Localization and action of adenosine A2a receptors in regions of the brainstem important in cardiovascular control

In vitro autoradiography and central microinjections of a P₁ adenosine A_2a receptor antagonist have been employed to investigate a possible role for centrally located adenosine A_2a receptors in modulation of the baroreceptor reflex. In vitro autoradiography using [¹²⁵I]4-(2-[7-amino-2-[2-furyl][1,2,4]triazolol[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol ([¹²⁵I]ZM241385), the high-affinity adenosine A_2a receptor antagonist, revealed a heterogeneous distribution of adenosine A_2a binding sites within the lower brainstem of the rat. Image analysis showed high levels of binding in rostral regions of both the nucleus tractus solitarius and the ventrolateral medulla. Intermediate levels of binding were observed in the commissural nucleus tractus solitarius and the dorsal vagal motor nucleus, with low levels of binding in caudal regions of the nucleus tractus solitarius and the ventrolateral medulla, and the hypoglossal nucleus. Unilateral microinjections of unlabelled ZM241385 into the nucleus tractus solitarius had no effect on baseline levels of arterial pressure, heart rate and phrenic nerve activity recorded in anaesthetized, artificially ventilated rats. However, microinjections of ZM241385 reduced the bradycardia evoked by stimulation of the ipsilateral aortic nerve. In contrast, ZM241385 had no effect on the depressor response or the reduction in phrenic nerve activity evoked by aortic nerve stimulation.Our results indicate that adenosine A_2a binding sites are located in a number of brainstem regions involved in autonomic function, consistent with the idea that adenosine acts as a neuromodulator of a variety of cardiorespiratory reflexes. Specifically, the data support the hypothesis that adenosine A_2a receptors located within the nucleus tractus solitarius are activated during baroreceptor stimulation and have an important modulatory role in the pattern of cardiovascular changes associated with this reflex.     

Baroreceptor reflex inhibition induced by the stimulation of serotonin3 receptors in the nucleus tractus solitarius of the rat.

Previous studies suggested that in the nucleus tractus solitarius, cardiovascular responses to serotonin may involve the simultaneous activation of more than one receptor subtype. In the present study, the cardiovascular effects of the local application of serotonin and different serotonin3 agonists and antagonists into the nucleus tractus solitarius were analysed in intact and unilaterally ganglionectomized rats. Unilateral injections of serotonin (5-15 nmol) produced a dose-dependent increase in blood pressure and partially antagonized the arterial baroreflex responses evoked by an i.v. injection of phenylephrine. Similar blood pressures response were obtained after unilateral microinjections of phenylbiguanide (5 nmol) and 2-methyl-serotonin (5 nmol), two serotonin3 receptor agonists. Bilateral microinjections of serotonin or phenylbiguanide produced more pronounced blood pressure effects and antagonized completely the baroreflex responses. Both blood pressure and baroreflex effects were antagonized by prior injections of specific serotonin3 antagonists such as zacopride (100 pmol) and ondansetron (100 pmol). Concomitant autoradiographic studies performed in intact and ganglionectomized rats, using [125I]iodozacopride, confirmed that serotonin3 receptors in the nucleus tractus solitarius are mainly located on vagal afferent fibers. In addition, serotonin microinjections made in the nucleus tractus solitarius ipsilateral to the ganglionectomy revealed a significant reduction in cardiovascular responses compared to intact animals. These results suggest that in the nucleus tractus solitarius of the rat, serotonin is involved in the reflex regulation of blood pressure through the stimulation of serotonin3 receptors presumably located on vagal afferent fibers. Since bicuculline antagonized the serotonin-mediated pressor responses, a serotonin3-dependent activation of an inhibitory GABAergic system within the nucleus tractus solitarius might be involved in blood pressure regulatory mechanisms.     

Barosensory cells in the nucleus tractus solitarius receive convergent input from group III muscle afferents and central command

Some neural mechanism must prevent the full expression of the baroreceptor reflex during static exercise because arterial blood pressure increases even though the baroreceptors are functioning. Two likely candidates are central command and input from the thin fiber muscle afferents evoking the exercise pressor reflex. Recently, activation of the mesencephalic locomotor region, an anatomical locus for central command, was found to inhibit the discharge of nucleus tractus solitarius cells that were stimulated by arterial baroreceptors in decerebrated cats. In contrast, the effect of thin fiber muscle afferent input on the discharge of nucleus tractus solitarius cells stimulated by baroreceptors is not known. Consequently in decerebrated unanesthetized cats, we examined the responses of barosensory nucleus tractus solitarius cells to stimulation of thin fiber muscle afferents and to stimulation of the mesencephalic locomotor region, a maneuver which evoked fictive locomotion. We found that electrical stimulation of either the mesencephalic locomotor region or the gastrocnemius nerve at current intensities that recruited group III afferents inhibited the discharge of nucleus tractus solitarius cells receiving baroreceptor input. We also found that the inhibitory effects of both gastrocnemius nerve stimulation and mesencephalic locomotor region stimulation converged onto the same barosensory nucleus tractus solitarius cells. We conclude that the nucleus tractus solitarius is probably the site whereby input from both central command and thin fiber muscle afferents function to reset the baroreceptor reflex during exercise.     

Carotid sinus nerve terminals which are tyrosine hydroxylase immunoreactive are found in the commissural nucleus of the tractus solitarius

Summary Tyrosine hydroxylase immunoreactive sensory neurons in the petrosal ganglion selectively innervate the carotid body via the carotid sinus nerve. Central projections of the carotid sinus nerve were traced with horseradish peroxidase. The commissural nucleus of the tractus solitarius was examined by dual labelling light and electron microscopy. Dense bilateral labelling with horseradish peroxidase was found in the tractus solitarius and commissural nucleus of the tractus solitarius. Horseradish peroxidase was found in unmyelinated axons, myelinated axons, and nerve terminals. About 88% of horseradish peroxidaselabelled carotid sinus nerve axons were unmyelinated. Tyrosine hydroxylase immunoreactivity was identified in unmyelinated axons, myelinated axons, dendrites, perikarya, and nerve terminals. Most tyrosine hydroxylase immunoreactive axons (93%) in the commissural nucleus of the tractus solitarius were unmyelinated. Tyrosine hydroxylase immunoreactivity was simultaneously identified in carotid sinus nerve unmyelinated axons, myelinated axons, and nerve terminals. These double-labelled terminals comprised 28% of the number of tyrosine hydroxylase immunoreactive terminals in the commissural nucleus of the tractus solitarius, and 55% of transganglionically-labelled terminals. Therefore, there are both central and peripheral sources of tyrosine hydroxylase immunoreactive nerve terminals in the commissural nucleus of the tractus solitarius. These data support the hypothesis that peripheral tyrosine hydroxylase immunoreactive neurons are involved in the origination of the chemoreceptor reflex. Axo-axonic synapses between peripheral carotid sinus nerve afferent terminals and central terminals containing tyrosine hydroxylase immunoreactivity were observed in 22% of the axo-axonic synapses observed. Thus, central tyrosine hydroxylase immunoreactivity neurons are involved in the modulation of the chemo- and/or baroreceptor reflexes. Synaptic contacts were not observed between carotid sinus nerve afferents and tyrosine hydroxylase immunoreactive perikarya of dendrites. Catecholaminergic neurons are thus unlikely to be the second order neurons of either the chemo- or baroreceptor reflex in the commissural nucleus of the tractus solitarius.     

L-dopaergic components in the caudal ventrolateral medulla in baroreflex neurotransmission.

L-3,4-Dihydroxyphenylalanine (L-DOPA) is probably a transmitter of the primary baroreceptor afferents terminating in the nucleus tractus solitarii; L-DOPA functions tonically to activate depressor sites of the caudal ventrolateral medulla, which receives input from the nucleus tractus solitarii [Misu Y. et al. (1996) Prog. Neurobiol. 49, 415-454]. We have attempted to clarify whether or not L-DOPAergic components within the caudal ventrolateral medulla are involved in baroreflex neurotransmission in anesthetized rats. Electrolytic lesions of the right nucleus tractus solitarii (1 mA d.c. for 10 s, 10 days before measurement) selectively decreased by 45% the tissue content of L-DOPA in the dissected ipsilateral caudal ventrolateral medulla. Electrolytic lesions did not decrease dopamine, norepinephrine and epinephrine levels. During microdialysis of the right caudal ventrolateral medulla, extracellular levels of L-DOPA, norepinephrine, epinephrine and 3,4-dihydroxyphenylacetic acid were consistently detectable using high-performance liquid chromatography with electrochemical detection. However, extracellular dopamine levels were lower than the assay limit. Baroreceptor activation by i.v. phenylephrine selectively evoked L-DOPA without increasing the levels of norepinephrine, epinephrine and 3,4-dihydroxyphenylacetic acid. This L-DOPA release was suppressed by acute lesion in the ipsilateral nucleus tractus solitarii. Intermittent stimulation of the right aortic depressor nerve (20 Hz, 3 V, 0.3 ms duration, for 30 min) repetitively and constantly caused L-DOPA release, hypotension and bradycardia, without increases in levels of norepinephrine, epinephrine and 3,4-dihydroxyphenylacetic acid. Local inhibition of L-DOPA synthesis with alpha-methyl-p-tyrosine (30 microM) infused into the ipsilateral caudal ventrolateral medulla gradually decreased basal levels of L-DOPA and 3,4-dihydroxyphenylacetic acid without decreasing norepinephrine and epinephrine. The inhibition of L-DOPA synthesis interrupted L-DOPA release and decreased by 65% depressor responses elicited by aortic nerve stimulation; however, it produced no effect on bradycardic responses. CoCl2 (119 ng), a mainly presynaptic inhibitory transmission marker, and L-DOPA methyl ester (1 microg), a competitive L-DOPA antagonist, when microinjected into depressor sites of the right caudal ventrolateral medulla, reduced by 60% depressor responses to transient ipsilateral stimulation of the aortic nerve (20 Hz, 3 V, 0.1 ms duration, for 10 s). No changes in bradycardic responses were observed. There may exist an L-DOPAergic relay from the nucleus tractus solitarii to the caudal ventrolateral medulla. L-DOPAergic components in the caudal ventrolateral medulla are involved in baroreflex neurotransmission via a baroreceptor-aortic depressor nerve-nucleus tractus solitarii-caudal ventrolateral medulla relay in the rat.     

Projections of medullary and pontine noradrenergic neurons to the horizontal limb of the nucleus of diagonal band in the rat.

Recent investigations in the rat have implicated a noradrenergic innervation to the horizontal nucleus of the diagonal band of Broca as a critical link in a neural circuit that conveys baroreceptor information centrally to inhibit the firing of vasopressin-secreting neurons in the hypothalamic supraoptic nucleus. In this study we used small intra-diagonal band injections of a retrograde tracer, rhodamine latex microspheres, in combination with tyrosine hydroxylase histochemistry to identify brainstem noradrenergic cells contributing to this innervation. In three cases where tracer injections were limited to the horizontal limb of the diagonal band, we observed 20-50 double-labelled neurons ipsilaterally in the dorsal part of the locus coeruleus (A6) and the caudal nucleus tractus solitarius (A2), and bilaterally in the caudal ventrolateral medulla (A1). Double-labelled neurons were also noted in the ventral tegmental area (dopaminergic A10 cell group). Although all major brainstem noradrenergic cell groups contribute fibers to the horizontal limb of the nucleus of diagonal band, data from physiological studies suggest that the noradrenergic A2 neurons in the nucleus tractus solitarius are the most likely pathway through which it receives this baroreceptor information.     

Morphology and electrophysiology of superior laryngeal nerve afferents and postsynaptic neurons in the medulla oblongata of the cat.

Intra-axonal recordings were made from 24 afferent fibres of the superior laryngeal nerve in and around the nucleus tractus solitarius, in 26 pentobarbitone-anaesthetized cats. Conduction velocity ranged from 15 to 38 m/s. Four afferents were injected with horseradish peroxidase. They showed dense terminal arborization in the region of the ventral and ventrolateral subnuclei of the nucleus tractus solitarius, both rostral and caudal to the obex. Six other intra-axonal recordings were thought to originate from axons of neurons postsynaptic to superior laryngeal afferents; one of these was injected with horseradish peroxidase and showed a similar arborization pattern to that of the afferent axons. In the same region, intracellular recordings were made from 124 neurons which responded to superior laryngeal nerve stimulation with excitatory postsynaptic potentials (mean latency 2.7 +/- 1.0 ms). Ninety-nine of these neurons were thought to receive a monosynaptic input. The stimulation threshold evoking these responses was similar to that which inhibited phrenic nerve discharge. Eleven of the monosynaptically excited neurons were injected with horseradish peroxidase. They had fusiform or stellate somata and simple dendritic trees, radiating mainly in the transverse plane. In one experiment, in which both a superior laryngeal nerve afferent fibre and a neuron were labelled, afferent terminal varicosities were found in close apposition with the postsynaptic membrane of the injected neuron. Four of 14 (29%) tested neurons could be antidromically activated from the C3 spinal segment. The stimulus thresholds and onset latencies of the responses of superior laryngeal nerve afferents and medullary neurons to stimulation of the superior laryngeal nerve are consistent with their involvement in the reflex inhibition of respiratory neurons evoked by superior laryngeal nerve stimulation.     

Neurons in the rat medulla oblongata containing neuropeptide Y-, angiotensin II-, or galanin-like immunoreactivity project to the parabrachial nucleus.

Projections from the medulla to the parabrachial complex of the rat were examined for their content of neuropeptide Y-, angiotensin II- or galanin-like immunoreactivity using combined retrograde tracing and immunohistochemical techniques. Rhodamine-labelled latex microspheres were stereotaxically injected into discrete nuclei of the parabrachial complex. After survival of two to five days, colchicine (100 micrograms in 10 microliters saline) was injected into the cisterna magna. One day later, rats were perfused and the brainstems were prepared for visualization of the retrograde tracer and immunoreactivity of one of the three peptides. Retrograde labelling verified that the area postrema, nucleus of the tractus solitarius, caudal spinal nucleus of the trigeminal nerve, parvocellular reticular nucleus, and ventrolateral medulla including the rostral ventrolateral medulla and nucleus paragigantocellularis project to the lateral parabrachial and K?lliker-Fuse nuclei. While most projections were primarily ipsilateral, a small proportion of the projections from the ventrolateral medulla was bilateral. Neurons containing neuropeptide Y-like immunoreactivity were found in the caudal and intermediate nucleus of the tractus solitarius, dorsal to the lateral reticular nucleus and in the nucleus paragigantocellularis. After bilateral microsphere injections into the lateral parabrachial and K?lliker-Fuse nuclei, double-labelled neurons were found dorsal to the lateral reticular nucleus of caudal and intermediate medullary levels, at the ventral surface of the medulla at intermediate levels and in the nucleus paragigantocellularis at rostral levels. Neurons with angiotensin II-like immunoreactivity were observed at the dorsomedial border of the caudal and intermediate nucleus of the tractus solitarius, in the area postrema and in the lateral reticular nucleus and nucleus paragigantocellularis. Of these neurons, small numbers in the nucleus of the tractus solitarius and ventrolateral medulla also projected to the lateral parabrachial and K?lliker-Fuse nuclei. Neurons containing galanin-like immunoreactivity were found in the caudal nucleus of the tractus solitarius, the area postrema, the spinal trigeminal nucleus, the raphe nuclei (pallidus and obscurus), the nucleus paragigantocellularis and dorsal to the lateral reticular nucleus. Of these cells, double-labelled neurons were found in the commissural and medial subdivisions of the caudal nucleus of the tractus solitarius and in the rostral ventrolateral medulla including the ventral surface and the nucleus paragigantocellularis. The results suggest that neuropeptide Y, angiotensin II and galanin may serve as neurochemical messengers in pathways from the medulla to the parabrachial complex. The location of double-labelled neurons suggests that the information relayed by these neurons is related to autonomic activity.     

Hypoxia-induced Fos expression in neurons projecting to the pressor region in the rostral ventrolateral medulla

Previous studies in anaesthetized animals have shown that the hypoxia-induced increase in sympathetic vasomotor activity is largely dependent on synaptic excitation of sympathoexcitatory pressor neurons in the rostral part of the ventrolateral medulla. The primary aim of this study was to determine, in conscious rabbits, the distribution of neurons within the brain that have properties characteristic of interneurons conveying excitatory inputs to the rostral ventrolateral medullary pressor region in response to systemic hypoxia. In a preliminary operation, a retrogradely-transported tracer, fluorescent-labelled microspheres, was injected into the physiologically-identified pressor region in the rostral ventrolateral medulla. After a waiting period of one to two weeks, the conscious rabbits were subjected to moderate hypoxia (induced by breathing 10% O₂ in N₂) for a period of 60 min. Control groups of animals were exposed to room air or to mild hypoxia (12% O₂ in N₂). Moderate hypoxia resulted in a modest hypertension of approximately 15 mmHg, and in the expression of Fos (a marker of neuronal activation) in many neurons in the nucleus tractus solitarius, the rostral, intermediate and caudal parts of the ventrolateral medulla, the Kölliker–Fuse nucleus, locus coeruleus, subcoeruleus and A5 area in the pons as well as in several midbrain and forebrain regions, including the periaqueductal grey in the midbrain and the paraventricular, supraoptic and arcuate nuclei in the hypothalamus. Fos expression was also observed in these regions in rabbits subjected to mild hypoxia or normoxia, but it was much reduced compared to rabbits subjected to moderate hypoxia. Approximately half of the neurons in the ventrolateral medulla, 27% of neurons in the nucleus tractus solitarius, and 49–81% of neurons in the locus coeruleus, sub-coeruleus and A5 area that expressed Fos following moderate hypoxia were also immunoreactive for tyrosine hydroxylase, and were therefore catecholamine cells. Approximately half of the neurons in the nucleus tractus solitarius and two-thirds of neurons in the Kölliker–Fuse nucleus that expressed Fos following moderate hypoxia were retrogradely labelled from the rostral ventrolateral medullary pressor region. Similarly, approximately one quarter of Fos-positive cells in the caudal and intermediate ventrolateral medulla were retrogradely labelled, but very few Fos-positive/retrogradely-labelled cells were found in other pontomedullary or suprapontine brain regions.

The results indicate that systemic hypoxia results in activation of neurons in several discrete nuclei in the brainstem and forebrain, including neurons in all the major pontomedullary catecholamine cell groups. However, neurons that are activated by systemic hypoxia and that also project to the rostral ventrolateral medullary pressor region are virtually confined to the lower brainstem, primarily in the nucleus tractus solitarius and Kölliker–Fuse nucleus and to a lesser extent the caudal/intermediate ventrolateral medulla. In a previous study from our laboratory, we determined the distribution of neurons in the brainstem that are activated by hypertension and that also project to the rostral ventrolateral medullary pressor region [Polson et al. (1995) Neuroscience 67, 107–123]. Comparison of the present results with those from this previous study indicates that the hypoxia-activated neurons in the nucleus tractus solitarius and Kölliker–Fuse nucleus that project to the rostral ventrolateral medulla are likely to be interneurons conveying excitatory chemoreceptor signals, while those in the caudal/intermediate ventrolateral medulla are likely to be mainly interneurons conveying inhibitory baroreceptor signals, activated by the rise in arterial blood pressure associated with the hypoxia-induced hypertension.     

The sinus nerve and baroreceptor input to the medulla of the cat.

Electrical stimulation within the medulla of cats revealed that myelinated primary afferent fibres of the sinus nerve terminated within the immediate vicinity of the tractus solitarius and its nucleus. 2. The activity of neurones within this area was also evoked on sinus nerve stimulation, although few (17%) were activated within a latency compatible with monosynaptic excitation. Additional projections over polysynaptic pathways have been shown to the parahypoglossal area and to the area of the nucleus ambiguus. 3. These three areas were shown to contain neurones whose activity was enhanced by stimulation of the baroreceptor endings of the ipsilateral carotid sinus. 4. No evidence for a projection of sinus nerve afferents to the medial reticular formation (an area extending medially from the hypoglossal nucleus and nerve tract and including the paramedian reticular nucleus) was obtained in either antidromic or orthodromic studies. 5. The organization of the central pathway of the carotid sinus baroreceptor reflex is discussed in the light of these results.     

Cardiovascular function of a glutamatergic projection from the hypothalamic paraventricular nucleus to the nucleus tractus solitarius in the rat

Experiments were done in urethane-anesthetized, barodenervated, male Wistar rats. Chemical stimulation of the hypothalamic paraventricular nucleus (PVN) by unilateral microinjections of N-methyl-d-aspartic acid (NMDA) elicited increases in mean arterial pressure (MAP) and greater splanchnic nerve activity (GSNA). The increases in the MAP and GSNA induced by chemical stimulation of the PVN were significantly exaggerated by bilateral microinjections of d(−)-2-amino-7-phosphono-heptanoic acid (d-AP7) and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydro-benzo[f]quinoxaline-7-sulfonamide disodium (NBQX) (ionotropic glutamate receptor antagonists) into the medial subnucleus of the nucleus tractus solitarius (mNTS). These results were confirmed by single unit recordings; i.e. excitation of mNTS barosensitive neurons caused by chemical stimulation of the ipsilateral PVN was blocked by application of d-AP7 and NBQX to these neurons. Bilateral microinjections of d-AP7 and NBQX into the mNTS elicited pressor responses which were significantly attenuated by inhibition of PVN neurons by bilateral microinjections of muscimol. Unilateral microinjections of fluorogold into the mNTS resulted in bilateral retrograde labeling of the PVN neurons. Unilateral microinjections of biotinylated dextran amine into the PVN resulted in anterograde labeling of axons and terminals in the mNTS bilaterally and the labeled terminals exhibited vesicular glutamate transporter-2 immunoreactivity. These results indicated that 1) a tonically active glutamatergic bilateral projection from the PVN to the mNTS exists; 2) bilateral blockade of ionotropic glutamate receptors in the mNTS exaggerates the increases in MAP and GSNA, but not heart rate, to the chemical stimulation of the PVN; and 3) this projection may serve as a restraint mechanism for excitatory cardiovascular effects of PVN stimulation.     

The localization of adrenoceptors and opiate receptors in regions of the cat central nervous system involved in cardiovascular control

The distribution of adrenoceptors and opiate receptors in the nucleus of the tractus solitarius and the intermediolateral cell column of the thoracic spinal cord of the cat have been investigated using an in vitro autoradiographic technique. Specific binding of [3H]yohimbine and [3H]rauwolscine (alpha 2-adrenoceptor ligands) was seen within the intermediolateral cell column but no obvious binding of [3H]prazosin, an alpha 1-ligand, was observed. No evidence of a significant population of opiate receptors was obtained in the intermediolateral cell column. Within the nucleus of the tractus solitarius a marked binding of [3H]yohimbine and [3H]rauwolscine was accompanied, however, by a more restricted binding of [3H]naloxone and [3H]dihydromorphine indicating the presence of both alpha 2-adrenoceptors and opiate receptors. As with the intermediolateral cell column no evidence of [3H]prazosin binding was seen. These observations may have particular relevance for the physiology and pharmacology of cardiovascular control. In the case of the intermediolateral cell column it is consistent with evidence of a catecholamine innervation originating from the brainstem. With regard to the nucleus of the tractus solitarius the location of the receptor groups is discussed in the light of the anatomy and physiology of its afferent innervation.     

Identification and distribution of 5-HT3 recognition sites within the human brainstem

The present studies demonstrate the presence of specific [3H]GR65630 binding sites within the human brainstem using the techniques of in vitro receptor autoradiography and ligand binding to homogenates. Autoradiography revealed the greatest accumulation of specific binding in the area postrema and subpostrema (AP/ASP). A lower level of specific binding was identified in the nucleus tractus solitarius (excluding area subpostrema). No specific binding was evident in the remainder of the hindbrain at this level. Discrete dissection followed by ligand binding to homogenates revealed that the specific binding of [3H]GR65630 (defined by the presence of 30 microM metoclopramide) was differentially distributed with highest levels in the AP/ASP (112.1 fmol/mg protein) and lower levels in the dorsal vagal complex (nucleus tractus solitarius--excluding the area subpostrema--dorsal motor nucleus of the vagus and hypoglossal nucleus) (DVC) and olivary nucleus (ON) (22.9 and 3.9 fmol/mg, respectively). No specific binding was detectable in the reticular formation (RF) located ventral to the dorsal vagal complex. The specific [3H]GR65630 binding site was pharmacologically similar to the 5-HT3 receptor since the potent and selective 5-HT3 receptor antagonists ICS 205-930 and zacopride (100 nM) and the agonist 5-HT (10 microM) inhibited binding to the same extent as metoclopramide in each of the individual areas (90, 60 and 20% in the AP/ASP, DVC and ON, respectively). The 5-HT1-like and 5-HT2 receptor antagonist methysergide (10 microM) failed to compete for the binding site. 5-HT3 receptor recognition sites within the AP/ASP and the DVC may be functionally involved in the ability of 5-HT3 receptor antagonists to control emesis.     

Hypotension activates neuropeptide Y-containing neurons in the rat medulla oblongata.

The present study was designed to determine whether neurons within cardiovascular control nuclei of the rat brainstem that become activated following a hypotensive insult also possess the capacity to utilize neuropeptide Y. Adult male Wistar-Kyoto rats were injected with glyceryl trinitrate (10 mg/kg, i.p.) or vehicle, and 4 h later anaesthetized (pentobarbitone, 60 mg/kg, i.p.) and transcardially perfused. The brains were removed and processed by standard two-colour peroxidase immunohistochemistry. Activated cells were determined by incubation with a primary antibody to Fos protein, which was followed by a second incubation with a primary antibody to neuropeptide Y for double labelling of Fos-positive cells. Compared to vehicle, glyceryl trinitrate-induced hypotension caused a marked induction of Fos protein in the caudal one-third of the nucleus tractus solitarius (bregma -14 to -13.3 mm), which tailed off rapidly in more rostral sections. Following hypotension, significant populations of activated cells were also observed in the rostral and caudal ventrolateral medulla. In the caudal nucleus tractus solitarius and the posterior part of the medial nucleus tractus solitarius, respectively, 15 of 104 and 40 of 120 Fos-positive cells exhibited cytoplasmic neuropeptide Y immunoreactivity following hypotension, compared to seven of 40 and 15 of 40 in vehicle-treated rats, indicating a significant (two- to three-fold) increase in double-labelled cells following systemic glyceryl trinitrate (P < 0.05, unpaired t-test). In contrast, in the anterior part of the medial nucleus tractus solitarius, the number of double-labelled cells did not change following hypotension. An increase in double-labelled cells was also observed in the rostral ventrolateral medulla (2.5-fold increase compared to vehicle) and caudal ventrolateral medulla (5.8-fold increase compared to vehicle) following hypotension. These data indicate that, in the rat, neuropeptide Y-containing neurons are involved in the central response to a hypotensive challenge. The primary regions where neuropeptide Y-containing neurons appear to be activated are the caudal one-third of the nucleus tractus solitarius and the caudal ventrolateral medulla/rostral ventrolateral medulla, which are key nuclei associated with the integration of the baroreceptor heart rate reflex and sympathetic vasomotor outflow.     

Expression of c-fos protein in the medulla oblongata of conscious rabbits in response to baroreceptor activation.

Neuronal expression of c-fos protein (Fos) in the medulla in response to baroreceptor activation was studied in conscious rabbits. Raising arterial pressure resulted in a marked increase, compared to control animals, in Fos immunoreactivity in the nucleus tractus solitarius, area postrema and ventrolateral medulla (VLM). Fos-immunoreactive neurons in the VLM extended from the level just rostral to the obex to 3 mm more caudal. Only a small proportion of these neurons showed tyrosine hydroxylase immunoreactivity. The results indicate that baroreceptor activation induces Fos expression in circumscribed medullary regions which have previously been shown to receive excitatory baroreceptor inputs.     

Autoradiographic localization of adenosine uptake sites in guinea pig brain using [3H]dipyridamole

Tritiated dipyridamole, a specific adenosine uptake inhibitor binds in a saturable and reversible fashion to high-affinity receptor sites in guinea pig brain sections (Kd = 10 +/- 1.5 nM; Bmax = 650 +/- 100 fmol/mg prot.). The anatomical distribution of [3H]dipyridamole binding sites obtained with autoradiographic techniques shows a widespread but heterogeneous distribution of the binding sites throughout the whole guinea pig brain. Very high densities of binding sites are observed in the cerebellar cortex (molecular layer), the pyriform cortex, the superior colliculus (superficial layer), the supraoptic nucleus and the nucleus of the tractus solitarius. The anatomical characterization of the adenosine uptake site, using [3H]dipyridamole as a probe, may be useful to determine the functional role of adenosine in the brain.     

Aortic nerve-activated cardioinhibitory neurons and interneurons.

Unit activity evoked by electrical stimulation of the aortic and vagus nerves was recorded in the dorsal motor nucleus and nucleus solitarius of unanesthetized rabbits. Cardioinhibitory cells which showed antidromic activation to stimulation of the vagus nerve and synaptic activation to stimulation of the aortic nerve were localized in lateral dorsal motor nucleus 0.5-0.8 mm anterior of the obex. Additionally, units were found that appeared to be interneurons in the medullary pathway subserving baroreceptor reflex effects on cardioinhibitory neurons. These cells were activated by aortic, and usually vagus, nerve stimulation, appeared to be polysynaptically activated, and were located in medial nucleus solitarius rostral to the obex. Neurons reflecting a cardiac rhythm but not activated by aortic nerve stimulation were also observed.     

Connections of the caudal ventrolateral medullary reticular formation in the cat brainstem

 A region of the caudal ventrolateral medullary reticular formation (CVLM) participates in baroreceptor, vestibulosympathetic, and somatosympathetic reflexes; the adjacent retroambigual area is involved in generating respiratory-related activity and is essential for control of the upper airway during vocalization. However, little is known about the connections of the CVLM in the cat. In order to determine the locations of terminations of CVLM neurons, the anterograde tracers Phaseolus vulgaris leucoagglutinin and tetramethylrhodamine dextran amine were injected into this region. These injections produced a dense concentration of labeled axons throughout the lateral medullary reticular formation (lateral tegmental field), including the retrofacial nucleus and nucleus ambiguus, regions of the rostral ventrolateral medulla, the lateral and ventrolateral aspects of the hypoglossal nucleus, nucleus intercalatus, and the facial nucleus. A smaller number of labeled axons were located in the medial, lateral, and commissural subnuclei of nucleus tractus solitarius, the A5 region of the pontine reticular formation, the ventral and medial portions of the spinal and motor trigeminal nuclei, locus coeruleus, and the parabrachial nucleus. We confirmed the projection from the CVLM to both the rostral ventrolateral medulla and lateral tegmental field using retrograde tracing. Injections of biotinylated dextran amine or Fluorogold into these regions resulted in retrogradely labeled cell bodies in the CVLM. However, the neurons projecting to the lateral tegmental field were located mainly dorsal to those projecting to the rostral ventrolateral medulla, suggesting that these neurons form two groups, possibly with different inputs. Injections of retrograde tracers into the lateral tegmental field and rostral ventrolateral medulla also produced labeled cell bodies in other regions, including the medial and inferior vestibular nuclei and nucleus solitarius. These data are consistent with the view that the CVLM of the cat is a multifunctional area that regulates blood pressure, produces vocalization, affects the shape of the oral cavity, and elicits contraction of particular facial muscles. Received: 18 February 1997 / Accepted: 27 March 1997