Noradrenergic excitation of magnocellular neurons in the rat hypothalamic paraventricular nucleus via intranuclear glutamatergic circuits

Abstract

Noradrenergic projections to the hypothalamus play a critical role in the afferent control of oxytocin and vasopressin release. Recent evidence for intrahypothalamic glutamatergic circuits prompted us to test the hypothesis that the excitatory effect of noradrenergic inputs on oxytocin and vasopressin release is mediated in part by local glutamatergic interneurons. The voltage response to norepinephrine (30-300 microM) was tested with whole-cell recordings in putative magnocellular neurons of the paraventricular nucleus (PVN) in hypothalamic slices (400 micrometers). Norepinephrine elicited an alpha1 receptor-mediated direct depolarization in 23% of the magnocellular neurons tested; however, the most prominent response, seen in 42% of the magnocellular neurons, was an alpha1 receptor-mediated increase in the frequency of EPSPs. The norepinephrine-induced increase in EPSPs was blocked by tetrodotoxin and by ionotropic glutamate receptor antagonists, suggesting that norepinephrine excited presynaptic glutamate neurons to cause an increase in spike-mediated transmitter release. The increase in EPSPs also was observed in a surgically isolated PVN preparation (64% of cells) and with microdrop applications of norepinephrine (1 mM, 33% of cells) and glutamate (0.5-1 mM, 28%) in the PVN, indicating that the norepinephrine-sensitive presynaptic glutamate neurons are located within the PVN. Biocytin injection and subsequent immunohistochemical labeling revealed that both oxytocin and vasopressin neurons responded to norepinephrine. Our data indicate that magnocellular neurons of the PVN receive excitatory inputs from intranuclear glutamatergic neurons that express alpha1-adrenoreceptors. These glutamatergic interneurons may serve as an excitatory relay in the afferent noradrenergic control of oxytocin and vasopressin release under certain physiological conditions.

Fig. 1.

Norepinephrine responses of PVN magnocellular neurons. A, Direct depolarization elicited by norepinephrine. A putative magnocellular neuron responded to bath application of norepinephrine (300 μm) with a 10 mV depolarization; no apparent change in input resistance was seen (data not shown). This cell had a resting membrane potential of −54 mV. Positive spikes are action potentials, which were truncated by digital filtering. Bar indicates the duration of the norepinephrine application. B, Increased EPSPs in norepinephrine. Bath application of norepinephrine (100 μm) elicited an increase in EPSPs (Norepinephrine) with a latency of ∼8 min in a putative magnocellular neuron recorded at resting potential. The response reversed after 20 min in normal aCSF (Wash). This cell had a resting membrane potential of −60 mV. Bottom traces are expanded recordings of the periods in the top traces indicated by thebars. C, Cumulative probability plots of the EPSP frequency and amplitude distributions in a representative neuron in control aCSF (•) and during norepinephrine application (▪). There is a significant shift toward higher instantaneous frequencies and larger amplitudes of the EPSPs in norepinephrine (p < 0.01; n = 5; Kolmogorov–Smirnov test). D, Changes in mean frequency and amplitude of EPSPs in norepinephrine. Mean frequencies and amplitudes were calculated in control medium and in 100 μm norepinephrine. These values were averaged, and the differences were expressed as percentage increase in norepinephrine. There was a 147% average increase in the mean frequency (n = 22) and a 53% average increase in the mean amplitude (n = 10) of EPSPs in norepinephrine.

Fig. 2.

The norepinephrine-induced increase in EPSPs is mediated by α₁-receptor activation. Bath application of norepinephrine (100 μm) caused a reversible increase in the frequency of EPSPs (Norepinephrine) in a putative magnocellular neuron. A second norepinephrine application in the presence of the α₁-receptor antagonist, prazosin hydrochloride (10 μm; Norepinephrine in Prazosin), failed to elicit the increase in EPSPs, suggesting that the response was mediated by the activation of α₁-adrenoreceptors. This cell had a resting membrane potential of −64 mV. The cell was subsequently found to be immunopositive for oxytocin and immunonegative for vasopressin, which is shown in Figure 7.

Fig. 3.

The norepinephrine-induced increase in EPSPs is caused by activation of local presynaptic excitatory neurons. Norepinephrine (100 μm) elicited a reversible increase in EPSPs (Norepinephrine) in a putative magnocellular neuron recorded at a resting membrane potential of −55 mV. The norepinephrine-induced EPSPs were blocked by TTX (Norepinephrine in TTX), suggesting that they were caused by the activation of presynaptic neuronal somata/dendrites, resulting in an increase in spike-mediated release of excitatory neurotransmitter. That the presynaptic neurons were intact in the slice suggests that they were cells with short axonal projections located in relative proximity to the recorded cell.

Fig. 4.

The norepinephrine-induced increase in EPSPs is mediated by presynaptic glutamate neurons. Norepinephrine (100 μm) caused an increase in the frequency of EPSPs in a magnocellular neuron. The response reversed after 21 min in wash (data not shown). The EPSPs elicited by norepinephrine were completely blocked by the NMDA and non-NMDA receptor antagonists AP5 (100 μm) and DNQX (50 μm), suggesting that they were mediated by the release of glutamate and the activation of ionotropic glutamate receptors. This cell had a resting membrane potential of −63 mV.

Fig. 5.

Norepinephrine-evoked increase in EPSPs in an isolated PVN preparation. A putative magnocellular neuron was recorded in a slice preparation in which the PVN had been surgically isolated from the rest of the slice (inset). Bath application of norepinephrine (100 μm) elicited a robust increase in the frequency of EPSPs (Norepinephrine), and the response reversed after 20 min in regular aCSF (Wash). This cell had a resting membrane potential of −65 mV. PVN, Paraventricular nucleus; Fx, fornix.

Fig. 6.

Norepinephrine microdrops in the PVN evoked an increase in EPSPs. Norepinephrine microdrop application in the PVN (inset) elicited a robust increase in EPSPs recorded in a putative magnocellular neuron. This effect was accompanied by a depolarization of the cell from its resting membrane potential of −64 to −55 mV. Bottom traces are expanded recordings of the periods in the top traces indicated by thebars. Top time calibration applies to top trace; bottom time calibration applies to expanded traces. NE, Norepinephrine; PVN, paraventricular nucleus;Fx, fornix; 3V, third ventricle.

Fig. 7.

Immunohistochemical double-labeling of norepinephrine-responsive oxytocin and vasopressin magnocellular neurons in the PVN. Magnocellular neurons that responded to norepinephrine with an increase in EPSPs were injected with biocytin and immunohistochemically processed with antibodies to oxytocin and vasopressin-associated neurophysin. A, A biocytin-injected, AMCA-labeled cell (A₁, arrow) was visualized under the AMCA filter combination (Biocytin). The same section was visualized under rhodamine filters to detect the vasopressinergic neurons (Vasopressin) and under FITC filters to see the oxytocinergic neurons (Oxytocin). This cell was immunopositive for vasopressin (A₂, arrow) and immunonegative for oxytocin (A₃, arrow), indicating that it was a vasopressinergic magnocellular neuron. B, Another magnocellular neuron that responded to norepinephrine with an increase in EPSPs (recordings shown in Fig. 2) and was labeled with biocytin (B₁, arrow) was immunonegative for vasopressin (B₂, arrow) and immunopositive for oxytocin (B₃, arrow), indicating that it was an oxytocinergic magnocellular neuron. The FITC–oxytocin label bleeds through the UV filters used to visualize the biocytin–AMCA label but is readily distinguished from the AMCA label by its intensity and its color (seen as gray scalehere). 200× magnification.

Fig. 8.

Model of the regulation of magnocellular neurons by norepinephrine. Norepinephrine projections, probably from the A1/A2 noradrenergic cell groups in the medulla, activate glutamatergic interneurons (GLU) within the PVN that send intranuclear excitatory projections to oxytocinergic and vasopressinergic magnocellular neurons (OXY/VP). Norepinephrine acts via α₁-adrenoreceptors to excite the glutamate interneurons, which leads to an increase in ionotropic receptor-mediated EPSPs in oxytocin and vasopressin neurons. PVN oxytocin and vasopressin neurons also receive direct noradrenergic inputs and express α₁-adrenoreceptors. 3V, Third ventricle.