Electrophysiological evidence that noradrenergic afferents selectively facilitate the activity of supraoptic vasopressin neurons

The functional role of the ascending projection from A1 noradrenergic neurons of the caudal ventrolateral medulla to the supraoptic nucleus of the hypothalamus was investigated by examining the effects of electrical stimulation of the A1 region on the activity of supraoptic neurons deemed to be vasopressinergic or oxytocinergic on the basis of basal firing patterns and responsivity to baroreceptor activation. A1 stimulation enhanced the activity of all putative vasopressin-secreting supraoptic neurons tested. This effect appeared to be selective in that no putative oxytocin-secreting neurons were excited by A1 stimulation. Destruction of the supraoptic noradrenergic terminal plexus by local application of the neurotoxin 6-hydroxydopamine abolished the facilitatory effects of A1 stimulation but did not noticeably alter basal activity patterns, nor the influence of baroreceptor inhibitory pathways. These findings suggest a facilitatory role for noradrenergic afferents in regulating the activity of neurohypophysially-projecting vasopressin neurons of the supraoptic nucleus.     

A1 noradrenergic neurons tonically inhibit sympathoexcitatory neurons of C1 area in rat brainstem

In rats anesthetized with urethane and paralyzed, bilateral microinjections of kainic acid (KA) into the region of caudal ventrolateral medulla (CVL) containing noradrenergic neurons of the A1 group (A1 area) elicited a decrease followed by an increase in arterial pressure (AP), heart rate (HR) and sympathetic renal nerve activity (RNA). The sympathoinhibitory and sympathoexcitatory effects of KA were prevented by bilateral microinjection of tetrodotoxin into an area of the rostral ventrolateral medulla (RVL) containing C1 adrenergic neurons (the C1 area). In contrast, the autonomic responses were not altered by interruption of the two other principal projections of A1 area neurons, namely to the hypothalamus or to the nucleus tractus solitarii. Bilateral microinjections of tyramine, clonidine, alpha-methylnoradrenaline or histamine into the C1 area elicited a dose-dependent, anatomically specific and reversible decrease in AP, HR and RNA. The effect of tyramine was blocked by previous microinjection of reserpine, 6-hydroxydopamine (6-OHDA), or phentolamine into the C1 area. Pretreatment with phentolamine unveiled a hypertensive effect of alpha-methylnoradrenaline. All effects of alpha-methylnoradrenaline were blocked by pretreatment of the C1 area with phentolamine plus DL-propranolol, whereas those elicited by histamine prevailed. Pretreatment of the C1 area with 6-OHDA abolished all changes in AP and HR elicited by microinjections of KA into the A1 area. We conclude that (1) neurons of the CVL tonically inhibit sympathetic activity, (2) this effect is mediated by an action upon vasomotor neurons of the C1 area of RVL, (3) the inhibition is mediated by noradrenergic projections from A1 neurons into the C1 area, and (4) this tonic sympathoinhibitory effect is independent of the baroreceptor reflex.     

The action of the A1 noradrenergic region on phasically firing neurons in the rat paraventricular nucleus

Electrical stimulation of the rat A1 noradrenergic region produced orthodromic inhibition (34%), excitation (23%) or excitation-inhibition sequence (25%) of the spontaneous activity of phasically firing units in the paraventricular nucleus of the hypothalamus (PVH). Excitatory responses could be distinguished into two types on the basis of their latency and duration. Both the primary and post-excitatory inhibitory responses were significantly attenuated by intravenous injection of propranolol, a beta-adrenoceptor antagonist, whereas neither type of excitatory response was affected.     

Inhibitory cardiovascular function of neurons in the caudal ventrolateral medulla of the rabbit: Relationship to the area containing A1 noradrenergic cells

Experiments were performed to test the hypothesis that A1 noradrenergic neurons, in the caudal ventrolateral medulla, have an inhibitory cardiovascular function. The lateral portion of the caudal medulla was systematically explored, using focal electrical stimulation, in anesthetized, paralyzed rabbits and arterial pressure and heart rate responses were recorded. Since electrical stimulation activates fibers of passage as well as neuronal cell bodies, we also determined cardiovascular responses to microinjections of L-glutamate, a neuroexcitatory amino acid with minimum effects on fibers of passage. Histological studies of stimulation sites were combined with catecholamine fluorescence histochemical studies to localize the A1 cells. A decrease in arterial pressure and heart rate, restricted to the region containing A1 cells, was observed with low frequency stimulation and with microinjections of L-glutamate. In contrast, when GABA, an inhibitory amino acid, was microinjected into the A1 region, we observed an increase in arterial pressure and heart rate. L-Glutamate and GABA responses were dose-related. The fall in pressure was independent of the fall in heart rate. Results support our hypothesis, providing evidence that neurons in the A1 area tonically inhibit sympathetic vasomotor tone.     

Prolactin-releasing peptide-immunoreactivity in A1 and A2 noradrenergic neurons of the rat medulla

Distribution of prolactin-releasing peptide-like immunoreactivity (PrRP-LI) was investigated in the rat medulla with the use of a rabbit polyclonal antiserum against the human PrRP-31 peptide. PrRP-positive neurons were noted mainly in two areas of the caudal medulla: ventrolateral reticular formation and commissural nucleus of the nucleus of the solitary tract (NTS), corresponding to the A1 and A2 areas. PrRP-LI neurons were absent in the medulla rostral to the area postrema. Double-labeling the sections with PrRP antisera and tyrosine hydroxylase (TH) monoclonal antibodies revealed extensive colocalization of PrRP- and TH-like immunoreactivity (TH-LI) in neurons of the A1 and A2 areas. Our results show that PrRP-LI is expressed in a population of A1 and A2 noradrenergic neurons of the rat caudal medulla.     

A1 noradrenergic action on medial preoptic-medial septal neurons: a neuropharmacological study

In an attempt to determine whether the excitatory and inhibitory orthodromic responses of single medial preoptic-medial septal (MPO-S) neurons to discrete electrical stimulation of the A1 noradrenergic region were mediated specifically by norepinephrine (NE) and involved different types of adrenoreceptors, a series of electrophysiological and neuropharmacological experiments was conducted. Extracellular single unit recording and local drug application techniques were used in female rats under urethane anesthesia. Chemical lesion of the catecholaminergic nerve terminal plexus in the medial preoptic area with 6-hydroxydopamine abolished both excitatory and inhibitory orthodromic effects of A1 region stimulation on MPO-S neurons, suggesting the noradrenergic nature of the effects. This conclusion was corroborated by the observation that the orthodromic effects were mimicked by locally applied exogenous NE. The excitatory effects were reliably mimicked by a low concentration of NE (0.5 mM; in-barrel concentration) and methoxamine (1.0 mM, an alpha-1 agonist), but not by either low or high concentrations (1 and 100 mM) of clonidine (an alpha-2 agonist) and isoproterenol (a beta agonist). The inhibitory orthodromic effects of A1 region stimulation were reliably mimicked by a high concentration of NE (50 mM), clonidine (100 mM) and isoproterenol (100 mM), but not by a low concentration of NE (0.5 mM), methoxamine (1 mM), clonidine (1 mM) or isoproterenol (1 mM). A high concentration (100 mM) of methoxamine mimicked the inhibitory effects less than 40% of the time. The low concentration (0.5 mM) NE-induced excitation that matched the excitatory orthodromic effect of A1 region stimulation was blocked by phentolamine (100 mM), an alpha blocker, but not by timolol (100 mM), a beta blocker. On the other hand, the high concentration (50 mM) NE-induced inhibition that matched the inhibitory orthodromic effect of A1 region stimulation was blocked by timolol, but not by phentolamine. Taken together, the present results are consistent with the hypotheses that the ascending noradrenergic projections from the A1 region affect the excitability of MPO-S neurons directly through NE and that the excitatory and inhibitory orthodromic effects involve different types of adrenoreceptors, i.e., alpha-1 and beta receptors, respectively.     

Effects of interleukin-1 and arachidonate on the preoptic and anterior hypothalamic neurons

Effects of microelectrophoretic application of ultrapure human interleukin-1 (IL-1), an endogenous pyrogen, on the activity of 80 neurons in the preoptic and anterior hypothalamus (PO/AH) were investigated in the urethane anesthetized rat. IL-1 predominantly decreased the activity of warm-sensitive neurons (15 of 19) and increased the activity of cold-sensitive neurons (10 of 12), but had no effect on 37 of 49 thermally insensitive neurons. The neuronal responses to IL-1 were blocked or attenuated by concurrent application of mepacrine (a phospholipase inhibitor) or sodium salicylate (a cyclooxygenase inhibitor). Local application of sodium arachidonate decreased the activity in 17 of 28 warm-units and excited 12 of 16 cold-units, and the effects of arachidonate were blocked by sodium salicylate. The results are compatible with the view that one or more cyclooxygenase metabolites of arachidonic acid are involved in the IL-1 induced fever.     

DSP-4: A novel compound with neurotoxic effects on noradrenergic neurons of adult and developing rats

The pharmacological actions of the compound N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP-4) are compatible with a specific neurotoxic effect on both peripheral and central noradrenergic neurons. The systemic injection of DSP-4 to adult rats transiently alters sympathetic neurons in the periphery but in the central nervous system the compound determines a marked and prolonged reduction of noradrenaline (NA) levels in all brain regions studied. When DSP-4 was injected systemically to rats at birth in doses ranging from 6.25 to 100 μg/g, no changes were found in peripheral sympathetic neurons 40 days later. On the contrary, in the same conditions and in relation to the dose injected, there were marked and persistent changes in the levels of NA in different regions of the brain. In the cerebral cortex and the spinal cord, the neonatal injection of DSP-4 produced a marked and long-lasting depletion of NA levels, similar to that observed after injection of the compound to adult rats. These changes were accompanied by a moderate increase in brain stem NA and a marked elevation of the amine in the cerebellum. These changes, different from the depletion observed in both regions when the compound was given to adult rats, are however similar to those observed after the neonatal injection of the neurotoxic compounds 6-hydroxydopamine or its precursor amino acid, 6-hydroxydopa. This indicates that probably central noradrenergic neurons respond in the same manner after different chemical injuries. DSP-4 crosses the placental barrier because when it was given to pregnant rats at the end of gestation, long-term changes were found in brain NA levels in their offspring, similar to those produced by the neonatal administration of the compound. This new neurotoxic compound provides a very useful tool for the study of noradrenergic neurons both in adult animals and during ontogenesis.     

Inhibitory action of the ventral noradrenergic bundle on the lateral hypothalamic neurons through alpha-noradrenergic mechanisms in the rat

Electrical stimulation of the ventral noradrenergic bundle (V-NA bundle) produced 3 types of responses in lateral hypothalamic neurons: IPSPs, a polysynaptic EPSP-IPSP sequence and antidromic spikes. The IPSPs were considered to be monosynaptic due to the fixed latencies seen at stimulus intensities. Iontophoretic application of an alpha-NA antagonist blocked only the presumed monosynaptic inhibition. Most of the glucose-sensitive neurons were inhibited by stimulation of the V-NA bundle. These results may account for the hyperphagia and obesity produced by selective lesions of the V-NA bundle.     

Effects of ovariectomy and hyperprolactinemia on tyrosine hydroxylase and dopamine-beta-hydroxylase activity in various limbic and hypothalamic structures

Rat pups were injected intracisternally with 20, 40 or 80 μg of 6-hydroxydopamine (6-OHDA) at various ages over the first 12 postnatal days in order to determine the critical period of the noradrenergic regenerative-sprouting response in the cerebellum. Twenty-four hours after the treatment NE fibers in the cerebellum had become extensively degenerated. NE levels were reduced by greater than 90–95% and histofluorescence microscopy revealed an absence of innervation except for lesioned axon stumps in the basal white matter and peduncles. The 80 μg dose produced considerable cellular degeneration in the locus coeruleus and no regenerative growth was seen to follow this treatment. Following the two lower doses, however, regenerative growth did occur. This was maximal in those rats treated closest to birth and declined progressively to become insignificant in rats which were treated on postnatal days 5–12, depending upon the cerebellar subregion. This decline in regenerative potential paralleled the time course for development of NE levels in control cerebella. For this reason the mechanism(s) controlling noradrenergic developmental and regenerative growth in the cerebellum appear to be similar. Such regenerative growth may thus be a useful model for the study of developmental growth of locus coeruleus axons. Contrary to the cerebellar projection, regenerative growth of the forebrain noradrenergic projection was not detected until the rats were between 7 and 12 days old at the time of treatment. This regeneration in the cerebral cortex was preceded by incomplete initial destruction of NE fibers there, in apparent similarity to regenerative growth described to occur in the adult rat forebrain ^4,7,32.     

Collateral axonal projections from ventrolateral medullary non-catecholaminergic neurons to central nucleus of the amygdala

Retrograde tract-tracing techniques were used to investigate whether catecholaminergic neurons in the ventrolateral medulla (VLM) send collateral axonal projections to both central nuclei of the amygdala (ACe) in the rat. Rhodamine-labelled latex microspheres or fluorogold (2%) were microinjected into the region of either the right or left ACe. After a survival period of 10–12 days, the rats were sacrificed and transverse sections of the brainstem were processed immunohistochemically for the identification of cell bodies containing the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH) or phenylethanolamine-N-methyltransferase (PNMT). Neuronal perikarya containing the retrogradely transported tracers were observed throughout the rostrocaudal extent of VLM, bilaterally. Approximately 10% of the retrogradely labelled neurons were observed to contain both retrograde tracers. The majority (79 ± 6.8%) of these double labelled neurons were located within the caudal VLM and their number decreased rostrally. In addition, the proportion of double labelled neurons to single labelled neurons in VLM decreased rostrally; approximately 11% in the caudal VLM and 6% in the rostral VLM. Furthermore, approximately 21% of all VLM neurons that projected to ACe were found to be catecholaminergic: 75% of these were immunoreactive to TH and 25% to PNMT. However, no neurons were found in VLM that contained both retrograde tracers and immunoreactivity to TH or PNMT. These data demonstrate that axons originating from non-catecholaminergic neurons in VLM bifurcate to innervate ACe bilaterally. Although the function of these VLM neurons that project to both ACe is not known, they may be the anatomical substrate by which VLM neurons relay simultaneously autonomic and/or visceral sensory information to influence the activity of ACe.     

Antidromic identification of neurons in the ventrolateral part of the medulla oblongata with ascending projections to the preoptic and anterior hypothalamic area (POA/AHA)

Seventy neurons in the ventrolateral medulla oblongata were antidromically activated by electrical stimulation of the preoptic and anterior hypothalamic area (POA/AHA) in female rats under urethane anesthesia. These identified cells were located within and adjacent to the nucleus reticularis lateralis and could be readily distinguished into at least two types of neurons, designated as ‘fast’ and ‘slow’ cells, on the basis of their waveform and conduction velocity.     

Medullary noradrenergic neurons projecting to the bed nucleus of the stria terminalis express mRNA for the NMDA-NR1 receptor

The bed nucleus of the stria terminalis pars ventralis (vBNST) receives dense noradrenergic terminals and contains the highest concentration of noradrenaline (NA) in the brain. We used autoradiography following retrograde axonal transport of [(3)H]-NA to identify selectively whether noradrenergic neurons innervating the vBNST originate in the medulla oblongata and/or the locus coeruleus. In combination with this technique, non-isotopic in situ hybridization for the NMDA-NR1 receptor subunit mRNA was used to examine, on the same brain sections, its expression in noradrenergic neurons that innervate the vBNST. The results showed that 60 +/- 6% and 35 +/- 7% of the total number of radiolabeled cells detected after injection of [(3)H]-NA in the vBNST were located in brainstems A1 and A2 noradrenergic cell groups, respectively. In addition, 18.5 +/- 4.2% of radiolabeled cells in A1 and 15.7 +/- 5% in A2 also expressed the mRNA for the NMDA-NR1 receptor subunit. In contrast, only 4 +/- 3% of the radiolabeled cells were present in the locus coeruleus, and none of these cells was positive to NMDA-NR1 receptor subunit mRNA. The present results provide evidence that BNST noradrenergic fibers and terminals originate predominantly from A1 and A2 noradrenergic cell groups, and that a significant number of these noradrenergic neurons also express the mRNA for the NMDA-NR1 receptor subunit. The observation that brainstem noradrenergic neurons innervating the vBNST express NMDA receptor mRNA gives anatomical support to the regulation of NA release by NMDA presynaptic receptors.     

Comparative effects of sciatic nerve stimulation, blood pressure, and morphine on the activity of A5 and A6 pontine noradrenergic neurons

The changes in the firing rate of A5 and A6 (locus coeruleus, LC) noradrenergic neurons induced by sciatic nerve stimulation, norepinephrine-induced elevations of blood pressure (BP) and systemic administration of morphine were studied in rats anesthetized with urethane, paralyzed and ventilated. Stimulation of the contralateral sciatic nerve with single shocks of low intensity (0.2 ms, 0.5 mA) produced a strong excitation of LC neurons with a latency of 14-18 ms. By contrast, shocks of similar intensities were ineffective in driving A5 cells. Higher stimulus intensities produced a low efficacy driving of A5 cells with a very long latency (120-250 ms). The efficacy of the stimulation could be increased by delivering trains of 4-8 stimuli but the latency remained very long. Norepinephrine-induced elevation of arterial pressure (140-160 mm Hg range) silenced the vast majority of A5 neurons. By contrast, the effects of blood pressure on locus coeruleus cells were of small amplitude, poorly reproducible and their time course was generally not correlated with the blood pressure alterations. Finally, the administration of 5 mg/kg of morphine i.v. silenced virtually all LC neurons while the majority of A5 cells were excited by the drug. These results provide evidence for the existence of a differential innervation of the two groups of pontine noradrenergic neurons investigated.     

Efferent connections from the lateral hypothalamic region and the lateral preoptic area to the hypothalamic paraventricular nucleus of the rat

The lateral preoptic and lateral hypothalamic regions contain the majority of the cell groups embedded in the fibre trajectories of the medial forebrain bundle on its course through the hypothalamus. Recent studies have extended considerably the parcellation of the lateral hypothalamic region, and, therefore, the need to emphasize new insights into the anatomical organisation of projections from the neurons of the lateral hypothalamic region. In the present study we describe the anatomical organisation of efferent projections from the lateral preoptic and lateral hypothalamic regions to the hypothalamic paraventricular nucleus (PVN) on the basis of retrograde- and anterograde-tracing techniques. Iontophoretic injections of the retrograde tracer, cholera toxin subunit B, into the PVN revealed that most hypothalamic nuclei project to the PVN. Within the lateral hypothalamic region, retrogradely labelled cells were concentrated in the intermediate hypothalamic area, the lateral hypothalamic area, and the perifornical nucleus, whereas fewer retrogradely labelled cells were found in the lateral preoptic area. To determine the distribution of terminating fibres in subnuclei of the heterogeneous PVN, iontophoretic injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin were delivered into distinct areas of the lateral hypothalamic region. Neurons of the intermediate hypothalamic area projected mainly to the PVN subnuclei, which contained parvicellular neuroendocrine cells. In contrast, neurons of the rostral and tuberal parts of the lateral hypothalamic area and the perifornical nucleus projected to the PVN subnuclei, which contained parvicellular neurons that send descending projections to preganglionic cell groups in the medulla and spinal cord. The perifornical nucleus was the only area within the lateral hypothalamic region that consistently innervated magnocellular perikarya of the PVN. Finally, all areas of the lateral hypothalamic region contributed substantially to fibres terminating in the perinuclear shell of the PVN. These results demonstrate that anatomically distinct areas of the lateral hypothalamic region have distinct projections to subnuclei of the PVN and further substantiate the view that the lateral hypothalamic region as well as the PVN constitute anatomically and functionally heterogeneous structures. © 1994 Wiley-Liss, Inc.     

Oxytocin Neurotransmission in the A1-area of the Brainstem Induces Hormonal Vasopressin Release in Rats

To investigate the role of the oxytocin innervation of the caudal ventrolateral medulla, immunocytochemical techniques were used to demonstrate the presence of oxytocin fibres and terminals in close apposition to noradrenergic neurons of the A1-area. Subsequently, in freely moving animals fitted with an indwelling jugular venous catheter and a bilaterally implanted chronic cannula in the A1-area, it was examined whether infusions of oxytocin in this area were able to influence hormonal vasopressin release. It appeared that nanomolar (50-500 nM) concentrations of oxytocin induce a fourfold rise in plasma vasopressin values. The specificity of this effect was established with control infusions of Ringer, vasopressin, and the addition of an antagonist to oxytocin. It was not possible to demonstrate a major role for oxytocin in the A1-area in the release of hormonal vasopressin occurring during haemorrhage. These data permit us to conclude that oxytocin acts on presumably noradrenergic neurons of the A1-area leading to the release of vasopressin into the peripheral circulation. The circumstances under which oxytocin is released in this area remain to be established.     

腺苷A1受体在双相呼气和吸气神经元电活动中的作用

目的探讨腺苷A1受体对双相呼气神经元和吸气神经元电活动的影响。方法制作新生大鼠体外延髓脑片标本，主要包含面神经后核内侧区(the medialregion of the nucleus retrofacialis，mNRF)，并保留舌下神经根的完整，以改良Kreb’s液灌流脑片，同步记录舌下神经根和双相呼气神经元/吸气神经元的放电活动。在灌流液中分别单独给予腺苷A1受体的特异性拮抗剂8-环戊-1，3-二丙基黄嘌呤(8-cyclopentyl-1，3-dipropylxa nthine，DPCPX)和特异性激动剂R-苯异丙基-腺苷(R—phenylisoprpyl—adeno sine，R-PIA)观察对神经元放电的影响。结果给予腺苷A1受体拮抗剂DPCPX后，双相呼气神经元/吸气神经元的呼吸周期和呼气时程明显缩短，单位放电峰频率显著性增大；给予相应激动剂R-PIA后，双相呼气神经元的呼气时程明显延长，放电频率和积分幅度显著降低，吸气神经元的放电时程和中期放电的频率和峰频率显著性降低，而早期和晚期的放电频率无明显改变。结论腺苷A1受体可能通过影响双相呼气神经元的电活动参与了呼吸时相的转换，并可能介导了吸气神经元的抑制性突触输入。     

Identification and characterization of estrogen receptor alpha-containing neurons projecting to the vicinity of the gonadotropin-releasing hormone perikarya in the rostral preoptic area of the rat.

Gonadal steroids exert a powerful regulatory influence upon the functioning of gonadotropin-releasing hormone (GnRH) neurons despite the apparent absence of gonadal steroid receptors in these cells. By using retrograde-tracing techniques combined with dual-labeling immunocytochemistry, we show here that distinct populations of estrogen receptor alpha (ERalpha)-containing neurons located in the hypothalamus and caudal brainstem project to the vicinity of the GnRH perikarya located in the rostral preoptic area (rPOA). The strongest estrogen-receptive afferent projection to this area originated from neurons located in the anteroventral periventricular and medial preoptic nuclei of the preoptic area. Approximately 50% of arcuate nucleus neurons projecting to the rPOA were demonstrated to synthesize either neuropeptide Y or beta-endorphin, but little evidence was found for ERalpha immunoreactivity in either of these specific subpopulations. Over 80% of all tyrosine hydroxylase-expressing neurons in the arcuate nucleus expressed ERalpha, but none projected to the rPOA. In the caudal brainstem, the A1 and A2 norepinephrine neurons comprised nearly all of the retrogradely labeled neurons. However, only the A2 afferents expressed ERalpha immunoreactivity, whereas the A1 afferents coexpressed neuropeptide Y. These observations, combined with the anterograde labeling data of others, provide neuroanatomical evidence for the existence of specific estrogen-receptive neuronal cell populations that project to the rPOA and may be involved in the estrogen-dependent transsynaptic regulation of GnRH neurons in the rat.