Changes in oestrogen receptor alpha expression in the nucleus of the solitary tract of the rat over the oestrous cycle and following ovariectomy.

Oestrogen is capable of modulating autonomic outflow and baroreflex function via actions on groups of neurones in the brainstem. We investigated the presence of oestrogen receptor (ER) alpha in a part of the nucleus of the solitary tract (NTS) associated with central cardiovascular control, aiming to determine whether ERalpha mRNA and protein expression is correlated with levels of circulating oestrogen during the oestrous cycle. Polymerase chain reaction (PCR) detected ERalpha mRNA in the NTS at each stage of the oestrous cycle, from ovariectomised, sham-operated and male rats. Real-time PCR showed variations in ERalpha mRNA expression during the oestrous cycle, with the highest levels seen in oestrus, and lowest levels in metoestrus (P < 0.05 versus oestrus) and proestrus (P < 0.05 versus oestrus). Expression in males was lower than in dioestrus and oestrus females (P < 0.05). After ovariectomy, ERalpha mRNA levels were decreased compared to sham-operated animals (P < 0.01). Confocal fluorescence immunohistochemistry with stereological analysis showed that numbers of ERalpha immunoreactive cell nuclei per mm(3) of tissue in the caudal NTS were significantly greater in proestrus than in other groups of rats (P < 0.05). There were also differences among the groups in the extent of colocalisation of ERalpha in neurones immunoreactive for tyrosine hydroxylase and nitric oxide synthase. These results imply a complex pattern of region-specific oestrogen signalling in the NTS and suggest that ERalpha expression in this important autonomic nucleus may be related to circulating oestrogen levels. This may have consequences for the regulation of autonomic tone and baroreflex sensitivity when oestrogen levels decline, for example following menopause.

Inhibitory transmission in locus coeruleus neurons expressing GABAA receptor epsilon subunit has a number of unique properties.

Fast inhibitory synaptic transmission in the brain relies on ionotropic GABA(A) receptors (GABA(A)R). Eighteen genes code for GABA(A)R subunits, but little is known about the epsilon subunit. Our aim was to identify the synaptic transmission properties displayed by native receptors incorporating epsilon. Immunogold localization detected epsilon at synaptic sites on locus coeruleus (LC) neurons. In situ hybridization revealed prominent signals from epsilon, and mRNAs, some low beta1 and beta3 signals, and no gamma signal. Using in vivo extracellular and in vitro patch-clamp recordings in LC, we established that neuron firing rates, GABA-activated currents, and mIPSC charge were insensitive to the benzodiazepine flunitrazepam (FLU), in agreement with the characteristics of recombinant receptors including an epsilon subunit. Surprisingly, LC provided binding sites for benzodiazepines, and GABA-induced currents were potentiated by diazepam (DZP) in the micromolar range. A number of GABA(A)R ligands significantly potentiated GABA-induced currents, and zinc ions were only active at concentrations above 1 muM, further indicating that receptors were not composed of only alpha and beta subunits, but included an epsilon subunit. In contrast to recombinant receptors including an epsilon subunit, GABA(A)R in LC showed no agonist-independent opening. Finally, we determined that mIPSCs, as well as ensemble currents induced by ultra-fast GABA application, exhibited surprisingly slow rise times. Our work thus defines the signature of native GABA(A)R with a subunit composition including epsilon: differential sensitivity to FLU and DZP and slow rise time of currents. We further propose that alpha(3,) beta(1/3,) and epsilon subunits compose GABA(A)R in LC.

Differential expression of vesicular glutamate transporters by vagal afferent terminals in rat nucleus of the solitary tract: projections from the heart preferentially express vesicular glutamate transporter 1.

The central projections and neurochemistry of vagal afferent neurones supplying the heart in the rat were investigated by injecting cholera toxin B-subunit into the pericardium. Transganglionically transported cholera toxin B-subunit was visualized in the medulla oblongata in axons and varicosities that were predominantly aggregated in the dorsomedial, dorsolateral, ventrolateral and commissural subnuclei of the caudal nucleus of the solitary tract. Unilateral vagal section in control rats prevented cholera toxin B-subunit labeling on the ipsilateral side of the nucleus of the solitary tract. Fluorescent and electron microscopic dual labeling showed colocalization of immunoreactivity for vesicular glutamate transporter 1, but only rarely vesicular glutamate transporters 2 or 3 with cholera toxin B-subunit in terminals in nucleus of the solitary tract, suggesting that cardiac vagal axons release glutamate as a neurotransmitter. In contrast, populations of vagal afferent fibers labeled by injection of cholera toxin B-subunit, tetra-methylrhodamine dextran or biotin dextran amine into the aortic nerve, stomach or nodose ganglion colocalized vesicular glutamate transporter 2 more frequently than vesicular glutamate transporter 1. The presence of other neurochemical markers of primary afferent neurones was examined in nucleus of the solitary tract axons and nodose ganglion cells labeled by pericardial cholera toxin B-subunit injections. Immunoreactivity for a 200-kDa neurofilament protein in many large, cholera toxin B-subunit-labeled nodose ganglion cells indicated that the cardiac afferent fibers labeled are mostly myelinated, whereas binding of Griffonia simplicifolia isolectin B4 to fewer small cholera toxin B-subunit-labeled ganglion cells suggested that tracer was also taken up by some non-myelinated axons. A few labeled nucleus of the solitary tract axons and ganglion cells were positive for substance P and calcitonin gene-related peptide, which are considered as peptide markers of nociceptive afferent neurones. These data suggest that the population of cardiac vagal afferents labeled by pericardial cholera toxin B-subunit injection is neurochemically varied, which may be related to a functional heterogeneity of baroreceptive, chemoreceptive and nociceptive afferent fibers. A high proportion of cardiac neurones appear to be glutamatergic, but differ from other vagal afferents in expressing vesicular glutamate transporter 1.

Association of potassium channel Kv3.4 subunits with pre- and post-synaptic structures in brainstem and spinal cord.

Voltage-gated K+ channels (Kv) are divided into eight subfamilies (Kv1-8) and play a major role in determining the excitability of neurones. Members of the Kv3 subfamily are highly abundant in the CNS, with each Kv3 gene (Kv3.1-Kv3.4) exhibiting a unique pattern of expression, although single neurones can express more than one subtype. Of the Kv3 subunits relatively little is known of the Kv3.4 subunit distribution in the nervous system, particularly in the brainstem and spinal cord of the rat. We performed immunohistochemistry to determine both the cellular and sub-cellular distribution of the Kv3.4 subunit in these areas. Kv3.4 subunit immunoreactivity (Kv3.4-IR) was widespread, with dense, punctate staining in many regions including the intermediolateral cell column (IML) and the dorsal vagal nucleus (DVN), nucleus ambiguus (NA) and nucleus tractus solitarius (NTS). In the ventral horn a presynaptic location was confirmed by co-localization of Kv3.4-IR with the synaptic vesicle protein, SV2 and also with the glutamate vesicle markers vesicular glutamate transporter (VGluT) 1, VGluT2 or the glycine transporter GlyT2, suggesting a role for the channel in both excitatory and inhibitory neurotransmission. Electron microscopy confirmed a presynaptic terminal location of Kv3.4-IR in the VH, IML, DVN, NA and NTS. Interestingly however, patches of Kv3.4-IR were also revealed postsynaptically in dendritic and somatic structures throughout these areas. This staining was striking due to its localization at synaptic junctions at terminals with morphological features consistent with excitatory functions, suggesting an association with the postsynaptic density. Therefore the pre and postsynaptic localization of Kv3.4-IR suggests a role both in the control of transmitter release and in regulating neuronal excitability.

Differential co-localisation of the P2X7 receptor subunit with vesicular glutamate transporters VGLUT1 and VGLUT2 in rat CNS.

Presynaptic P2X(7) receptors are thought to play a role in the modulation of transmitter release and have been localised to terminals with the location and morphology typical of excitatory boutons. To test the hypothesis that this receptor is preferentially associated with excitatory terminals we combined immunohistochemistry for the P2X(7) receptor subunit (P2X(7)R) with that for two vesicular glutamate transporters (VGLUT1 and VGLUT2) in the rat CNS. This confirmed that P2X(7)R immunoreactivity (IR) is present in glutamatergic terminals; however, whether it was co-localised with VGLUT1-IR or VGLUT2-IR depended on the CNS region examined. In the spinal cord, P2X(7)R-IR co-localised with VGLUT2-IR. In the brainstem, co-localisation of P2X(7)R-IR with VGLUT2-IR was widespread, but co-localisation with VGLUT1-IR was seen only in the external cuneate nucleus and spinocerebellar tract region of the ventral medulla. In the cerebellum, P2X(7)R-IR co-localised with both VGLUT1 and VGLUT2-IR in the granular layer. In the hippocampus it was co-localised only with VGLUT1-IR, including in the polymorphic layer of the dentate gyrus and the substantia radiatum of the CA3 region. In other forebrain areas, P2X(7)R-IR co-localised with VGLUT1-IR throughout the amygdala, caudate putamen, striatum, reticular thalamic nucleus and cortex and with VGLUT2-IR in the dorsal lateral geniculate nucleus, amygdala and hypothalamus. Dual labelling studies performed using markers for cholinergic, monoaminergic, GABAergic and glycinergic terminals indicated that in certain brainstem and spinal cord nuclei the P2X(7)R is also expressed by subpopulations of cholinergic and GABAergic/glycinergic terminals. These data support our previous hypothesis that the P2X(7)R may play a role in modulating glutamate release in functionally different systems throughout the CNS but further suggest a role in modulating release of inhibitory transmitters in some regions.

Subcellular localization of neuronal nitric oxide synthase in the rat nucleus of the solitary tract in relation to vagal afferent inputs.

In the nucleus of the solitary tract (NTS), nitric oxide (NO) modulates neuronal circuits controlling autonomic functions. A proposed source of this NO is via nitric oxide synthase (NOS) present in vagal afferent fibre terminals, which convey visceral afferent information to the NTS. Here, we first determined with electron microscopy that neuronal NOS (nNOS) is present in both presynaptic and postsynaptic structures in the NTS. To examine the relationship of nNOS to vagal afferent fibres the anterograde tracer biotinylated dextran amine was injected into the nodose ganglion and detected in brainstem sections using peroxidase-based methods. nNOS was subsequently visualised using a pre-embedding immunogold procedure. Ultrastructural examination revealed nNOS immunoreactivity in dendrites receiving vagal afferent input. However, although nNOS-immunoreactive terminals were frequently evident in the NTS, none were vagal afferent in origin. Dual immunofluorescence also confirmed lack of co-localisation. Nevertheless, nNOS immunoreactivity was observed in vagal afferent neurone cell bodies of the nodose ganglion. To determine if these labelled cells in the nodose ganglion were indeed vagal afferent neurones nodose ganglion sections were immunostained following application of cholera toxin B subunit to the heart. Whilst some cardiac-innervating neurones were also nNOS immunoreactive, nNOS was never detected in the central terminals of these neurones. These data show that nNOS is present in the NTS in both pre- and postsynaptic structures. However, these presynaptic structures are unlikely to be of vagal afferent origin. The lack of nNOS in vagal afferent terminals in the NTS, yet the presence in some vagal afferent cell bodies, suggests it is selectively targeted to specific regions of the same neurones.

Somatostatin immunoreactivity in axon terminals in rat nucleus tractus solitarii arising from central nucleus of amygdala: coexistence with GABA and postsynaptic expression of sst2A receptor.

Axon terminals synapsing on neurones in the nucleus tractus solitarii (NTS) that originate from the central nucleus of the amygdala (CeA) have been shown to contain gamma-aminobutyric acid (GABA) immunoreactivity. Here we investigated whether such terminals also contain somatostatin (SOM), a neuropeptide found in axons distributed throughout the NTS and in somata in the CeA, and known to modulate cardiovascular reflexes when microinjected into the NTS. With fluorescence microscopy, SOM immunoreactivity was seen in the varicosities of some axons throughout the NTS that were anterogradely labelled with biotin dextran amine injected into the CeA. Such varicosities were frequently observed in close proximity to dendrites of NTS neurones that were immunoreactive for the SOM receptor sst(2A) subtype, and in many cases also for catecholamine synthesising enzymes. In the caudal, cardioregulatory zone of NTS, SOM immunoreactivity was localised by electron microscopic pre-embedding gold labelling to boutons containing dense-cored and clear pleomorphic vesicles and forming symmetrical synapses, mostly onto dendrites. Additional post-embedding gold labelling for GABA suggested that a subpopulation (29%) of GABAergic terminals sampled in this area of NTS contained SOM. Almost all boutons anterogradely labelled from the amygdala were GABA-immunoreactive (-IR) and 21% of these were SOM-IR. A similar proportion of these boutons (22%) formed synapses onto dendrites containing immunoreactivity for the SOM receptor sst(2A) subtype. These observations provide evidence that some of the GABAergic projection neurones in the CeA that inhibit baroreceptor reflex responses in the NTS in response to fear or emotional stimuli could release SOM, which might modulate the activity of NTS neurones via an action on sst(2A) receptors.

Projection sites of superficial and deep spinal dorsal horn cells in the nucleus tractus solitarii of the rat.

By using anterograde transport of biotin dextran amine injected into the cervical spinal dorsal horn, we have shown that fibres from superficial and deep dorsal horn project to the nucleus tractus solitarii via two distinct pathways. Afferent fibres from the superficial lamina (I-III) were found to course in the dorsal funiculus and terminate bilaterally in the caudal zone of the nucleus tractus solitarii (NTS), mainly within the commissural subnucleus. In contrast, afferents from the deeper dorsal horn laminae (IV-V) were found to course in the dorsolateral fasciculus and terminate ipsilaterally, mostly in the lateral areas of the caudal nucleus tractus solitarii. Similar, but more extensive patterns of labelled fibres were produced by injections into the white matter of the dorsal funiculus and dorsolateral fasciculus, respectively. These observations suggest that the caudal NTS not only serves as a location of visceral afferent convergence and integration, but may also be a receptive area for monosynaptic projections from dorsal horn neurons receiving sensory afferent inputs. Such projections may represent pathways through which NTS neurons are influenced by nociceptive and non-nociceptive information from the dorsal horn and thereby can co-ordinate the appropriate autonomic response, including adjustments in cardiorespiratory reflex output.

Neuronal P2X7 receptors are targeted to presynaptic terminals in the central and peripheral nervous systems.

The ionotropic ATP receptor subunits P2X(1-6) receptors play important roles in synaptic transmission, yet the P2X(7) receptor has been reported as absent from neurons in the normal adult brain. Here we use RT-PCR to demonstrate that transcripts for the P2X(7) receptor are present in extracts from the medulla oblongata, spinal cord, and nodose ganglion. Using in situ hybridization mRNA encoding, the P2X(7) receptor was detected in numerous neurons throughout the medulla oblongata and spinal cord. Localizing the P2X(7) receptor protein with immunohistochemistry and electron microscopy revealed that it is targeted to presynaptic terminals in the CNS. Anterograde labeling of vagal afferent terminals before immunohistochemistry confirmed the presence of the receptor in excitatory terminals. Pharmacological activation of the receptor in spinal cord slices by addition of 2'- and 3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP; 30 microm) resulted in glutamate mediated excitation of recorded neurons, blocked by P2X(7) receptor antagonists oxidized ATP (100 microm) and Brilliant Blue G (2 microm). At the neuromuscular junction (NMJ) immunohistochemistry revealed that the P2X(7) receptor was present in motor nerve terminals. Furthermore, motor nerve terminals loaded with the vital dye FM1-43 in isolated NMJ preparations destained after application of BzATP (30 microm). This BzATP evoked destaining is blocked by oxidized ATP (100 microm) and Brilliant Blue G (1 microm). This indicates that activation of the P2X(7) receptor promotes release of vesicular contents from presynaptic terminals. Such a widespread distribution and functional role suggests that the receptor may be involved in the fundamental regulation of synaptic transmission at the presynaptic site.

Immunohistochemical profiles of spinal lamina I neurones retrogradely labelled from the nucleus tractus solitarii in rat suggest excitatory projections.

Three morphologically distinct types of lamina I neurones, fusiform, flattened and pyramidal, project from the spinal cord to the caudal part of the nucleus tractus solitarii in the rat, and may represent a pathway whereby peripheral stimuli can modify autonomic functions. The neurochemistry of these three types of projection neurones was investigated using retrograde neuronal tracing with cholera toxin B-subunit combined with dual and triple immunofluorescence labelling for different neuroactive substances. None of the lamina I neurones with immunoreactivity for GABA or glycine were found to project to the nucleus tractus solitarii, whereas high levels of glutamate immunoreactivity, which may indicate a glutamatergic phenotype, were found in 18.4% of fusiform, 9.6% of pyramidal and 2.1% of flattened projection neurones. Immunoreactivity for calbindin-D28K was present in 34.9% of fusiform cells, 18.3% of pyramidal cells and 10.5% of flattened cells, and nitric oxide synthase immunoreactivity was detected in 13.8% of fusiform cells, 1.1% of pyramidal cells and 4.2% of flattened cells that had projections to the nucleus tractus solitarii. Calbindin immunoreactivity was co-localised in major subpopulations of projection neurones of each morphological type that contained glutamate immunoreactivity, whereas co-localisation of nitric oxide synthase immunoreactivity in these neurones was relatively uncommon. The pyramidal cell was the only retrogradely labelled cell type found to be immunoreactive for substance P, but few (<5%) of these neurones were immunolabelled. These data are consistent with the hypothesis that lamina I neurones projecting to the dorsal vagal complex are not inhibitory, and that some of them, belonging mostly to the fusiform and pyramidal types, may exert excitatory, glutamate- or substance P-mediated effects upon inhibitory interneurones in the nucleus tractus solitarii. Such excitatory pathways could be involved in the attenuation of the reflex control of blood pressure by both painful and innocuous peripheral stimuli, such as those arising in injury and exercise.

Gamma-aminobutyric acid receptor (GABA(A)) subunits in rat nucleus tractus solitarii (NTS) revealed by polymerase chain reaction (PCR) and immunohistochemistry.

Expression of mRNAs encoding seven GABA(A) receptor subunits (alpha1, alpha2, alpha3, alpha5, beta2, beta3, gamma2) in the nucleus tractus solitarii (NTS) of rat medulla oblongata was examined by reverse transcription-polymerase chain reaction (RT-PCR). All subunit mRNAs, except alpha5, were clearly detected. Band densities produced by alpha1, alpha3, beta3, and gamma2 subunits were greater than those corresponding to beta2 and alpha2 transcripts. The localization of these subunits in tissue sections through NTS was examined by immunohistochemistry. The differential patterns of immunoreactivity in neuronal somata and dendrites of NTS neurons were generally in agreement with the PCR results, confirming that mRNA expression is correlated with receptor protein synthesis. At ultrastructural level, alpha1, alpha3, beta2/3, and gamma2 subunits were localized in both cytoplasmic and subsynaptic sites, the latter often apposed to GABA immunoreactive synapses. These results suggest that ionotropic receptors comprising the alpha1, alpha3, beta2/3, and gamma2 may mediate inhibitory GABA responses in the NTS.

Neurochemistry of superficial spinal neurones projecting to nucleus of the solitary tract that express c-fos on chemical somatic and visceral nociceptive input in the rat.

We have investigated the presence of three neurochemical markers, glutamate, calbindin-D28k, and nitric oxide synthase, in spinal neurones that transmit chemical noxious inputs from both the skin and the viscera, by combining retrograde labelling with the fluorescent tracer Fluorogold with dual labelling immunohistochemistry. Neurones projecting to the nucleus of the solitary tract (NTS) that expressed Fos protein in response to cutaneous or visceral noxious stimulation were concentrated in lamina I of the cervical and lumbosacral segments, respectively. Although both labelled neuronal populations were numerous, the spino-solitary cells that transmit visceral nociceptive input predominated over those transmitting cutaneous nociceptive input. Calbindin-D28k-immunoreactivity was observed in neurones of three morphological types (fusiform, flattened, and pyramidal) projecting to the NTS that were activated by somatic or visceral nociceptive neurones. Nitric oxide synthase and glutamate immunoreactivities were present only in viscerally activated nociceptive neurones projecting to the NTS. Glutamate-immunopositive NTS-projecting cells were exclusively of the flattened type, and the nitric oxide synthase-immunolabelled NTS-projecting cells comprised 75%/fusiform cells and 25% flattened cells. These data suggest that the involvement of excitatory spinal lamina I projection neurones in the transmission of peripheral chemical nociceptive inputs to the NTS may be restricted to information of visceral origin.

Nitric oxide producing neurones in the rat medulla oblongata that project to nucleus tractus solitarii.

The production of nitric oxide in neurones of the rat medulla oblongata that project to the nucleus tractus solitarii (NTS) was examined by simultaneous immunohistochemical detection of nitric oxide synthase (NOS) and of cholera toxin B-subunit (CTb), which was injected into the caudal zone of the NTS. Neurones immunoreactive for CTb and neurones immunoreactive for NOS were widely co-distributed and found in almost all the anatomical divisions of the medulla. Dual-labelled cells, containing both CTb and NOS immunoreactivities were more numerous ipsilaterally to the injection sites. They were concentrated principally in the more rostral zone of the NTS, raphé nuclei, dorsal, intermediate and lateral reticular areas, spinal trigeminal and paratrigeminal nuclei and the external cuneate and medial vestibular nuclei. Isolated dual-labelled neurones were also scattered throughout most of the divisions of the reticular formation. These observations indicate that many areas of the medulla that are known to relay somatosensory and viscerosensory inputs contain NOS immunoreactive neurones that project to the NTS, and may, therefore, contribute to the dense NOS-immunoreactive innervation of the NTS. The release of nitric oxide from the axon terminals of these neurones may modulate autonomic responses generated by NTS neurones in relation to peripheral sensory stimuli, and thus ultimately regulate sympathetic and/or parasympathetic outflow.

A GABAergic projection from the central nucleus of the amygdala to the nucleus of the solitary tract: a combined anterograde tracing and electron microscopic immunohistochemical study.

The central nucleus of the amygdala is involved in the modulation of autonomic, somatic and endocrine functions, as well as behavioural responses to stressful stimuli. Anatomical and physiological studies have suggested that this nucleus sends projections to the nucleus of the solitary tract, the primary site of termination of vagal and glossopharyngeal afferent fibres in the brain stem. To determine the neurochemical nature of the amygdaloid input to the nucleus of the solitary tract, anterograde tracing with biotinylated dextran amine was combined with post-embedding immunogold labelling for GABA and glutamate immunoreactivities and with pre-embedding labelling for the vesicular GABA transporter. Following injection of biotin dextran amine into the central nucleus of the amygdala, anterogradely labelled axons and varicosities were found throughout the rostrocaudal extent of the nucleus of the solitary tract, particularly in the medial, ventral and ventrolateral subnuclei. The anterogradely labelled terminals were found to make predominantly symmetrical synaptic contacts with dendrites, and occasionally onto cell bodies and dendritic spines, and to contain immunoreactivity for GABA and for the vesicular GABA transporter. Immunolabelling of serial sections with antibodies to glutamate showed that none of these axon terminals contained high enough densities of gold particle labelling to suggest that they contained other than low metabolic levels of glutamate immunoreactivity. These results provide conclusive evidence for a GABAergic pathway from the central nucleus of the amygdala to the nucleus of the solitary tract. This GABAergic projection may provide a substrate for inhibition of lower brain stem visceral reflexes, including baroreflex inhibition, through which the central nucleus of the amygdala could participate in cardiovascular regulation related to emotional behaviour and the defence reaction.

P2X(2) receptor immunoreactivity in the dorsal vagal complex and area postrema of the rat.

Adenosine 5'-triphosphate (ATP) can function as a fast synaptic transmitter through its actions on ionotropic (P2X) and metabotropic (P2Y) receptors in neuronal tissue. The ionotropic receptors have been classified into seven subtypes (P2X(1)-P2X(7)) by molecular cloning. However, they are difficult to distinguish pharmacologically owing to an absence of specific agonists and antagonists. In this study we used neuroanatomical methods to determine the origin and neurochemical phenotype of the P2X(2) subtype of purinoceptor in the dorsal medulla of the rat. Using immunohistochemistry we observed dense networks of P2X(2) receptor immunoreactive labelled fibres and terminals in the dorsal vagal complex and area postrema, as well as labelled cell bodies in the dorsal vagal nucleus and the area postrema. The P2X(2) receptor was localized presynaptically in vagal afferent fibres and terminals in the nucleus tractus solitarius at the ultrastructural level by combining injections of an anterograde tracer (biotin dextran amine) into the nodose ganglion with pre-embedding immunogold visualization of P2X(2) immunoreactivity. Terminals immunoreactive for the P2X(2) receptor in the nucleus tractus solitarius were found to contain glutamate, but not GABA immunoreactivity by post-embedding immunogold-labelling techniques. In cell bodies in the area postrema, dual immunofluorescence also indicated that P2X(2) receptor immunoreactive cells are glutamatergic but not GABAergic. The P2X(2) receptor was localized to vagal preganglionic neurons in the dorsal vagal nucleus that were identified by prior intraperitoneal injections of the retrograde tracer FluoroGold. No cells immunoreactive for the P2X(2) receptor were observed in the nucleus tractus solitarius. The localization of P2X(2) receptor immunoreactivity presynaptically in vagal afferent terminals indicates that the receptor may be involved in modulating transmitter release from vagal afferent fibres. Furthermore, the presence of the P2X(2) receptor in vagal preganglionic cells and in glutamatergic cells of the area postrema implies that it may, respectively, play a role in regulation of vagal efferent cell activity and modulation of excitatory outputs from the area postrema to other brain regions.

Hypoxic enhancement of quantal catecholamine secretion. Evidence for the involvement of amyloid beta-peptides.

Prolonged exposure to hypoxia (10% O(2)) enhanced quantal catecholamine release evoked from O(2)-sensing pheochromocytoma (PC12) cells, as monitored using single-cell amperometric recordings. The enhancement of exocytosis was apparent after 12 h of hypoxia and was maximal at 24 h. Elevated levels of secretion were due to the emergence of a Ca(2+) influx pathway that persisted during complete blockade of known voltage-gated Ca(2+) channels. Secretion triggered by this Ca(2+) influx was severely reduced by known inhibitors of Alzheimer's amyloid beta-peptides (AbetaPs), including an N terminus-directed monoclonal antibody. The enhancing effect on secretion of chronic hypoxia was mimicked closely by direct application of AbetaP to cells under normoxic conditions, although the effects of AbetaP were more rapid at onset, being maximal after only 6 h. The present results suggest that prolonged hypoxia can induce formation of Ca(2+)-permeable AbetaP channels and that such induction can lead directly to excessive neurosecretion. This is a potential contributory factor to AbetaP pathophysiology following cerebral ischemia.

Glycine-immunoreactive synaptic terminals in the nucleus tractus solitarii of the cat: ultrastructure and relationship to GABA-immunoreactive terminals.

Postembedding immunogold labeling methods applied to ultrathin and semithin sections of cat dorsomedial medulla showed that neuronal perikarya, dendrites, myelinated and nonmyelinated axons, and axon terminals in the nucleus tractus solitarii contain glycine immunoreactivity. Light microscopic observations on semithin sections revealed that these immunoreactive structures were unevenly distributed throughout the entire nucleus. At the electron microscopic level, synaptic terminals with high levels of glycine-immunoreactivity, assumed to represent those releasing glycine as a neurotransmitter, were discriminated from terminals containing low, probably metabolic levels of glycine-immunoreactivity, by a quantitative analysis method. This compared the immunolabeling of randomly sampled terminals with a reference level of labeling derived from sampling the perikarya of dorsal vagal neurones. The vast majority of these "glycinergic" terminals contained pleomorphic vesicles, formed symmetrical synaptic active zones, and targeted dendrites. They appeared to be more numerous in areas of the nucleus tractus solitarii adjoining the tractus solitarius, but rather scarce caudally, medially, ventrally, and in the dorsal motor vagal nucleus. In a random analysis of the entire nucleus tractus solitarii, 26.2% of sampled terminals were found to qualify as glycine-immunoreactive. In contrast, boutons immunoreactive for gamma-aminobutyric acid (GABA) were more evenly distributed throughout the dorsal vagal complex and accounted for 33.7% of the synaptic terminals sampled. A comparison of serial ultrathin sections suggested three subpopulations of synaptic terminals: one containing high levels of both GABA- and glycine-immunoreactivities (21% of all terminals sampled), one containing only GABA-immunoreactivity (12.7%), and relatively few terminals (5.2%) that were immunoreactive for glycine alone. These results were confirmed by dual labeling of sections using gold particles of different sizes. This study reports the first analysis of the ultrastructure of glycinergic nerve terminals in the cat dorsal vagal complex, and the pattern of coexistence of glycine and GABA observed provides an anatomical explanation for our previously reported inhibitory effects of glycine and GABA on neurones with cardiovascular and respiratory functions in the nucleus tractus solitarii.

Labelling of rat vagal preganglionic neurones by carbocyanine dye DiI applied to the heart.

We describe a method for applying the carbocyanine dye DiI to the rat heart that takes advantage of the dye's lipophilic properties and its ability to diffuse easily into tissues, and results in specific retrograde labelling of cardiac vagal preganglionic neurones in the medulla oblongata. Most of the labelled neurones were found bilaterally in the nucleus ambiguus (81%), with a few sparsely distributed in the dorsal motor vagal nucleus (6.5%), and in an intermediate area located between these two nuclei (12.5%). We contend that the method of applying DiI crystals to the surface of the heart is a more efficient, accurate and reproducible method of retrograde labelling than the injection of tracers into this very delicate tissue.

Redistribution of F-actin and large dense-cored vesicles in the human neuroblastoma SH-SY5Y in response to secretagogues and protein kinase Calpha activation.

Our previous studies have shown that noradrenaline release is enhanced by activation of protein kinase Calpha in SH-SY5Y cells. In the present study, we report that activation of protein kinase Calpha leads to (a) partial redistribution of the F-actin cytoskeleton and (b) a 2.5-fold increase in the number of large dense-cored vesicles within 100 nm of the plasma membrane. This redistribution can be prevented by down-regulation of protein kinase Calpha by up to 48 h exposure to phorbol dibutyrate. Treatment with the secretagogues 100 mM KCl, the Ca2+ ionophore A23187 (20 microM) and 1 mM carbachol also leads to a partial disassembly of the F-actin cytoskeleton. This is accompanied by an increase in the number of large dense cored vesicles at the plasma membrane following exposure to KCl and A23187 but not following exposure to carbachol. These results are discussed in relation to the hypothesis that a key step in the enhancement of noradrenaline release following activation of protein kinase Calpha and elevation of intracellular calcium is the movement of large dense cored vesicles to the plasma membrane following partial disassembly of the F-actin cytoskeleton.

Innervation and control of the adenohypophysis by hypothalamic peptidergic neurons in teleost fishes: EM immunohistochemical evidence.

Previous light microscopic studies have revealed neuropeptide-immunoreactive neurosecretory fibers in the teleostean neurohypophysis, and ultrastructural work has reported direct innervation of endocrine cells by the terminals of fibers penetrating the adenohypophysis. This paper reviews our recent data from ultrastructural, immunohistochemical, receptor localization, and superfusion studies, which suggest a role for neuropeptides in the control of teleost pituitary secretion. We have used a combination of pre- and post-embedding electron microscopic immunolabeling methods to determine which neuropeptides are present in fibers innervating the pituitaries of three species: Poecilia latipinna, Dicentrarchus labrax, and Clarias gariepinus. Numerous axon profiles with immunoreactivity for the neurosecretory peptides vasotocin and isotocin formed large Herring bodies and terminal-like boutons in contact with corticotropic, growth hormone, thyrotropic, and pars intermedia cells. Numerous melanin-concentrating hormone-immunoreactive fibers and scarcer neurotensin and corticotropin-releasing factor-immunoreactive fibers showed similar distributions, terminating close to pars intermedia and corticotropic cells. Somatostatin, cholecystokinin, galanin, substance P, neuropeptide Y, growth hormone-releasing factor, thyrotropin-releasing hormone, and gonadotropin-releasing hormone-immunoreactivities were found in small calibre fibers penetrating among growth hormone, thyrotropic, and gonadotropic cells. These morphological findings have been supplemented by autoradiographic studies, which showed the distribution of binding sites for vasotocin, isotocin, galanin, and neuropeptide Y ligands over specific groups of pituitary cells, and superfusion studies that showed growth hormone release was stimulated by growth hormone-releasing factor and thyrotropin-releasing hormone, but inhibited by somatostatin. The implications of these results for neuropeptidergic control of teleostean pituitary secretions are discussed.