supraoptic magnocellular neurons

Pharmacological dissection of high-voltage-activated Ca2+ current types in acutely dissociated rat supraoptic magnocellular neurons.

1. We used pharmacological blockers and whole cell voltage-clamp recordings from acutely dissociated magnocellular neurons from the rat supraoptic nucleus to test for the presence of L-, N-, P-, Q-, and R-type calcium currents. 2. We found clear evidence for L-type (dihydropyridine sensitive), N-type (blocked by omega-conotoxin (GVIA), and P-type (blocked by omega-agatoxin IVA) currents. Nifedipine blocked 28 +/- 2% (mean +/- SE) of the current, omega-conotoxin GVIA blocked 26 +/- 4%, and omega-agatoxin IVA (25-30 nM) blocked 17 +/- 4%. In addition, omega-conotoxin MVIIC blocked about half (54 +/- 2%) of the current remaining after L-, N-, and P-type currents were blocked, suggesting the presence of Q-type currents. 3. About 20% of the whole cell current was unblocked by the combination of all four blockers, suggesting the presence of a fifth current type. This residual current inactivated slowly at -10 mV.

Central inhibitory effects of muscimol and bicuculline on the milk ejection reflex in the anaesthetized rat.

1. In order to determine whether GABAergic mechanisms are involved in the control of the milk ejection reflex in the rat, we examined the effects of central administration of a GABAA receptor agonist (muscimol) and antagonist (bicuculline) on the milk ejection reflex in the urethane-anaesthetized rat. 2. Intracerebroventricular (i.c.v.) injection of both muscimol (n = 17), at doses of 5, 10 and 20 ng, and bicuculline (n = 15), at doses of 0.01, 0.1 and 0.3 microgram, inhibited the milk ejection reflex in a dose-dependent manner. The bicuculline-induced inhibition was accompanied by desynchronization of the electroencephalogram and, at the highest dose, by alteration in the sensitivity of the mammary gland to oxytocin. No significant effect on the milk ejection reflex was seen with i.c.v. isotonic saline (n = 5). 3. Injection of 20 (n = 5) or 40 ng (n = 2) muscimol or 0.1 microgram bicuculline (n = 5) i.c.v. did not significantly alter the rise in intramammary pressure evoked by electrical stimulation of the neurohypophysis. 4. Bilateral 400 nl microinfusions directly into the supraoptic nuclei of either muscimol (20-100 ng microliter(-1); n = 10) or bicuculline (0.15 micrograms microliter(-1); n = 5) [corrected] resulted in an inhibition of the milk ejection reflex, which was not accompanied by desynchronization of the electroencephalogram. 5. The effects of i.c.v. injections of muscimol (15 and 20 ng) and bicuculline (0.01, 0.12 and 0.3 microgram) on the electrical activity of twenty-seven antidromically identified supraoptic magnocellular neurones were examined. Both compounds resulted in an inhibition of the background firing of oxytocinergic and vasopressinergic cells, and delayed the occurrence of high frequency bursts in oxytocin neurones. In five supraoptic neurones, bicuculline induced a transient activation before inhibition. 6. The powerful inhibitory action on the milk ejection reflex of both muscimol and bicuculline provides evidence for the importance of GABA neurones in maintaining the functional integrity of the mechanisms which allow the intermittent and pulsatile release of oxytocin during suckling.

Properties of supraoptic magnocellular neurones isolated from the adult rat.

1. Magnocellular neurosecretory cells (MNCs) were isolated from the supraoptic nucleus of adult Long-Evans rats using an enzymatic procedure. Immunocytochemical staining with antibodies against vasopressin and oxytocin revealed that MNCs can be identified by size. The membrane properties of these cells were examined at 32-34 degrees C using intracellular recording methods. 2. Isolated MNCs displayed a mean (+/- S.E.M.; n = 109) resting membrane potential of -64.1 +/- 1.0 mV, an input resistance of 571 +/- 34 M omega, and a time constant of 8.7 +/- 0.4 ms. Measurements of specific resistivity and input capacitance revealed that the soma of these cells accounts for a mere 20% of their total somato-dendritic membrane in situ. 3. Voltage-current relations measured near -60 mV were linear negative to spike threshold. From more hyperpolarized membrane potentials, voltage responses to depolarizing current steps displayed transient outward rectification and delayed impulse discharge. 4. Action potentials (76.6 +/- 0.9 mV) triggered from an apparent threshold of -59.3 +/- 0.1 mV broadened progressively at the onset of spontaneous or current-evoked spike trains. Steady-state spike duration increased as a logarithmic function of firing frequency with a maximum near 25 Hz. These effects were abolished in Ca(2+)-free solutions. 5. In all cells, evoked spike trains were followed by a prolonged Ca(2+)-sensitive after-hyperpolarization. In contrast, only a small proportion (16%) of MNCs displayed spontaneous bursting activity or depolarizing after-potentials following brief current-evoked bursts. 6. Isolated MNCs responded to amino acids (glutamate and GABA) and to the neuropeptide cholecystokinin, indicating that receptors for these neurotransmitters are expressed postsynaptically by MNCs and are retained following dissociation. 7. Increasing the osmolality of the superfusing solution by 5-30 mosmol kg-1 caused a membrane depolarization associated with a decrease of input resistance and accelerated spontaneous spike discharge in each of thirty-six MNCs tested. Current-clamp analysis suggested that these responses resulted from the activation of a cationic conductance. Excitatory effects of hyperosmolality were not observed in non-magnocellular neurones (n = 6).

Chronic stress elevates enkephalin expression in the rat paraventricular and supraoptic nuclei

Numerous studies have implicated opioids in the regulation of hypothalamic functions. Dynorphin, which is co-expressed with vasopressin in the magnocellular neurons of the paraventricular and supraoptic nuclei, is co-regulated with vasopressin in response to hyperosmolality and appears to inhibit vasopressin and oxytocin release from the posterior pituitary. Enkephalin is present in paraventricular parvocellular neurons and its expression is elevated in response to various stresses. However, enkephalin's presence and roles in paraventricular and supraoptic magnocellular neurons are uncertain. By giving rats daily intraperitoneal injections of hypertonic saline for up to 12 days, we induced a marked increase in enkephalin expression in magnocellular neurons of the paraventricular and supraoptic nuclei, beyond what develops from drinking hypertonic saline. Our results suggest that enkephalin expression in both vasopressin and oxytocin neurons may increase in response to chronic stresses and provide another source of enkephalin in addition to the parvocellular neurons.

Ultrastructural changes in magnocellular neurons from the supraoptic nucleus of aged rats

Magnocellular neurosecretory neurons in the supraoptic nucleus were examined in aged and young subjects to assess the ultrastructural correlates of cellular activity including cell, nuclear and nucleolar size, as well as the percentage of the cell occupied by Golgi, mitochondrial and rough endoplasmic reticulum compartments. Morphometric analysis was performed on arbitrarily selected cell profiles using a computerized morphometry protocol. Approximately 50% of those neurons examined from aged subjects exhibited a statistically significant increase relative to the young rats in total cell area and all cellular organelles examined except nuclear area. Moreover, an increase in the number and density (per unit membrane area) of synaptic contacts onto those neurons was observed. These findings suggest an increase in synthetic activity by a subpopulation of supraoptic magnocellular neurons, and support the concept of neuronal plasticity in aged rats.

Ontogeny of Diabetes Insipidus in the Brattleboro Rat: Morphological and Physiological Correlates

Abstract The homozygous Brattleboro rat is a mutant of the Long Evans rat which fails to produce assayable quantities of vasopressin. Somata of supraoptic magnocellular neurons from adult Brattleboro rats are hypertrophied relative to those from normally hydrated adult Long Evans rats. We have investigated, by light microscopic morphometric analysis of immunoperoxidase-labelled vibratome sections, the postnatal growth of magnocellular neurons in normal Long Evans rats, and the relative hypertrophy of these cells in Brattleboro rats. Morphometric analysis of the somata of immunoidentified oxytocinergic and vasopressinergic supraoptic magnocellular neurons from Long Evans rats aged between 1 and 140 days postnatum revealed that their somata increased rapidly in size only after 14 days; a time that coincides with the start of weaning, with a transient increase in serum osmolality, and with the onset of ability to produce hyperosmotic urine. Oxytocin- and vasopressin-containing neurons in Long Evans rats achieved adult dimensions by 45 days postnatum. By contrast, somata of oxytocin neurons in the Brattleboro rat already showed significant hypertrophy relative to those in Long Evans rats at 7 days postnatum; hypertrophy continued until at least 140 days. The hypertrophy in the Brattleboro rat at 7 days was associated with markedly raised serum osmolality relative to that of age-matched Long Evans rats between 1 and 14 days.

Fetal hypothalamic transplants promote survival and functional regeneration of axotomized adult supraoptic magnocellular neurons

This study investigated the mechanisms by which fetal hypothalamic transplants promote functional recovery in neurohypophysectomized rats. Seven days after neurohypophysectomy (resulting in urine osmolalities of about 800 mOsm), young adult male Long-Evans rats received either fetal hypothalamic grafts ( n = 10) or sham transplants ( n = 7). Recovery from the lesioned-induced diabetes insipidus was monitored for 6 months and then the transplant sites were evaluated by immunocytochemistry. Surviving host supraoptic magnocellular neurons and neurophysin-positive grafted neurons were counted and their formation of neurohemal contacts evaluated by retrograde transport of systemically injected horseradish peroxidase (HRP). There were significantly more surviving supraoptic magnocellular neurons in neurohypophysectomized animals with median eminence-placed grafts (2236 ± 261 neurons/animal) than in animals with ectopic tissue grafts (895 ± 142 neurons/animal) or sham implants (1052 ± 92 neurons/animal). Almost all surviving host magnocellular neurons were labeled with retrogradely transported HRP while virtually none of the grafted neurophysin positive cells showed evidence of HRP uptake. The degree of functional recovery was directly correlated with the increased survival of host neurons. By 8 weeks post-transplantation, animals with median eminence-placed grafts had recovered from their diabetes insipidus and could concentrate their urine to within normal limits (2,120 ± 110 mOsm). This recovery was stable for the remainder of the 6 month test period. In contrast, animals with ectopic grafts and sham transplants had permanent deficits in fluid regulation. Our results provide evidence for the long-term capacity of fetal neural tissue implants to rescue host neurons from the cell death that typically occurs in the mature central nervous system after axotomy. In addition, these results demonstrate that the surviving neurons can regenerate and establish functionally appropriate connections in the adult brain.

Differential distribution of acetylcholinesterase activity among vasopressin- and oxytocin-containing supraoptic magnocellular neurons

Acetylcholinesterase activity was demonstrated histochemically at light- and electron-microscopic levels, in Vibratome sections of the supraoptic nucleus of fixed hypothalami derived from osmotically stimulated and unstimulated Long-Evans rats, from homozygous Brattleboro rats with hypothalamic diabetes insipidus, from lactating rats, from normal adult male house mice ( Mus musculus) and from mice with hereditary nephrogenic diabetes insipidus ( di/di). Reaction product was located in supraoptic magnocellular neurons; in dorsal and rostral aspects of the supraoptic nuclei lightly stained cells predominate, whereas in ventral and caudal regions densely staining perikarya predominate. Pre- and post-embedding immunocytochemical detection of oxytocin-neurophysin or vasopressin-neurophysin, combined with acetylcholinesterase histochemistry, showed that the lightly staining cells are oxytocinergic, and the densely staining cells vasopressinergic. Osmotic stimulation of the animals, either by substitution of drinking water for 3 days with 2.5% saline or by reason of genetic defects which result in diabetes insipidus, enhanced the acetylcholinesterase activity of the vasopressin neurons but had little effect on the weakly acetylcholinesterase-reactive oxytocin cells. Acetylcholinesterase activity was particularly marked in the hypertrophied abnormal magnocellular neurons of homozygous Brattleboro rats which do not release significant amounts of vasopressin. The increased acetylcholinesterase activity in osmotically stimulated animals cannot, therefore, be a function of vasopressin. Acetylcholinesterase activity was also detected in large multipolar neurons lying dorsolateral to the supraoptic nucleus, and in their fine axonal processes which project towards the supraoptic nucleus. A very few synaptic boutons surrounded by acetylcholinesterase reaction product were found in contact with magnocellular neuron basal dendrites. However, much of the punctate acetylcholinesterase reactivity observed at the light microscopic level and previously interpreted as representing the loci of cholinergic synaptic boutons was shown to be intracellular, and probably caused by acetylcholinesterase activity in some large, secondary lysosomes.

Microelectrophoresis of cholinergic and aminergic drugs on paraventricular neurons

Microelectrophoresis of cholinergic and aminergic drugs on paraventricular neurons