Reciprocal synaptic relationships between angiotensin II-containing neurons and enkephalinergic neurons in the rat area postrema

A preembedding double immunostaining technique was used to study synaptic relationships between angiotensin-II-like immunoreactive and enkephalin-like immunoreactive neurons in the rat area postrema. The angiotensin-II-like immunoreactive neurons were detected by silver-gold intensification of the DAB reaction results while the enkephalin-like immunoreactive neurons were detected by simple ABC-DAB reaction. The synaptic relationships were reciprocal between the two neurons. Most of the synapses found between these two neurons were the presynaptic enkephalin-like immunoreactive axon terminals that made synapses on the angiotensin-II-like immunoreactive perikarya and dendrites. Both the axo-somatic and axo-dendritic synapses were symmetrical. However, although angiotensin-II-like immunoreactive axon terminals also made synapses on enkephalin-like perikarya and dendrites, the axo-somatic synapses were symmetrical, while the axo-dendritic synapses were asymmetrical. The present results confirm the presence of angiotensin-II-like immunoreactive neurons in the area postrema and suggest that these angiotensinergic neurons in the area postrema may play a role in the regulation of blood pressure via coordinated synaptic interactions with enkephalinergic neurons.     

Reciprocal synaptic relations between enkephalinergic and GABAergic neurons in the area postrema of the rat

A preembedding, double immunostaining technique was used to study synaptic relations between enkephalinergic and GABAergic neurons in the area postrema of the rat. As a main result, the enkephalinergic dendrites received many synapses from GABAergic axon terminals, and most of the synapses were symmetrical. Enkephalinergic neuronal perikarya received a few synapses from GABAergic axon terminals, and a few enkephalinergic axon terminals were found presynaptic to GABAergic neurons. Synapses between enkephalinergic profiles were frequent, but no axo-axonic synapses were seen. These findings suggest that GABAergic innervation of enkephalinergic neurons is the main relation between the two kinds of neurons in the area postrema. The synapses between the ankephalinergic axon terminals and GABAergic neurons might be explained as being part of the local servo system of the area postrema.     

Neural and vascular ultrastructure of the area postrema in the rat

The early development of the disynaptic cutaneous reflex pathway in the brachial spinal cord of rat fetuses was investigated both light and electron microscopically. The spinal cord areas which contain the neurons (association interneurons and lateral motor nucleus neurons) involved in this reflex pathway were identified in both neurofibrillar (silver-stained) and semithin plastic embedded specimens. Equivalent areas were identified in adjacent ultrathin preparations for electron microscopy and micrographs of these sample areas were taken of specimens at embryonic days 13.5–19.5 as well as for postnatal day 4 and adult specimens. The relative volume of association and dorsolateral motor neuropils occupied by synaptic boutons in these micrographs was determined using stereological methods. The mean number of synaptic junctions and boutons per 100 μ² was also determined for the two neuropil areas at each developmental stage. These analyses show that synaptic junctions and boutons occur in the dorsolateral motor neuropil before they can be found in association neuropil. Synapses within motor neuropil are first seen at embryonic day 13.5. Synaptogenesis in the dorsolateral motor neuropil is temporally correlated with the presence of numerous fibers which, in silver-stained specimens, can be seen to arise from the association interneurons and to course into the dorsolateral aspects of the motor nucleus. Synaptic junctions and boutons are not observed in the association neuropil until embryonic day 14.5 and this correlates well with the time when collaterals of the primary afferent fibers can first be observed penetrating into the dorsal half of the intermediate zone from the anlage of the dorsal funiculus. The precocious development of synaptic junctions in dorsolateral motor neuropil in comparison to the association neuropil continues throughout the developmental period examined. These data support a retrograde pattern of synaptogenesis in the early development of the spinal cutaneous reflex pathway. Association interneurons make synaptic contacts with motor neurons before synapses are formed between primary sensory neurons and association cells. This sequence is the reverse of the normal flow of nerve impulses through spinal reflex pathways.     

Actions of angiotensin on area postrema of the rat

Previous studies have reported that rats drink more saline after area postrema has been removed. The results presented here indicate that prolonged administration of angiotensin II into area postrema of unrestrained rats of 4 pmol/h also caused them to drink more saline. They drank more when angiotensin was released in the anterolateral part of the organ than when it was released anteromedially. Diurnal variation of drinking was not disordered. Dose-response curves showed that rats lacking area postrema drank more saline in response to systemic angiotensin than sham operated animals. The very large ‘spontaneous’ consumption of saline by rats lacking area postrema was not diminished by saralasin, an angiotensin antagonist. It is concluded that area postrema is a site where systemic angiotensin can act to promote sodium consumption: and that although removing area postrema increases the sensitivity of the drinking response to systemic angiotensin, this enhanced sensitivity is not the cause of the increased sodium consumption.     

The reciprocal synaptic relations between enkephalinergic neurons and catecholaminergic neurons in the area postrema

A preembedding double immunostaining technique using antibodies against methionine-enkephalin and tyrosine hydroxylase was used to study synaptic relations between enkephalinergic and catecholaminergic neurons in the area postrema of the rat at the electron microscopic level. The large nuclei-containing cell bodies of the catecholaminergic neurons displayed well-developed Golgi apparatus. The catecholaminergic somata and dendrites received synapses from ankephalinergic axon terminals, and most of the synapses were symmetrical. Occasionally, the catecholaminergic axon terminals were also found to be presynaptic to the enkephalinergic dendrites. Because the enkephalinergic neurons have been reported to be involved in cardiovascular function and the catecholaminergic neurons involved in the vomiting behavior, the synapses observed in this study may provide morphological evidence of the relationship between the vomiting and cardiovascular functions that are triggered in the area postrema.     

GABAergic neurons in the rat hippocampal formation: ultrastructure and synaptic relationships with catecholaminergic terminals

Numerous studies indicate that gamma-aminobutyric acid (GABA) can either hyperpolarize or depolarize hippocampal pyramidal and granule cells. While the inhibitory action of GABA may occur directly on these cells, the excitatory action may be mediated by interactions of GABAergic neurons with each other or with catecholaminergic afferents. We sought to examine the cellular basis for these interactions and their relative frequency. Thus, the ultrastructural morphology of GABAergic neurons and their relation to terminals exhibiting immunoreactivity for the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) were examined in the rat hippocampal formation using combined immunoautoradiographic and peroxidase-antiperoxidase labeling methods. By light microscopy, GABAergic perikarya and processes codistributed most noticeably with TH-containing processes in the hilus of the dentate gyrus (DG) and in strata lucidum, radiatum, and lacunosum-moleculare of the CA3 region of the hippocampus. Thus, these regions were examined further by electron microscopy. In the ultrastructural analysis, GABA-like immunoreactivity (GABA-LI) was detected in neuronal perikarya, dendrites, axons, and axon terminals. The GABA-containing perikarya were large, ovoid (20-40 microns in diameter), and contained abundant cytoplasm and an indented nucleus with one nucleolus. Synaptic junctions on the perikarya and dendrites with GABA-LI were both symmetric and asymmetric. Approximately equal numbers of TH-labeled terminals (19% of 133 in DG; 39% of 26 in CA3) and GABA-containing terminals (19% DG, 15% CA3) formed synapses with GABA-labeled perikarya. The remainder of the presynaptic terminals (62% DG, 46% CA3) were unlabeled, i.e., contained unidentified transmitters. Terminals with GABA-LI (0.5-1.6 microns) contained numerous small clear vesicles and from 0 to 2 large dense-core vesicles. The types of associations formed by terminals with GABA-LI were remarkably similar in the DG and hippocampus proper despite differences in intrinsic cell type and function. Terminals with GABA-LI formed associations with unlabeled perikarya and dendrites (24% of 151 in DG, 25% of 75 in CA3) and synapses with GABA-containing perikarya and dendrites (18% DG, 5% CA3). Additionally, GABAergic terminals converged upon the same perikarya or dendrite as a TH-containing terminal (15% DG, 21% CA3) and were in direct apposition to TH-labeled terminals (19% DG, 20% CA3). The remaining GABAergic terminals (24% DG, 28% CA3) were without any apparent synaptic relations. In both the DG and CA3, the junctions formed by GABAergic terminals were symmetric. Terminals showing colocalization of GABA-LI and TH-I were also detected although rarely.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of parabrachial stimulation on angiotensin and blood pressure sensitive area postrema neurons

Subpopulations of neurons in the area postrema (AP) and commissural nucleus tractus solitarius (NTS) have been identified according to their responses to systemic angiotensin-II (ANG-II) and increases in blood pressure (BP). In order to further define the functional connections of these subpopulations of cells, electrophysiological single unit recording studies have been done to determine the orthodromic effects of parabrachial nucleus (PBN) stimulation on these functionally defined cell groups. Orthodromic effects were seen in a similar proportion of ANG-II sensitive neurons in the AP (31.5%) and NTS (31%). PBN stimulation influenced a similar percentage of BP sensitive neurons in the AP (35%), although a larger proportion of this group of NTS cells was affected (55.5%). Twenty-five percent of ANG-II/BP sensitive neurons in the AP were orthodromically influenced, and 71.5% of this group of NTS neurons were affected by PBN stimulation. Small proportions of the neurons in the unaffected subpopulation of AP (10%) and NTS (27%) were also orthodromically affected by PBN stimulation. The remaining neurons in each group were not affected. This study suggests that there is no apparent preferential distribution of excitatory or inhibitory PBN efferents to any of the identified subpopulations of AP and NTS neurons.     

Inhibitory synaptic transmission in area postrema neurons of the rat showing robust presynaptic facilitation mediated by nicotinic ACh receptors

Inhibitory synaptic transmission and its modulation in neurons of the area postrema (AP), one of autonomic nuclei in the medulla, were studied using whole-cell patch-electrodes applied to slices from rats on postnatal days 10-24. When glycine (100 microM) or GABA (10 microM) was applied to AP neurons from a "Y tube", large outward currents that showed reversal potential of - 67 mV (approximate Cl- equilibrium potential estimated) were induced. At a holding potential of - 10 mV, application of high K+ to the AP neurons evoked massive inhibitory postsynaptic currents (IPSCs) in the neurons. Most of the evoked synaptic currents were blocked by bicuculline, while the remaining currents were sensitive to strychnine, indicating that the major inhibitory transmission in the area postrema was GABAergic. When nicotine (5-100 microM) was applied to AP neurons, robust IPSCs having GABAergic identity were evoked. Even in the presence of tetrodotoxin, nicotine could induce GABAergic IPSCs, most of which, however, disappeared in the presence of 5 mM Mg2+. Presynaptic facilitation was also induced by other nicotinic agonists, including cytisine, 1,1-dimethyl-4-phenyl-piperazinium iodide, ACh and choline. The nicotine-induced presynaptic facilitation was inhibited by mecamylamine and slightly inhibited by dihydro-beta-erythroidine or alpha-Bungarotoxin. These results indicate that nicotinic receptors are expressed at GABAergic presynaptic terminals in the area postrema and induce Ca2+ influx to trigger vesicular release. The major nicotinic receptors involved are thought to be heteromeric subtypes such as alpha3beta4 receptors, which may regulate inhibitory transmission potently responding to endogenous or exogenous nicotinic agents appeared in this area.     

Chemosensitive neurons in the area postrema of the rat and their possible functions

The present study is an attempt to demonstrate chemosensitive neurons within the area postrema (AP) electrophysiologically. Three types of chemosensitive neurons were identified: 1) glucose-responsive neurons that may participate in control of blood glucose and satiation, 2) sodium (osmotic pressure)-responsive neurons that may contribute to control of sodium and water balance of the body fluid and may be involved in salt appetite, 3) nausea-related neurons which respond to excess distension of stomach and LiCl as well. They may play a role in formation of conditioned taste aversion.     

Membrane properties of area postrema neurons

Intrinsic membrane properties, voltage-dependent sodium and voltage-dependent potassium currents of area postrema neurons in culture have been characterized with respect to their voltage dependence, time dependence and sensitivity to specific blocking agents. The area postrema is a hindbrain circumventricular organ which is known to have an important role in the central regulation of cardiovascular function. This study is the first to describe the biophysical properties of ion channels present in rat area postrema neurons. Recordings in current-clamp mode revealed a mean resting membrane potential of -55.0 ± 1.6 (n = 24) mV and an input resistance of 213.6 ± 23 M Ω. For the 24 neurons tested, the evoked action potential had a mean threshold of 38.8 ± 2 mV and a mean amplitude of 107.3 ± 15 mV. Our results show that the area postrema possesses only one principle sodium current which is completely abolished by 5 μM tetrodotoxin (TTX) (n = 28). This current activated near −50 mV and reached peak amplitude at −30 mV. The area postrema does not possess a TTX insensitive sodium current. The area postrema has at least two types of potassium currents. All area postrema neurons studied with tetraethylamonium (TEA) (n = 40) showed the presence of a slowly activating outward current which was present at voltages greater than −40 mV and was blocked by 10 mM TEA. In addition, 75% of the neurons studied (n = 30/40) also showed a rapidly inactivating, 4-AP sensitive IA type current which activated near −30 mV. Angiotensin II attenuated both the peak and the steady-state potassium currents, suggesting that angiotensin 11 may modulate area postrema activity by inhibiting voltage-gated potassium channels.     

Catecholamine and serotonin colocalization in projection neurons of the area postrema

A large population of the rat area postrema (AP) neurons which project to the parabrachial area (PBA) are serotonergic. Many AP neurons contain tyrosine hydroxylase (TH) and are presumed to be noradrenergic. However, it was not previously known whether TH-containing AP neurons also have projections to the PBA. TH-containing and serotonin (5-HT)-containing neurons have wide and overlapping distributions within the AP, and it was therefore possible that TH and 5-HT may be contained within the same AP neurons. In the present study immunohistochemical and retrograde axonal transport techniques were combined to determine whether TH-containing AP neurons project to the PBA and whether TH and 5-HT coexist in AP neurons. Adult male rats were given bilateral injections of the retrograde transport tracer, True blue (TB), into the PBA. After a 4-day survival period, vibratome sections of the caudal brainstem were processed for both TH and 5-HT immunohistochemistry. Examination of the sections revealed that over 25% of the 5-HT and over 30% of the TH-containing AP neurons were retrogradely labelled with TB. More surprising were our findings that many AP neurons displayed both TH and 5-HT immunoreactivity and that almost 40% of these double-labelled cells project to the PBA. Our results indicate that serotonin and noradrenaline coexist in a substantial proportion of the neurons of a major ascending viscerosensory pathway from the AP to the PBA.     

Direct synaptic contacts of medial septal efferents with somatostatin immunoreactive neurons in the rat hippocampus

Anterogradely labeled projections from the medial septum to hippocampal somatostatin immunoreactive (SOM-i) neurons were studied with double-label immunocytochemistry under light (LM) and electron microscopic (EM) conditions. Medial septal projections were identified after injecting the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) followed by immunohistochemical visualization of PHA-L presynaptic terminal labeling and concurrent immunocytochemical staining of SOM in hippocampal target cell bodies. This double-label procedure yielded blue-black nickel enhanced DAB stained, PHA-L-immunoreactive terminals on light brown SOM-i neurons that were investigated by correlative LM and EM observations. PHA-L-labeled terminal contacts with often basket-like appearance were localized with highest densities on soma and proximal dendrites of SOM-i neurons in stratum oriens of Ammon's horn and hilus of dentate gyrus, and some minor projections to stratum pyramidale and radiatum. Most double-labeled contacts could be identified as symmetric type synapses equally divided over soma and proximal dendrites of several forms of SOM-i neurons. These data indicate monosynaptic regulation of the hippocampal intrinsic SOM system by septal input, which probably represents a peptidergic subpopulation of the hippocampal GABAergic system.     

Extracellular recording in rat area postrema in vitro and the effects of cholinergic drugs, serotonin and angiotensin II

Spontaneous extracellular action potentials were recorded from rat area postrema explants in vitro for up to 6 h at 35 degrees C. Their geometric mean frequency was 4.4 +/- 1.7-11 Hz (n = 120) and they were most often recorded caudal to the obex. The frequency of spontaneously discharging units could be increased three-fold by raising the KCl concentration from 5 to 15 X 10(-3) M but a claimed non-specific excitant of neurones, L-glutamic acid at 10(-7)-10(-3) M was without effect. Carbamylcholine at 10(-9)-10(-7) M increased the frequency of spontaneous units (12/13 trials) as did 10(-7) M neostigmine sulphate (14/14 trials). The effects of carbamylcholine and neostigmine were additive and were blocked by atropine sulphate at 10(-6) M (18/18 trials). Atropine also stopped the discharge of spontaneous units while D-tubocurarine did not affect unit discharge frequency. It is suggested that units responding to cholinergic drugs have an afferent input from the dorsal vagus. A number of putative transmitters, serotonin (10(-9)-10(-7) M), angiotensin II (0.5 X 10(-10)-0.5 X 10(-9) M) and dopamine (10(-9)-10(-5) M) which on indirect grounds are thought to affect area postrema neurones, were without effect on unit discharge frequency.     

The microcirculation of the area postrema in the rat

The validity of current theories concerning the function of the area postrema ultimately rests on the nature of its microcirculation. Therefore a detailed study of the vasculature of the postremal region was undertaken in the rat. The findings indicate a singular arteriolar supply from branches of the posterior inferior cerebellar arteries. These vessels are caudal, intracranial branches of the vertebral arteries. Networks of enlarged capillaries are prominent features of sections taken through the area postrema. These enlarged capillary channels are re-entrant. At the borders of the structure they are joined by short interconnecting vessels to capillaries of smaller caliber typical of the medullary tissue. The bed is considered to be a portal system because two distinctive, serially connected capillary beds are interposed between artery and vein. No arteriovenous shunts or thoroughfare channels are observed in the rat, contrary to reports of this type of vessel in other species. This microcirculatory pattern would seem to be adequate to current theories of postremal function, which attribute neurosecretory or chemosensory functions to this region.     

Two distinct types of transient outward currents in area postrema neurons in rat brain slices

We investigated the electrophysiological properties of the area postrema neurons in acutely prepared rat brain slices using the whole-cell patch-clamp technique. Two different types of transient outward potassium current (I(to)), fast and slow, were found in the area postrema. Both the decay time constant and rise time were significantly faster in the fast I(to) than in the slow I(to). Both current-clamp and voltage-clamp recordings revealed that the activation of fast and slow I(to) contributes to generation of the different spiking patterns, late spiking and interrupted spiking, respectively. The activation and inactivation of both I(to) were strongly voltage-dependent. Curve fitting by the Boltzmann equation revealed no significant difference in the activation and inactivation curves for each I(to) except that the slope factor of inactivation was larger for fast I(to). Both I(to) were suppressed dose-dependently by application of 4-aminopyridine. Each spiking pattern was enhanced when cells were held at a more hyperpolarized membrane potential, i.e. a longer latency of the first spike or longer interspike interval between the first and second spikes. The voltage-dependent modulation of the spiking pattern was consistent with the voltage-dependent activation of I(to). The present study shows significant subdivisions of the area postrema neurons distinguished by a difference in the kinetics of I(to) and spiking patterns. We discuss the role of I(to) as the ionic current underlying neuronal excitability.     

Systematic glutamate induces degeneration of a subpopulation of tyrosine hydroxylase-immunoreactive neurons in the rat area postrema

Neuronal damage in the area postrema (AP) of 12–14-week-old male rats was induced by subcutaneous administration of monosodium glutamate (MSG). An immunocytochemical method was used to visualize catecholaminergic neurons in the AP after MSG-treatment. Some tyrosine hydroxylase-immunoreactive neurons exhibited marked signs of degeneration, while others appeared undamaged. We conclude that catecholamine-synthesizing neurons in the AP are differentially sensitive to the neuroexcitotoxic effect of systemic glutamate.     

Systemic glutamate induces degeneration of a subpopulation of tyrosine hydroxylase-immunoreactive neurons in the rat area postrema

Neuronal damage in the area postrema (AP) of 12-14-week-old male rats was induced by subcutaneous administration of monosodium glutamate (MSG). An immunocytochemical method was used to visualize catecholaminergic neurons in the AP after MSG-treatment. Some tyrosine hydroxylase-immunoreactive neurons exhibited marked signs of degeneration, while others appeared undamaged. We conclude that catecholamine-synthesizing neurons in the AP are differentially sensitive to the neuroexcitotoxic effect of systemic glutamate.     

Amylin receptor components and the leptin receptor are co‐expressed in single rat area postrema neurons

Amylin is a pancreatic β‐cell hormone that acts as a satiating signal to inhibit food intake by binding to amylin receptors (AMYs) and activating a specific neuronal population in the area postrema (AP). AMYs are heterodimers that include a calcitonin receptor (CTR) subunit [CTR isoform a or b (CTRa or CTRb)] and a member of the receptor activity‐modifying proteins (RAMPs). Here, we used single‐cell quantitative polymerase chain reaction to assess co‐expression of AMY subunits in AP neurons from rats that were injected with amylin or vehicle. Because amylin interacts synergistically with the adipokine leptin to reduce body weight, we also assessed the co‐expression of AMY and the leptin receptor isoform b (LepRb) in amylin‐activated AP neurons. Single cells were collected from Wistar rats and from transgenic Fos‐GFP rats that express green fluorescent protein (GFP) under the control of the Fos promoter. We found that the mRNAs of CTRa, RAMP1, RAMP2 and RAMP3 were all co‐expressed in single AP neurons. Moreover, most of the CTRa+ cells co‐expressed more than one of the RAMPs. Amylin down‐regulated RAMP1 and RAMP3 but not CTR mRNAs in AMY+ neurons, suggesting a possible negative feedback mechanism of amylin at its own primary receptors. Interestingly, amylin up‐regulated RAMP2 mRNA. We also found that a high percentage of single cells that co‐expressed all components of a functional AMY expressed LepRb mRNA. Thus, single AP cells expressed both AMY and LepRb, which formed a population of first‐order neurons that presumably can be directly activated by amylin and, at least in part, also by leptin.     

The sensitivity of hyperpolarization-activated cation current (Ih) to propofol in rat area postrema neurons

Area postrema neurons mediate various autonomic responses, including emesis. We examined the effects of propofol, a widely used anesthetic with antiemetic properties, on the hyperpolarization-activated cation current (Ih) in rat area postrema neurons using a slice patch-clamp technique. Although propofol suppressed Ih of area postrema neurons in a dose-dependent manner that was similar to what we observed for the hippocampal CA1 neurons, the IC50 for Ih in area postrema neurons (38 microM) was more than six times less than that found for hippocampal CA1 neurons (235 microM). We conclude that rat area postrema neurons are exquisitely sensitive to propofol. Given that reductions of Ih are associated with decreased excitability in neurons, we believe that the known antiemetic effects of propofol anesthesia are at least partly a result of a direct action on area postrema neurons to lower their excitability.     

Calcitonin gene-related peptide-like immunoreactive sensory fibers form synaptic contact with sympathetic neurons in the rat celiac ganglion

The present study demonstrates synaptic contact between calcitonin gene-related peptide (CGRP)-like immunoreactive axon terminals and sympathetic neurons in the rat celiac ganglion. Our observations suggest that sensory ganglion neurons directly regulate the sympathetic activity via synapses, because CGRP immunoreactive (CGRPI) fibers in this ganglion are supplied by the sensory ganglia.