Javier E. Stern

Electrophysiological and morphological properties of pre‐autonomic neurones in the rat hypothalamic paraventricular nucleus

1 The cellular properties of pre‐autonomic neurones in the hypothalamic paraventricular nucleus (PVN) were characterized by combining in vivo retrograde tracing techniques, in vitro patch‐clamp recordings and three‐dimensional reconstruction of recorded neurones in adult hypothalamic slices. 2 The results showed that PVN pre‐autonomic neurones constitute a heterogeneous neuronal population. Based on morphological criteria, neurones were classified into three subgroups. Type A neurones (52 %) were located in the ventral parvocellular (PaV) subnucleus, and showed an oblique orientation with respect to the third ventricle (3V). Type B neurones (25 %) were located in the posterior parvocellular (PaPo) subnucleus, and were oriented perpendicularly with respect to the 3V. Type C neurones (23 %) were located in both the PaPo (82 %) and the PaV (18 %) subnuclei, and displayed a concentric dendritic configuration. 3 A morphometric analysis revealed significant differences in the dendritic configuration among neuronal types. Type B neurones had the most complex dendritic arborization, with longer and more branching dendritic trees. 4 Several electrophysiological properties, including cell input resistance and action potential waveforms, differed between cell types, suggesting that the expression and/or properties of a variety of ion channels differ between neuronal types. 5 Common features of PVN pre‐autonomic neurones included the expression of a low‐threshold spike and strong inward rectification. These properties distinguished them from neighbouring magnocellular vasopressin neurones. 6 In summary, these results indicate that PVN pre‐autonomic neurones constitute a heterogeneous neuronal population, and provide a cellular basis for the study of their involvement in the pathophysiology of hypertension and congestive heart failure disorders.

Nitric oxide inhibits the firing activity of hypothalamic paraventricular neurons that innervate the medulla oblongata: role of GABA.

Nitric oxide (NO) has been shown to modulate autonomic function by acting both peripherally and centrally. A growing body of evidence indicates that the paraventricular nucleus of the hypothalamus (PVN), an important site for autonomic and endocrine homeostasis, constitutes an important locus mediating central NO actions. However, the cellular targets and mechanisms mediating NO actions within the PVN are not completely understood. Here, we examined whether NO influences the firing activity of identified PVN neurons that innervate two functionally different autonomic centers, the dorsal vagal complex (DVC) and the rostral ventrolateral medulla (RVLM). Perforated patch-clamp recordings were performed in hypothalamic slices containing retrogradely labeled PVN neurons innervating the DVC or the RVLM. Application of the NO donors dyethylamine- or 1-propanamine, 3-(2-hydroxy-2-nitroso-1-propylhydrazino) NONOate inhibited the firing activity of both DVC- and RVLM-projecting PVN neurons. Furthermore, application of 2-(4-carboxypheny)-4,4,5,5,-tetramethilimidazoline-1-oxyl-3-oxide (carboxy-PTIO), or the relatively selective neuronal nitric oxide synthase (nNOS) inhibitor 7-nitroindazole alone, increased their basal firing activity, suggesting the presence of an endogenous NO inhibitory tone. GABAergic synaptic activity in PVN neurons was potentiated by NO donors, an action that involved a presynaptic mechanism. Furthermore, the NO-mediated inhibition of firing activity was blocked by the GABA(A) receptor antagonist bicuculline, suggesting that NO-inhibitory actions involved potentiation of local GABAergic synaptic activity. Immunohistochemical studies showed that approximately 25% of DVC- and RVLM-projecting PVN neurons express nNOS, suggesting that a proportion of these medullary-projecting PVN neurons contribute to the cellular source of NO within the PVN. In summary, NO has been identified as an important molecule controlling autonomic function under physiological and pathological conditions. Here, we provide information on the cellular mechanisms mediating central NO actions. Our results demonstrate for the first time that NO modulates the activity of identified populations of PVN neurons that innervate the medulla oblongata, an action that is likely mediated by enhancing synaptic GABAergic function. This work suggests that NO-GABA interaction in PVN neurons that innervate the medulla constitutes an efficient cellular mechanism mediating NO central regulation of autonomic function.

Electrophysiological differences between oxytocin and vasopressin neurones recorded from female rats in vitro.

1. Intracellular recordings in vitro from immunochemically identified oxytocin (OT) and vasopressin (VP) neurones in the supraoptic nucleus (SON) of virgin or lactating female rats revealed no differences between neurone types in membrane potential (Vm), input resistance and current‐voltage relationships (I‐V), when taken at resting membrane potentials. 2. When OT (94%), but not VP, neurones (93%) were current clamped at depolarized voltages (above ‐50 mV), small hyperpolarizing pulses revealed a time‐ and voltage‐dependent outward rectification that was present above ‐75 mV and that decreased in amplitude as Vm approached the equilibrium potential for potassium (EK). The rectification was more pronounced when the neurones were held at a more depolarized membrane potential, and was larger the longer the neurone was held depolarized, reaching a maximum at 0.6‐0.9 s. 3. A rebound depolarization followed the offset of hyperpolarizing pulses that were associated with the rectification. The peak amplitude of the rebound showed a time and a voltage dependence. It followed a bell‐shaped curve as the hyperpolarizing commands were made larger, attaining a peak at ‐65 +/‐ 1.5 mV. The rebound amplitude increased with pulse duration, achieving a half‐maximal amplitude at 0.5 +/‐ 0.1 s. 4. The expression of the sustained outward rectification and the rebound in OT neurones was similar in virgin and lactating female rats. 5. These results indicate the presence of significant differences in the intrinsic membrane properties, probably K+ currents, between OT and VP neurones in both lactating and virgin female rats.

SUSTAINED OUTWARD RECTIFICATION OF OXYTOCINERGIC NEURONES IN THE RAT SUPRAOPTIC NUCLEUS : IONIC DEPENDENCE AND PHARMACOLOGY

1. Intracellular recordings were obtained in vitro from oxytocin and vasopressin neurones from dioestrous and lactating female rats. Oxytocin neurones were characterized under current clamp by the expression of a depolarization‐activated, sustained outward rectification (SOR) and a rebound depolarization (RD). 2. An increment in extracellular K+ shifted the expression of the SOR and RD towards a more depolarized membrane potential, indicating that the mechanisms underlying these events are dependent on extracellular potassium. 3. The SOR and RD were blocked by external tetraethylammonium (10 mM) and Ba2+ (0.1‐0.5 mM). Cs+ (2 mM) blocked the hyperpolarization‐activated inward rectification without affecting the expression of the SOR and RD. 4. The SOR was not affected by 4‐aminopyridine (6 mM). However, the rebound amplitude was significantly enhanced, indicating that the activation of a transient outward current interacts with the expression of the rebound. 5. Iberiotoxin (100 nM) and apamin (50 nM), toxins known to block some calcium‐dependent potassium conductances, did not affect the expression of the SOR and RD. 6. The SOR and RD were significantly reduced by Cd2+ (0.5 mM) but not by Ni2+ (0.25 mM). 7. Muscarine (10 microM) did not affect the SOR or the RD. 8. These results indicate that the SOR and RD depend upon a depolarization‐activated, sustained outward potassium current, which might be calcium dependent. A current with these characteristics has never been described before in the magnocellular system. Voltage‐clamp experiments are needed to completely characterize this potassium conductance selectively expressed by oxytocin neurones.

Enhanced neurotransmitter release at glutamatergic synapses on oxytocin neurones during lactation in the rat

1 The increased release of oxytocin during lactation has been shown to be dependent upon glutamatergic transmission and is associated with an increased synaptic innervation of the supraoptic nucleus (SON). 2 To determine whether the glutamatergic synaptic properties of oxytocin neurones are changed during lactation, we recorded excitatory postsynaptic currents (EPSCs) from identified oxytocin neurones in the SON of slices taken from adult virgin and lactating rats. 3 The frequency of AMPA‐mediated miniature EPSCs (mEPSCs) more than doubled during lactation. In addition, the decay time constant, but not the amplitude of the mEPSCs was significantly increased in both vasopressin and oxytocin neurones. 4 Paired‐pulse facilitation (PPF) was significantly reduced in oxytocin neurones during lactation, whereas no change was observed in vasopressin neurones. Elevating Ca2+ reduced PPF in oxytocin neurones in virgin rats but did not alter PPF in oxytocin neurones from lactating rats. 5 Collectively, our results suggest that excitatory glutamatergic transmission is strengthened in oxytocin neurones during lactation, probably by a combination of an increased number of terminals, slower decay kinetics, and an increase in the probability of release.

Electrophysiological Distinctions Between Oxytocin and Vasopressin Neurons in the Supraoptic Nucleus

Oxytocin and vasopressin neurons can be differentiated from one another, and from neurons in the immediately adjacent perinuclear zone, by their electrophysiological properties. In both sexes, oxytocin and vasopressin neurons are characterized by a prominent transient outward rectification which is conspicuously lacking in most perinuclear neurons. In addition, perinuclear neurons, some of which project to the supraoptic nucleus, exhibit a transient depolarization which underlies short bursts of spikes. Oxytocin neurons are characterized by: 1) the presence of a sustained outward rectifier above -50 mV, active below spike threshold; 2) a rebound depolarization following deactivation of the sustained rectification which can sustain short spike trains; and 3) a smaller transient outward rectification, probably associated with the potassium current, Ia. Vasopressin neurons show little of the sustained outward rectification and rebound depolarization, but have a stronger transient outward rectification. Although both cell types exhibit depolarizing afterpotentials, in vasopressin neurons these lead to plateau potentials underlying prolonged discharges. In oxytocin neurons, the depolarizing potential usually sustains a short spike discharge, but less often leads to prolonged bursts. These data suggest that the intrinsic properties of oxytocin and vasopressin neurons lead to quantitatively different forms of burst discharges, both of which may facilitate hormone release.

Chapter 2.1.3 Phenotypic and state-dependent expression of the electrical and morphological properties of oxytocin and vasopressin neurones

Oxytocin and vasopressin secreting neurones of the hypothalamic supraoptic nucleus share many membrane characteristics and a roughly similar morphology. However, these two neurone types differ in the relative expression of some intrinsic and synaptic currents, and in the extent of their respective dendritic arbors. Spike depolarizing afterpotentials are present in both types, but more frequently give rise to prolonged burst discharges in vasopressin neurones. Oxytocin, but not vasopressin neurones, are characterized by a depolarization-activated, sustained outward rectifier which turns on near spike threshold, and which can produce prolonged spike frequency adaptation. When this sustained current is deactivated by small hyperpolarizing pulses, a rebound depolarization sufficient to evoke short spike trains follows the offset of these pulses. Both oxytocin and vasopressin neurones exhibit a transient outward rectification underlain by an Ia-type current. This transient rectifier delays spiking to depolarizing stimuli from a relatively hyperpolarized baseline, and is more prominent in vasopressin neurones. As a result, oxytocin neurones may be more reactive to depolarizing inputs. Both cell types receive glutamatergic, excitatory synaptic inputs and both possess R,S- alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor subtypes. The AMPA receptor channel on both cell types is characterized by a relatively high calcium permeability and voltage-dependent rectification, characteristic of a diminished presence of the GluR2 AMPA subunit. However, AMPA-mediated synaptic transients are larger, and decay faster, in oxytocin compared with vasopressin neurones, suggesting a potential difference for synaptic integration. The characteristics of NMDA-mediated synaptic transients are similar in oxytocin and vasopressin neurones, but some data suggest NMDA receptors may be less involved in the glutamatergic activation of oxytocin neurones. In both cell types, synaptic release of glutamate often coactivates AMPA and NMDA receptors. The dendritic morphology of oxytocin and vasopressin neurones in female rats differs from one another and exhibits considerable plasticity as a function of endocrine state. In virgin rats, oxytocin neurones have more dendritic branches and a greater total dendritic length compared with lactation, when the arbor is much less extensive. A complementary change occurs in vasopressin dendrites, which are more extensive during lactation. This reorganization suggests that oxytocin neurones may be more electronically compact during lactation. In addition, such dramatic shifts in overall dendritic length imply that significant gains and losses in either the total number of synapses, or in synaptic density, are incurred by both cell types as a function of reproductive state.

Activation of postsynaptic GABAB receptors modulate the firing activity of supraoptic oxytocin and vasopressin neurones: role of calcium channels.

Oxytocin and vasopressin release from neurohypophysial terminals is closely related to the firing activity of magnocellular neurones in the supraoptic (SON) and paraventricular nuclei. It is well established that activation of GABAA receptors potently inhibits the activity of SON neurones and, thus, hormone release. However, whether postsynaptic GABAB receptors are expressed in magnocellular neurones, and the role they play in controlling their firing activity, is still controversial. In the present work, we combined immunohistochemical and electrophysiological techniques to determine whether activation of GABAB receptors in identified oxytocin and vasopressin neurones modulates their firing activity. Patch‐clamp recordings from SON neurones were obtained either in the slice preparation or from acutely dissociated neurones. Activation of GABAB receptors with the selective agonist baclofen (10u2003µm) inhibited voltage‐gated Ca2+ currents, reduced the duration of individual action potentials, as well as the magnitude of the hyperpolarizing after‐potential. SON firing activity was reduced by baclofen, and effect that was accompanied by a small membrane hyperpolarization. The inhibition of firing discharge persisted in the presence of synaptic blockade media, and was also observed in acutely dissociated SON neurones. Finally, GABAB‐mediated modulation of firing activity was largely blocked by the Ca2+ channel blocker Co2+ (2u2003mm). In general, baclofen modulatory actions were significantly larger, or observed more predominantly, in vasopressin neurones. In summary, these results support the expression of functional postsynaptic GABAB receptors in SON neurones, activation of which efficiently modulates neuronal excitability, in a Ca2+‐ and cell‐type dependent manner.

Cellular Properties of Autonomic-Related Neurons in the Paraventricular Nucleus of the Hypothalamus

The paraventricular nucleus of the hypothalamus (PVN) is a complex area composed of functionally different subsets of neurons, including magnocellular neuroendocrine, parvocellular neuroendocrine and parvocellular pre-autonomic neurons. Accumulating evidence indicates that PVN pre-autonomic neurons play important roles in cardiovascular control, both under physiological and pathological conditions. Despite this growing evidence, fundamental information on the cellular mechanisms controlling neuronal excitability in PVN pre-autonomic neurons is still missing. Using a multilevel experimental approach that combines neuronal tract tracing, in vitro patch clamp recordings, three dimensional neuronal reconstruction and immunohistochemistry, we have recently started to characterize the cellular properties of pre-autonomic PVN neurons that innervate medullary autonomic centers. PVN neurons innervating the dorsal vagal complex are characterized by a prominent low threshold spike (LTS), mediated by the activation of Ni2+sensitive, low-threshold Ca2+ conductance (IT). The LTS can efficiently modulate the ability of these neurons to fire in different pattern modes. Morphologically, PVN pre-autonomic neurons have medium to large somata and a bipolar or multipolar dendritic configuration with a relatively high degree of branching. Interestingly, the axons of the majority of these neurons originated from a primary dendrite, instead of originating from the soma with a typical axon hillock. In summary, these studies provide for the first time a detailed characterization of the basic cellular properties underlying neuronal excitability in PVN pre-autonomic neurons, providing a basis for the study of their involvement in the patho-physiology of hypertension and congestive heart failure disorders.

Postsynaptic GABAB receptors in supraoptic oxytocin and vasopressin neurons.

Publisher Summary This chapter describes the expression and function of postsynaptic gamma-aminobutyric acid (GABA B ) receptors in magnocellular neurons, using a combination of in vitro patch-clamp recordings and immunohistochemical approaches. GABA B receptor is an unusual G-protein-coupled receptor, composed of two seven transmembrane domain subunits, GABA B R1 and GABA B R2, respectively. Heteromerization of these subunits results in a functional receptor at the cell surface. Immunohistochemical techniques were used to study the expression of GABA B receptor subunits in identified oxtytocin (OT) and vasopressin (VP) neurons. Triple fluorescent immunohistochemical experiments were performed in 30 μm coronal hypothalamic brain slices obtained from Sprague–Dawley male and female rats. Antibodies raised against GABA B R1 and/or GABA B R2 receptor subunits were combined simultaneously with antibodies raised against OT and VP neurophysins. Colocalization of the three antibodies in individual supraoptic (SON) and paraventrieular nuclei (PVN) neurons was assessed. GABAB receptor subunit immunoreactivity was quantified using an optical density analysis. The electrical activity of SON neurons was recorded using the perforated patch-clamp configuration, which allows electrical access to the cell, preventing the dialysis of the intracellular environment. GABA B receptor immunoreactivity was significantly stronger in VP, as compared to OT neurons, suggesting that the expression of this receptor in the magnocellular neuroendocrine system is cell-type dependent.