Dorsomedial Medulla Research Articles

Genetic and pharmacological dissection of pathways involved in the angiotensin II-mediated depression of baroreflex function.

Heart failure and hypertension are associated with increases in angiotensin II (ANG II) activity. One brain area where ANG II effects may be particularly important in these situations is the nucleus of the solitary tract (NTS). Located in the dorsomedial medulla, the NTS is the termination site of baroreceptor afferents and is essential for mediating the baroreflex. In hypertensive animals the baroreflex is impaired; this may be reversed by antagonizing ANG II AT1 receptors in the NTS. Recently, we showed that the baroreflex depressant action of ANG II in the NTS is mediated by activation of endothelial nitric oxide synthase (eNOS) and enhanced release of GABA. Using conventional pharmacological tools and a range of adenoviral-mediated expression of dominant negative proteins, we have determined the intracellular pathway(s) in the NTS by which ANG II activates eNOS. Our data indicate that ANG II acting in the NTS depresses the baroreflex via a Gq protein-mediated activation of phospholipase C, which through 1,4,5-inositol triphosphate causes release of calcium from the IP3-sensitive intracellular stores and calcium-calmodulin formation. In contrast, multiple site disruption of a pathway leading to eNOS activation via the serine/threonine kinase Akt was ineffective

Angiotensin peptides as neurotransmitters/neuromodulators in the dorsomedial medulla.

1. The present review provides an update on evidence of the neurotransmitter pathways and location of receptors within the nucleus tractus solitarii (NTS) mediating the baroreflex and other haemodynamic actions of angiotensin (Ang) II. 2. A series of studies suggests a significant role for substance P in the acute cardiovascular and carotid sinus chemoreceptor facilitatory actions of AngII in the NTS. The use of antisense oligonucleotides to AT1 receptors indicates both pre- and post-synaptic AngII receptors are likely to be involved in these actions. 3. With respect to baroreceptor reflex actions, it is clear that endogenous AngII impairs the gain for operation of the baroreceptor reflex, because AT1 receptor antagonists facilitate reflex function. This effect is either independent of substance P or involves inhibition of release. Moreover, initial data obtained using antisense oligonucleotides to AT1 receptors suggest that, in the NTS, the effect of endogenous AngII on the baroreceptor reflex is mainly due to presynaptic actions on vagal or carotid sinus afferent fibres. In contrast, the level of endogenous AngII within the NTS appears to have variable effects on activation of cardiopulmonary vagal afferent fibres by phenylbiguanide. These results indicate a divergence of effects of AngII on reflexes evoked by these two different types of sensory input. 4. Use of transgenic rats with alterations in brain angiotensin peptides allowed us to assess the effect of long-term alterations in brain Ang peptides on reflex function. We studied (mRen2)27 transgenic rats (TGR(mRen2)) with high brain medulla AngII levels and transgenic rats with angiotensinogen (Aogen) antisense linked to glial fibrillary acidic protein promoter (TGR(ASrAogen)) with greatly reduced brain Aogen. The reflex evoked by activation of cardiac vagal chemosensitive afferent fibres was enhanced in TGR(ASrAogen), whereas the baroreceptor reflex control of heart rate was attenuated in TGR(mRen2), further confirming a divergence of effects of AngII on these two sensory modalities. 5. The overall results are consistent with a sustained inhibitory effect of AngII on the baroreceptor reflexes, with dose-dependent or activation-dependent effects on cardiac vagal afferent fibre activation. Moreover, alterations in substance P pathways may contribute to the actions of AngII on reflex function.

Opposing roles for medial and central amygdala in the initiation of noradrenergic cell responses to a psychological stressor.

Psychological stressors trigger the activation of medullary noradrenergic cells, an effect that has been shown to depend upon yet-to-be-identified structures located higher in the brain. To test whether the amygdala is important in this regard, we examined the effects of amygdala lesions on noradrenergic cell responses to restraint, and also looked at whether any amygdala cells that respond to restraint project directly to the medulla. Ibotenic acid lesions of the medial amygdala completely abolished restraint-induced Fos expression in A1 and A2 noradrenergic cells. In contrast, lesions of the central amygdala actually facilitated noradrenergic cell responses to restraint. Tracer deposits in the dorsomedial (but not ventrolateral) medulla retrogradely labelled many cells in the central nucleus of the amygdala, but none of these cells expressed Fos in response to restraint. These data suggest for the first time that the medial amygdala is critical to the activation of medullary noradrenergic cells by a psychological stressor whereas the central nucleus exerts an opposing, inhibitory influence upon noradrenergic cell recruitment. The initiation of noradrenergic cell responses by the medial amygdala does not involve a direct projection to the medulla. Accordingly, a relay through some other structure, such as the hypothalamic paraventricular nucleus, warrants careful consideration.

Mediation by nucleus tractus solitarii glutamatergic neurotransmission of the cardiovascular reflex evoked by distal esophageal distension

Distention of the rat distal esophagus evokes an arterial pressor and a cardioaccelerator response that depends upon activation of a vagal afferent projection to the nucleus tractus solitarii (NTS). The present study aimed to determine in urethane-anesthetized rats if the afferent limb of this reflex (a) relays in the NTS subdivision known ro receive esophageal afferents, and (b) utilizes glutamatergic synapses. To this end, tetrodotoxin or the glutamate antagonists γ- d-glutamyl-glycine, 6-7-dinitroquinoxaline-2,3-dione (DNQX) and 2-amino-5-phosphonovaleric acid (AP-5) were applied to the NTS extraventricular surface rostral to the obex. All four agents inhibited both components of the reflex. DNQX or AP-5 produced a similar reversible inhibition upon pressure ejection in the vagal esophageal afferent projection area. Application of tetrodotoxin to the dorsomedial medulla oblongata caudal to the area postrema (AP) was ineffective. Basal heart rate (HR) (except in the case of AP-5) and blood pressure increased upon NTS surface application of the antagonists but not after intra-NTS ejection. At corresponding dorsal NTS sites, focal excitation of solitarial neurons with glutamate evoked hypotension and cardiac slowing. At adjacent ventral sites in the NTS subnucleus centralis (NTSc) and/or its immediate vicinity, glutamate elicited an arterial pressor response that coincided with an esophageal contraction in most but not all cases. In conclusion, afferent fibers of the esophago-cardiovascular reflex (ECVR) probably (1) terminate in the vicinity of esophageal premotor neurons comprising the NTSc and (2) activate second-order neurons via glutamate receptors of both the NMDA and non-NMDA subtype.

Catecholaminergic innervation of the sympathetic preganglionic cell column of the filefish Stephanolepis cirrhifer.

Nerve fibers immunoreactive for enzymes synthesizing catecholamines were examined in the central autonomic nucleus, a column of sympathetic preganglionic neurons, in the filefish Stephanolepis cirrhifer. Varicose nerve fibers immunoreactive for tyrosine hydroxylase were densely distributed in the rostral part, sometimes in contact with perikarya but were sparse in the caudal part of this nucleus. Fluorescent double labeling distinguished noradrenergic nerve fibers immunoreactive for both tyrosine hydroxylase and dopamine beta hydroxylase, and dopaminergic fibers immunoreactive only for tyrosine hydroxylase. In the brainstem, catecholaminergic neurons were observed in the locus coeruleus, the caudal dorsomedial reticular zone of the medulla, and the area postrema. Double labeling of tyrosine hydroxylase and dopamine beta hydroxylase showed that the neurons in the locus coeruleus were all noradrenergic, and those in the caudal dorsomedial medulla were mostly noradrenergic, whereas the area postrema contained both noradrenergic and dopaminergic neurons. No catecholaminergic neurons were found in the ventral region of the brainstem. After application of DiI to the central autonomic nucleus, retrogradely labeled neurons were seen in the caudal dorsomedial medulla but not in the locus coeruleus or the area postrema. These findings suggest that the sympathetic preganglionic neurons of the filefish may receive noradrenergic axonal projections from neurons in the caudal dorsomedial medulla. In the light of previous studies, inputs of these catecholaminergic fibers to the central autonomic nucleus may be involved in regulation of sympathetic activity of peripheral organs, together with serotoninergic and peptidergic inputs to this nucleus.

Activation of noradrenergic mechanism attenuates glutamate-induced vasopressor responses in the pons and medulla of cats in vivo

1. Using anesthetized cats, the authors examined the noradrenergic modulation of the glutamate induced pressor and depressor responses in various brainstem areas, including pontine gigantocellular tegmental field (FTG), dorsomedial medulla (DM), rostral ventrolateral medulla (RVLM), and caudal ventrolateral medulla (CVLM). 2. Unilateral microinjection of L-glutamate (Glu, 3 nmol in 30 nL saline) into FTG, DM and RVLM produced an increased in systemic arterial presure (SAP) and a decrease in heart rate (HR), while into CVLM produced decreases of SAP and HR. 3. Application of norepinephrine (NE) into the pressor areas (005 to 5 nmol) did not alter the resting SAP and HR, but significantly attenauted the Glu-induced pressor response with an order of potency: FTG > DM > RVLM. In the depressor CVLM, NE alone produced a dose-dependent decrease of resting SAP and HR, but did not affect the Glu-induced depressor responses. 4. The involvement of different adrenoceptor subtypes was further investigated by application of selective adrenoceptor agonists including phenylephrine (α1), clonidine (α2), and isoproterenol (β). Responses to these agonists are similar to those elicited by NE, except that only α-adrenoceptor agonists could antagonize the Glu-induced pressor responses of the RVLM. 5. Our observations indicate that NE not only inhibits the pressor mechanisms in various brainstem areas but also elicits a direct depressor response in CVLM. These findings also suggest that NE acts more likely a neurotransmitter, rather than a modulator, in the CVLM.

Role of nitric oxide on pressor mechanisms within the dorsomedial and rostral ventrolateral medulla in anaesthetized cats.

1. The role of nitric oxide (NO) in central cardiovascular regulation and the correlation between NO and glutamate-induced mechanisms is not clear. Microinjection of glutamate (3 nmol/30 nL) into dorsomedial medulla (DM) and rostral ventrolateral medulla (RVLM) increased arterial blood pressure (BP) and sympathetic vertebral nerve activity (VNA). Thus, in the present study, we examined the modulation by NO of glutamate-induced pressor responses in the DM and RVLM of cats. 2. Histochemical methods using nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) as a marker to stain neurons containing NO synthase (NOS), showed positive findings of NOS in both the DM and RVLM. 3. Microinjection of N(G)-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor, into the DM or RVLM did not alter resting BP and VNA, but it did cause a dose-dependent attenuation of glutamate-induced pressor responses. Interestingly, the increase in NO levels that resulted from pretreatment with L-arginine (L-Arg) or sodium nitroprusside (SNP) did not alter resting BP and VNA, but still inhibited glutamate-induced pressor responses in the DM and RVLM in a dose-dependent manner. 4. We also examined whether NO modulated the pressor responses induced by activation of different excitatory amino acid receptors. N-Methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) were used. Consistent with the results from the initial glutamate studies, we observed that not only L-NAME, but also L-Arg and SNP attenuated pressor responses induced by NMDA and AMPA. No difference was found between the effects of NO on NMDA- and AMPA-induced pressor responses. 5. To investigate the possibility of a loss of agonist selectivity, the effects of D-2-amino-5-phosphonovalerate (D-AP5) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) on AMPA and NMDA responses in the DM were examined. The results showed that CNQX did not alter NMDA-induced pressor responses, while D-AP5 failed to alter AMPA-induced responses. 6. Our results suggest that activation of the glutamate-induced pressor mechanism is regulated by changes in NO levels in the DM and RVLM. This implies that NO may play a permissive role to allow operation of the glutamate-activation mechanism.

Fos protein is required for the re-expression of angiotensin II type 1 receptors in the nucleus tractus solitarii after baroreceptor activation in the rat.

We evaluated in Sprague--Dawley rats the hypothesis that Fos protein induced by baroreceptor activation in the nucleus tractus solitarii participates in transcriptional regulation of the expression of angiotensin receptor genes. Reverse transcription-polymerase chain reaction revealed that baroreceptor activation elicited by sustained hypertension resulted in a transient decrease in angiotensin II subtype 1, but not subtype 2, receptor messenger RNA, in the dorsomedial medulla, including the nucleus tractus solitarii. There was subsequently a transitory reduction in the pressor response elicited by microinjection bilaterally of angiotensin II (40 pmol) into the nucleus tractus solitarii, followed by an increase in c-fos messenger RNA and Fos immunoreactivity at the same nucleus. Both the re-expression of angiotensin II subtype 1 receptor messenger RNA and restoration of pressor response to angiotensin II after baroreceptor activation were significantly blunted by bilateral application into the nucleus tractus solitarii of an antisense oligonucleotide (50 pmol) that targets against the initiation codon of c-fos messenger RNA. Control pretreatment with the corresponding sense oligonucleotide (50 pmol), or an antisense c-fos oligonucleotide that targets against a different portion of the coding sequence of the c-fos messenger RNA (50 pmol), was ineffective. At the receptor level, the angiotensin II-induced pressor response was antagonized by the subtype 1 receptor antagonist losartan (1.6 nmol), but not by the subtype 2 receptor antagonist PD-123319 (1.6 nmol). These findings suggest that sustained hypertension down-regulates angiotensin II subtype 1 receptors at both messenger RNA and functional expression levels in the nucleus tractus solitarii. Furthermore, Fos protein induced in the nucleus tractus solitarii by baroreceptor activation may play a permissive role in the transcriptional regulation of the re-expression of this subtype of angiotensin receptors.

Dorsomedial medulla is more susceptible than rostral ventrolateral medulla to hypoxic insult in cats.

We investigated the responses of systemic arterial pressure and vertebral sympathetic nerve activity to glutamate microinjections (0. 1 M, 70 nl) in the dorsomedial (DM) and the rostral ventrolateral medulla (RVLM) before hypoxia and after reoxygenation (posthypoxia) after various degrees of hypoxia in anesthetized cats. Hypoxia was produced by ventilating 5% O(2) and 95% N(2) for different durations (hypoxia I-III). In intact cats, the glutamate-induced systemic arterial pressure and vertebral nerve activity responses of the DM were depressed after all degrees of hypoxia. Posthypoxic depression in the RVLM, however, was not observed until hypoxia II and III. Precollicular decerebration prevented depression in the RVLM, but, for the DM, it was effective only for hypoxia I. Baro- and chemoreceptor denervation abolished all posthypoxic depression in both the DM and the RVLM. Pressor responses to tyramine (100-400 microg/kg iv) remained unchanged after all degrees of hypoxia. These results suggest that the DM is more susceptible to hypoxia than the RVLM. The peripheral baro- and chemoreceptors and the suprapontine structures apparently play an important role in posthypoxic depression. Moreover, the depression is not due to the postganglionic norepinephrine depletion.

Dorsal medullary pathways subserving oromotor reflexes in the rat: Implications for the central neural control of swallowing

Retrograde and anterograde axonal transport techniques were used to investigate the organization of inputs from the dorsomedial medulla, a region known to elicit patterned swallowing reflexes following focal stimulation, to the fifth (MoV), seventh (VII), tenth (nucleus ambiguus, NA), and twelfth (XII) cranial nerve motor nuclei in the rat, those motor nuclei most directly involved in the control of deglutition. The results may be summarized as follows. 1) Dorsal medullary inputs to MoV, VII, and XII arise primarily from an extended region of the caudal reticular formation immediately ventral to the nucleus of the solitary tract (NTS), which we term the dorsal medullary reticular column (DMRC). Projections from the DMRC are largely bilateral and are distributed preferentially to the ventral subdivision of MoV, to the dorsal and intermediate subdivisions of VII, and to both the dorsal and the ventral subdivisions of XII. In addition, a subpopulation of large multipolar neurons embedded within the DMRC gives rise to a primarily crossed input to the dorsal subdivision of MoV. 2) Dorsal medullary inputs to the NA arise from the NTS, are largely uncrossed, and are organized such that the ventrolateral, intermediate, and interstitial subdivisions of the NTS project to the semicompact formation and to the rostral extension of the compact formation (which supplies the pharynx) and to the loose formation (larynx), whereas the central subdivision of the NTS provides input to the compact formation (esophagus). 3) Neither the NTS nor the DMRC gives rise to significant projections to the central subnucleus of the NTS. Together, these results provide evidence for discrete medullary pathways subserving sequential activation of swallowing reflexes. J. Comp. Neurol. 417:448–466, 2000. © 2000 Wiley-Liss, Inc.

Dorsal medullary pathways subserving oromotor reflexes in the rat: Implications for the central neural control of swallowing

Retrograde and anterograde axonal transport techniques were used to investigate the organization of inputs from the dorsomedial medulla, a region known to elicit patterned swallowing reflexes following focal stimulation, to the fifth (MoV), seventh (VII), tenth (nucleus ambiguus, NA), and twelfth (XII) cranial nerve motor nuclei in the rat, those motor nuclei most directly involved in the control of deglutition. The results may be summarized as follows. 1) Dorsal medullary inputs to MoV, VII, and XII arise primarily from an extended region of the caudal reticular formation immediately ventral to the nucleus of the solitary tract (NTS), which we term the dorsal medullary reticular column (DMRC). Projections from the DMRC are largely bilateral and are distributed preferentially to the ventral subdivision of MoV, to the dorsal and intermediate subdivisions of VII, and to both the dorsal and the ventral subdivisions of XII. In addition, a subpopulation of large multipolar neurons embedded within the DMRC gives rise to a primarily crossed input to the dorsal subdivision of MoV. 2) Dorsal medullary inputs to the NA arise from the NTS, are largely uncrossed, and are organized such that the ventrolateral, intermediate, and interstitial subdivisions of the NTS project to the semicompact formation and to the rostral extension of the compact formation (which supplies the pharynx) and to the loose formation (larynx), whereas the central subdivision of the NTS provides input to the compact formation (esophagus). 3) Neither the NTS nor the DMRC gives rise to significant projections to the central subnucleus of the NTS. Together, these results provide evidence for discrete medullary pathways subserving sequential activation of swallowing reflexes.

Localization of hindbrain glucoreceptive sites controlling food intake and blood glucose

Feeding and blood glucose responses to local injection of nanoliter volumes of 5-thio- d-glucose (5TG), a potent antimetabolic glucose analogue, were studied at 142 hindbrain and 61 hypothalamic cannula sites. A site was considered positive if 5TG elicited at least 1.5 g more food intake or a hyperglycemic response at least 25 mg/dl greater than the respective responses elicited by vehicle injection in the same rat. Of 61 hypothalamic cannula sites tested, none were positive for blood glucose and only one was positive for feeding. Increasing the 5TG dose to 48 ug did not produce additional positive results at hypothalamic sites. In contrast, 66 hindbrain sites were positive for feeding and 49 were positive for blood glucose, with 33 of these being positive for both responses. The distribution of positive sites for feeding and hyperglycemia overlapped almost completely. Positive sites were concentrated in two distinct zones: one in the ventrolateral and one in the dorsomedial medulla. In both locations, the glucoreceptive areas extended approximately from the level of the area postrema (AP) to the pontomedullary junction. Glucoreceptive zones were co-distributed with epinephrine cell groups C1–C3, suggesting that epinephrine neurons may be important components of the neural circuitry for glucoregulation. Localization of glucoreceptive sites will facilitate positive identification of glucoreceptor cells and the direct analysis of the neural mechanisms through which they influence food intake and metabolic responses.

Distribution of AMPA receptor subunits GluR1‐4 in the dorsal vagal complex of the rat: a light and electron microscope immunocytochemical study

The dorsal vagal complex, localized in the dorsomedial medulla, includes the nucleus tractus solitarii (NTS), the dorsal motor nucleus of the vagus nerve (DMN) and the area postrema (AP). The distribution of AMPA-preferring glutamate receptors (AMPA receptors) within this region was investigated using immunohistochemistry and antibodies recognizing either one (GluR1 or GluR4) or two (GluR2 and GluR3) AMPA receptors subunits. The distribution of GluR1 immunoreactivity showed high contrast of staining between strongly and lightly labeled areas. Labeling was intense in the AP and weak in the NTS, except for its medial and dorsalmost parts which exhibited moderate staining. Almost no GluR1 immunoreactivity was found in the DMN. GluR2/3 immunolabeling was present in the entire dorsal vagal complex. This labeling was strong in the AP, the DMN and the medial half of the NTS and moderate in the lateral half of the NTS, except for the interstitial subdivision which exhibited intense staining. Labeling induced by the GluR4 antibody was very weak throughout the dorsal vagal complex. Ultrastructural examination showed that GluR1 and GluR2/3 immunoreactivity was localized in neuronal cell bodies and dendrites. No labeled axon terminal or glial cell body was found. Immunoperoxidase staining in labeled cell bodies and dendrites was associated with intracellular organelles (microtubules, mitochondria, cisternae of the endoplasmic reticulum, . . .) and/or parts of the plasma membrane. Plasma membrane labeling was often associated with asymmetrical synaptic differentiations. No labeled symmetrical synapse was found using either GluR1 or GluR2/3 antibody. The present results show that AMPA receptors have a widespread distribution in neuronal perikarya and dendrites of the rat dorsal vagal complex. They suggest differences in subunit composition between AMPA receptors localized in the NTS, the DMN and the AP. Ultrastructural data are consistent with the fact that AMPA receptors associated with the plasma membrane are mostly synaptic receptors. However, they also suggest the existence of a large intracellular pool of receptor subunits in neuronal soma and dendrites. Synapse 34:55–67, 1999. © 1999 Wiley-Liss, Inc.

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.

Distribution of AMPA receptor subunits GluR1-4 in the dorsal vagal complex of the rat: a light and electron microscope immunocytochemical study.

The dorsal vagal complex, localized in the dorsomedial medulla, includes the nucleus tractus solitarii (NTS), the dorsal motor nucleus of the vagus nerve (DMN) and the area postrema (AP). The distribution of AMPA-preferring glutamate receptors (AMPA receptors) within this region was investigated using immunohistochemistry and antibodies recognizing either one (GluR1 or GluR4) or two (GluR2 and GluR3) AMPA receptors subunits. The distribution of GluR1 immunoreactivity showed high contrast of staining between strongly and lightly labeled areas. Labeling was intense in the AP and weak in the NTS, except for its medial and dorsalmost parts which exhibited moderate staining. Almost no GluR1 immunoreactivity was found in the DMN. GluR2/3 immunolabeling was present in the entire dorsal vagal complex. This labeling was strong in the AP, the DMN and the medial half of the NTS and moderate in the lateral half of the NTS, except for the interstitial subdivision which exhibited intense staining. Labeling induced by the GluR4 antibody was very weak throughout the dorsal vagal complex. Ultrastructural examination showed that GluR1 and GluR2/3 immunoreactivity was localized in neuronal cell bodies and dendrites. No labeled axon terminal or glial cell body was found. Immunoperoxidase staining in labeled cell bodies and dendrites was associated with intracellular organelles (microtubules, mitochondria, cisternae of the endoplasmic reticulum,.) and/or parts of the plasma membrane. Plasma membrane labeling was often associated with asymmetrical synaptic differentiations. No labeled symmetrical synapse was found using either GluR1 or GluR2/3 antibody. The present results show that AMPA receptors have a widespread distribution in neuronal perikarya and dendrites of the rat dorsal vagal complex. They suggest differences in subunit composition between AMPA receptors localized in the NTS, the DMN and the AP. Ultrastructural data are consistent with the fact that AMPA receptors associated with the plasma membrane are mostly synaptic receptors. However, they also suggest the existence of a large intracellular pool of receptor subunits in neuronal soma and dendrites.

Altered daily rhythms of brain and pituitary indolamines and neuropeptides in long-term hypoxic rats.

To determine whether sustained hypoxia alters daily rhythms in brain and pituitary neurotransmitters, the daily variations in vasoactive intestinal peptide-like immunoreactivity (VIP-LI), neuropeptide Y-like immunoreactivity (NPY-LI), serotonin (5-HT), and 5-hydroxyindole-3-acetic acid (5-HIAA) content were determined in discrete brain regions, pineal gland and anterior pituitary of hypoxic (10% O(2); 14 days) and normoxic rats. Hypoxia suppressed daily variations in VIP-LI in the suprachiasmatic nuclei (SCN) and the anterior pituitary, enhanced the daily rhythmicity in serotonergic elements of the caudal part of the dorsomedial medulla oblongata (DMMc), and even induced daily variations in NPY-LI in the DMMc as well as in the ventrolateral medulla oblongata. In addition, punctual alterations in the rhythmicity of 5-HT and 5-HIAA in the pineal gland and of plasma corticosterone were observed in hypoxic rats. Thus results of this study indicate that a permanent nonphotic stimulus, such as sustained hypoxia, may affect the functioning of the internal clock located in the SCN and may alter the daily rhythmicity in neurotransmitter content of some brain nuclei and the pituitary gland.

H +-Sensitivity of cultured neurons from the dorsomedial and ventrolateral medulla of neonate rats

The H +-sensitivity of neonate rat cultured neurons derived from the dorsomedial medulla (DMM) containing the nucleus tractus solitarii and the ventrolateral medulla (VLM) was determined by H +-sensitive fluorescent probe BCECF-AM and immunohistochemical methods. Against an extracellular pH as low as 7.2–7.3, H +-sensitivity was verified in 2.6% of the DMM neurons (46/1800) and 2.1% of the VLM neurons (38/1800). This H +-sensitive neurons of the DMM were immunoreactive to glutamate (52.4%) and glutamic acid decarboxylase (GAD) (28.6%), while those of the VLM were immunoreactive to glutamate (66.7%) and GAD (33.3%). There was no immunoreactivity to tyrosine hydroxylase, phenylethanolamine- N-methyltransferase or choline acetyltransferase in the H +-sensitive neurons are present in the DMM and VLM besides the ventral medullary surface, the site of the central chemoreceptors.

Importance of neurokinin-1 receptors in the nucleus tractus solitarii of mice for the integration of cardiac vagal inputs.

Unmyelinated vagal afferents from the heart terminate within the nucleus tractus solitarii (NTS) located in the dorsomedial medulla. The neurotransmitter and postsynaptic receptors mediating information from cardiac vagal receptors to the NTS are unknown. This study determined the effects of neurokinin-1 (NK1) receptor blockade on: (i) the reflex response evoked following aortic root injection of either veratridine (1-3 microg/kg) or bradykinin (80-300 ng/kg) to stimulate cardiac receptors in in vivo anaesthetized mice; and (ii) the evoked synaptic response of cardioreceptive NTS neurons following both intraleft-ventricular injection of veratridine or bradykinin, and electrical stimulation of the ipsilateral vagus nerve in an arterially perfused working heart-brainstem preparation of mouse. Administration of CP-99,994 (0.75-1.5 mg/kg i.v.), a specific NK1 antagonist, attenuated significantly the evoked reflex bradycardia and depressor response following cardiac receptor (n = 6), but not pulmonary chemoreflex stimulation in vivo. From extracellular recordings of cardioreceptive NTS neurons, CP-99,994 reduced reversibly the total number of evoked spikes, peak firing frequency and response duration evoked by intraventricular injections of veratridine (n = 5) or bradykinin (n = 5). The number of evoked action potentials following electrical stimulation of the vagus nerve was also reduced. In five whole cell recordings of NTS neurons, both the evoked depolarization following cardiac receptor stimulation, and the peak amplitude and duration of vagus nerve-evoked EPSPs were reduced by CP-99,994; synaptic inputs from both peripheral chemoreceptors or pulmonary C-fibres were unaffected. These data support a selective involvement of NK1 receptors in the transmission of cardiac vagal afferent inputs to NTS neurons integrating cardiorespiratory information.

The depressor caudal ventrolateral medulla: Its correlation with the pressor dorsomedial and ventrolateral medulla and the depressor paramedian reticular nucleus

The functional correlation of the depressor caudal ventrolateral medulla (CVLM) with the two pressor regions, i.e. rostral ventrolateral medulla (RVLM) and dorsomedial medulla (DM), and with another inhibitory region, i.e. the paramedian reticular nucleus (PRN), were studied in cats anesthetized intraperitoneally with chloralose (40 mg/kg) and urethane (400 mg/kg). Systemic arterial pressure (SAP), heart rate (HR) and the sympathetic vertebral nerve activity (VNA) were recorded. The correct location of CVLM, RVLM or DM was determined by their sepecific responses, i.e. decreases of SAP, HR and VNA, for CVLM increases of these parameters for RVLM and DM, elicited first by electrical stimulation (80 Hz, 0.5 ms, 50–100 μA) then followed by microinjection of glutamate (Glu, 0.25 M, 70 nl). The depressor action of PRN was produced by electrical stimulation only. It was found that the depressor responses caused by the CVLM stimulation were greatly reduced 2 h after lesioning either the RVLM or DM by microinjection of kainic acid (KA, 24 mM, 200 nl) ipsilateral to the side of CVLM stimulation. The CVLM responses were further reduced after the remaining side of RVLM or DM was lesioned. The reduction of the CVLM-depressor responses was more apparent after the RVLM than DM lesioning. Data suggest that the CVLM-depressor responses are mediated through inhibition of the sympathetic-pressor neurons in both RVLM and DM with predominance of the former. Lesioning the PRN by KA and/or combination with DC electrolytic lesion reduced the CVLM-induced depressor responses. In turn, lesioning the CVLM by KA reduced the PRN-induced depressor responses. The reduction in the later manipulation was more apparent in the PRN-depressor responses than the CVLM-depressor responses. Data suggest that part of the PRN depressor action is mediated through activation of the CVLM.

Characterization of [ 125I]β-Endorphin Binding Sites in the Rat Caudal Dorsomedial Medulla

[ 125I]β-endorphin bound to high affinity (K d = 0.25 n M) receptors in the caudal dorsomedial medulla of rats with a B max of 97 fmol/mg protein. The relative potency for displacement of [ 125I]β-endorphin binding was: β-endorphin 1–31 > β-endorphin 1–27 > DAMGO > naloxone > N-acetyl-β-endorphin 1–31 > U50488 > DPDPE. The B max for [ 3H]DAMGO binding was 81 fmol/mg protein, indicating that most [ 125I]β-endorphin binding corresponds to μ-opioid receptors. [ 3H]DAMGO binding was not influenced by lesioning noradrenergic nerve terminals in the caudal dorsomedial medulla. Our findings indicate that β-endorphin interacts primarily with μ-opioid receptors in the caudal dorsomedial medulla. These receptors are not affected by noradrenergic denervation.