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Clonidine-displacing substance (CDS) is biologically active in the brain, as well as the gastric fundus, platelets and vas deferens. We sought to determine whether CDS is contained within peripheral tissues in the rat. Using competitive radioimmunoassay with a clonidine-specific antiserum and 3H-p-aminoclonidine rat adrenal gland and gastric fundus were shown to contain significantly greater amounts of CDS-like radioimmunoactivity than the brain; intermediate-to-low activity was present in the heart, small intestine, serum, kidney and liver. Lung and skeletal muscle exhibited near-background levels. CDS may not be unique to the brain, but also may be synthesized and stored in peripheral organs.

Opioids and some alpha 2-adrenergic agonists are both known for their potent hypotensive actions following local application to the rostral ventrolateral medulla (RVL), in particular the region containing the C1 adrenergic neurons. We sought to determine whether coexistence and/or synaptic interactions might account for the commonality of cardiovascular responses to opioids and catecholamines in the RVL. Dual light and electron microscopic (EM) immunoperoxidase labeling of a rat monoclonal antibody against the opioid peptide Leucine5 (Leu5)-enkephalin and immunoautoradiographic localization of a rabbit antiserum against the catecholamine synthesizing enzyme tyrosine hydroxylase (TH) were examined in single sections through the RVL of adult colchicine-pretreated rats. Cross-reactivity of the enkephalin antibody was most intense with Leu5-enkephalin. Methionine5-enkephalin as well as dynorphin A, but not beta-endorphin, were also recognized by the antisera. By light microscopy, the Leu5-enkephalin-like immunoreactivity (LE-LI) was identified by peroxidase reaction product in perikarya and processes. Most of the perikarya containing LE-LI were located dorsolaterally or ventromedially to those showing immunoautoradiographic labeling for TH. However, a few perikarya appeared to contain both LE-LI and TH-immunoreactivity (TH-I) which were difficult to differentiate by light microscopy. By EM, perikarya and dendrites immunoreactive for LE, TH, and both LE and TH were readily distinguishable. Perikarya and dendrites immunoautoradiographically labeled for TH alone were more numerous than those containing LE-LI or TH-I and LE-LI. Axon terminals also were immunolabeled either for one or both reaction products. However, the TH-labeled neurons constituted one of the primary (42% from a total of 118) targets of terminals containing LE-LI. Additionally, some of these terminals containing LE-LI shared a common target with TH-labeled terminals. These common target neurons contained either TH-I or TH-I and LE-LI. In most cases, the identified junctions were symmetric and the terminals with LE-LI (0.4-1.2 microns in diameter) contained either (1) a few small clear vesicles (scv's) and numerous intensely immunoreactive large (100-150 nm) dense-core vesicles (dcv's); or (2) many scv's and from 0-6 dcv's of a somewhat smaller (80-120 nm) diameter. The latter type of terminal was more consistently dually labeled for TH. The remaining terminals containing LE-LI formed synaptic junctions with unlabeled perikarya or dendrites (32%), were in apposition to other unlabeled as well as TH or LE- and TH-containing terminals (4%) or were without recognizable specializations within the plane of section (22%).(ABSTRACT TRUNCATED AT 400 WORDS)

When A1 noradrenergic neurons in the caudal ventrolateral medulla of rabbits are destroyed electrolytically or by local injection of the neurotoxin kainic acid, the concentration of vasopressin in plasma increases, causing hypertension. The A1 neurons may tonically inhibit the activity of vasopressin-secreting neuroendocrine cells through a direct hypothalamic projection.

In this study, the distribution of neurons containing the adrenaline-synthesizing enzyme phenylethanolamine-N-methyltransferase (PNMT) was mapped in the medulla of the cat. Data from recent studies in the rat suggest that the anatomical structure responsible for cardiorespiratory changes that occur following application of neurotransmitters and drugs to Schlaefke's area on the ventral medullary surface is the nucleus reticularis rostroventrolateralis (RVL), which is distinguished from adjacent regions of the reticular formation, in part, by the presence of adrenaline-synthesizing neurons. To determine whether an equivalent adrenergic population is present in the RVL of the cat, we used antibodies raised against bovine adrenal PNMT to map the distribution of adrenaline-synthesizing neurons in the reticular formation. In the ventrolateral medulla, we found that labeled cells extended from the level of the retrofacial nucleus to the calamus scriptorius. The majority of labeled cells were seen in a nucleus designated RVL at the level of the rostral one-third of the inferior olive. In the dorsomedial medulla, cells were labeled in the caudal aspect of the nucleus tractus solitarii (NTS) and were especially dense in the subnucleus gelatinosus and commissural nucleus of the vagus. A few lightly labeled cells were also present in the rostral pole of the area postrema (AP). In contrast to the rat, few or no immunoreactive cells were found in the rostral NTS, medial longitudinal fasciculus, nucleus paragigantocellularis dorsalis, or periventricular gray. Our results are consistent with the notion that an area of the RVL containing adrenergic perikarya is the anatomical structure responsible for cardiovascular changes that occur when chemicals are applied to Schlaefke's area.

The somatic pressor reflex (SPR) elicited in anesthetized paralyzed rats by electrical stimulation of the sciatic or sural cutaneous afferent nerves produced an increase in arterial pressure ranging from 5 to 40 mmHg. Stimulation of femoral or tibial afferent nerves from muscle produced a depressor response. The SPR was not affected by midpontine transection but was eliminated either by hemisection of the lumbar spinal cord contralateral, but not ipsilateral, to the stimulated nerve or by electrolytic or kainic acid lesion of the contralateral, but not ipsilateral, rostral ventrolateral medulla (RVL). Stimulation of the brachial plexus elicited an SPR that was not eliminated by contralateral lumbar hemisection but was abolished by RVL lesion. RVL lesions consistently overlapped areas containing phenylethanolamine N-methyltransferase-labeled C1 adrenergic neurons. Kainic acid injections into the lateral reticular nucleus (LRN) did not affect the SPR. Neither contralateral nor ipsilateral electrolytic lesions of other autonomic areas including parabrachial nucleus, the nucleus tractus solitarii, the A5 region, or the inferior cerebellar peduncle (output pathway of the LRN) affected the reflex. In axonal transport studies using horseradish peroxidase, afferent terminals of the sciatic nerve were shown to overlap spinoreticular neurons in the dorsal horn retrogradely labeled from tracer injections in the RVL. We conclude that the SPR can be elicited in rats, that it is mediated by spinoreticular afferents traveling in the contralateral spinal cord, and that the C1 adrenergic area of the RVL is a critical region for the integration of the somatic pressor reflex.

We sought to characterize in detail neurons in rat retina that contain phenylethanolamine N-methyltransferase (PNMT), the epinephrine biosynthetic enzyme. Cell bodies and processes of PNMT-containing neurons in retina were identified by immunohistochemistry. The coexistence of other catecholamine biosynthetic enzymes in the same cells was also investigated. Biochemical, molecular biological and immunochemical methods were applied to determine whether retinal PNMT is similar to the adrenal enzyme, since regulation of PNMT in retina and adrenal appears to be different. The results show that there are two types of PNMT-containing cells: those containing PNMT exclusively and those containing PNMT with two other catecholamine-synthesizing enzymes, tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC), but not dopamine beta-hydroxylase (DBH). PNMT-only cell bodies are localized in the inner nuclear layer (INL) and the ganglion cell layer (GCL). Their processes are observed in outer and inner strata of the inner plexiform layer (IPL). Only a small fraction of PNMT neurons in INL also contain TH and AADC. These cells send their processes to the adjacent stratum of the IPL. Antibodies to bovine adrenal DBH, however, fail to localize DBH in any rat retinal cells. Immunochemical titration shows that PNMT from both retina and adrenal gland has the same immunoreactivity. Furthermore, a PNMT-cDNA probe hybridizes equally with PNMT-mRNA isolated from both the retina and the adrenal gland. These results indicate that PNMT is identical in these tissues.

To examine the biochemical events associated with the retrograde reaction in central noradrenergic neurons, changes in the activities of several enzymes subserving the metabolism of catecholamines, including tyrosine hydroxylase (TH),dopamine-beta-hydroxylase (DBH), DOPA decarboxylase (DDC), and monoamine oxidase (MAO), were measured in the nucleus locus coeruleus of rat brain following transection of the ascending axons from neurons in this nucleus by electrolytic lesions of the posterolateral hypothalamus. Such lesions produced a triphasic response in the activities of TH and DBH consisting of: (a) an increase to approximately 150 percent of control during the first 48 h followed by (b) a reduction reaching 60 percent of control by day 14, and (c) a full recovery of activity by day 21-28. In contrast, the activities of DDC and MAO, enzymes non-specific for catecholamine neurons, were unchanged. Immunochemical titration with specific antibodies to TH and DBH demonstrated that the fall in enzyme activity was entirely attributable to reduced accumulation of specific enzyme protein and not inhibition of pre-existing enzyme molecules. There was no reduction in the number of neurons in the nucleus locus coeruleus as a consequence of the lesion. We conclude that a reduction in the accumulation of specific enzymes subserving transmitter biosynthesis characterizes a reversible retrograde reaction of central noradrenergic neurons. The coincidence of the time course of reduced enzyme accumulation with regenerative sprouting from damaged noradrenergic axons and also the absence of classical signs of chromatolysis in locus coeruleus neurons following comparable lesions suggest that, first, during the retrograde reaction there may be a reordering of priorities governing accumulation of specific proteins favoring accumulation of those required for reconstitution of cellular processes by sprouting at the expense of proteins utilized in the synthesis of neurotransmitters, and second, some intrinsic neurons of the CNS may undergo reversible biochemical changes of a retrograde reaction in the absence of the classical morphological appearance of chromatolysis.

Pharmacological and biochemical studies suggest that interactions between cholinergic and catecholaminergic and catecholaminergic neurons, particularly those of the C1 adrenergic cell group, in the rostral ventrolateral medulla (RVL) may be important in cardiovascular control. Ultrastructural localization of choline acetyltransferase (ChAT), the biosynthetic enzyme for acetylcholine, and its relation to neurons exhibiting immunoreactivity for catecholamine- (tyrosine hydroxylase; TH) or adrenaline (phenylethanolamine-N-methyltransferase; PNMT) -synthesizing enzymes were examined in the RVL using dual immunoautoradiographic and peroxidase anti-peroxidase (PAP) labeling methods. By light microscopy, the ChAT-immunoreactive neurons were located both dorsally (i.e. the nucleus ambiguus) and ventromedially to those labeled with TH or PNMT (TH/PNMT). A few ChAT-labeled processes were dispersed among TH/PNMT-containing neurons with the majority of overlap immediately ventral to the nucleus ambiguus. By electron microscopy, ChAT-immunoreactivity (ChAT-I) was detected in neuronal perikarya, dendrites, axons and axon terminals and in the vascular endothelial cells of certain blood vessels. The ChAT-labeled perikarya in the ventromedial RVL were medium-sized (15-20 microns), elongated, contained abundant cytoplasm and had had slightly indented nuclei. Synaptic junctions on ChAT-immunoreactive perikarya and dendrites were primarily symmetric with 64% (45 out of 70) of the presynaptic terminals unlabeled. The remaining terminals were immunoreactive for ChAT (30%) or TH/PNMT (6%). Terminals with ChAT-I were large (0.8-2.0 microns) and contained numerous small clear vesicles and 1-2 dense core vesicles. Seventy-seven percent (112 out of 145) of the ChAT-labeled terminals formed symmetric synapses with unlabeled perikarya and dendrites, whereas only 8% were with TH/PNMT-labeled perikarya and dendrites, and 15% were with ChAT-immunoreactive perikarya and dendrites. We conclude (1) that cholinergic neurons in the RVL principally terminate on and receive input from non-catecholaminergic neurons, and (2) that the reported sympathetic activation following application of cholinergic agents to the RVL may be mediated by cholinergic inhibition of local inhibitory interneurons. The observed synapses between ChAT and TH/PNMT-containing neurons suggests that cholinergic and adrenergic neurons additionally may exert a minor reciprocal control on each other and thus may modulate their response to the more abundant input from afferents containing other transmitters.

We sought to determine the role in control of arterial pressure (AP) of the caudal ventrolateral medulla (CVL), a region containing noradrenergic neurons of the A1 group. Electrical stimulation of the A1 area in anaesthetized rats elicits a fall of AP and heart rate at low, but not high, stimulus frequencies. Electrolytic lesions of the A1 area produce fulminating hypertension and a 13-fold increase in plasma arginine vasopressin (AVP). A1-hypertension is attenuated by treatment with an AVP antagonist and is also diminished in rats of the Brattleboro strain. After AVP blockade, the residual hypertension is abolished by treatment with ganglionic or alpha adrenergic blockers, but not by adrenalectomy. We conclude that the region of the CVL containing the A1 noradrenergic cells tonically inhibits the discharge of sympathetic nerve activity as well as the release of AVP from the pituitary.

We examined the effects of electrical stimulation of a restricted area of the dorsal medullary reticular formation (DMRF) on regional cerebral blood flow (CBF) in anesthetized (by chloralose), paralyzed (by curare) rats. CBF was measured in tissue samples by the Kety principle, with 14C-iodoantipyrine as indicator. Stimulation of DMRF elicited a widespread, significant increase in CBF in 12 of 13 areas. The increase in flow was greatest in cerebral cortex, up to 240% of control. However, it was also substantially increased in selected regions of telencephalon, diencephalon, mesencephalon, and lower brainstem, but not cerebellum. In contrast, electrical stimulation of the midline (interstitial nucleus of the medial longitudinal fasciculus) 1 mm medial to the DMRF did not change CBF. The increase in CBF evoked by DMRF stimulation persisted after transection of the spinal cord at C1 or cervical sympathetic trunk. We conclude that excitation of neurons originating in or passing through the DMRF can elicit a potent and virtually global increase of CBF. The effect appears to be mediated by intrinsic pathways of the central nervous system.

Electrical stimulation of the cerebellar fastigial nucleus (FN) in anesthetized, paralyzed, and artificially ventilated rat with a 10-s stimulus train (50 Hz) resulted in a stimulus-locked elevation in arterial pressure (AP) and heart rate, the fastigial pressor response (FPR). Blockade of autonomic effectors by chemosympathectomy (produced by treatment with 6-hydroxydopamine) combined with adrenalectomy, or by spinal cord transection at C1, abolished the FPR but unmasked an elevation of AP with longer latency (10-12 s) and duration (2-4 min), termed the residual FPR. The residual FPR was 1) abolished by midbrain transection, 2) blocked by administration of a specific antagonist of the vasopressor response to arginine vasopressin (AVP) [1,d(CH2)5Tyr(Me)AVP], and 3) was absent in homozygous and attenuated in heterozygous rats of the Brattleboro strain. FN stimulation elevated AVP threefold (from 13 +/- 1 to 38 +/- 8 pg/ml, P less than 0.02; n = 6) in intact rats and sevenfold in rats with combined chemosympathectomy and adrenalectomy (from 14 +/- 1 to 96 +/- 11 pg/ml, P less than 0.001; n = 9). Stimulation of the cerebellar FN can release AVP. In the absence of sympathoadrenal effectors, the amount so released is enhanced and capable of elevating AP.

Horseradish peroxidase was injected unilaterally into the intermediate portion of the nucleus of the tractus solitarius. The most extensive retrograde labeling was present contralaterally in the rostral fastigial nucleus, bilaterally in the Koelliker-Fuse nucleus, and bilaterally with ipsilateral predominance in the posterolateral hypothalamus and paraventricular hypothalamic nucleus. These projections may represent the anatomical substrates by which these brain areas modulate baroreceptor reflex function.

In anesthetized, paralyzed rats, bilateral microinjections of kainic acid (KA) into an area of caudal ventrolateral medulla containing A1 noradrenergic neurons of the A1 group (A1 area) first reduced and then elevated arterial pressure (AP), heart rate (HR) and sympathetic renal nerve activity. These effects are attributable to initial excitation and then paralysis of local neurons by KA. The microinjection of tetrodotoxin into an area of rostral ventrolateral medulla containing adrenaline neurons of the C1 group (C1 area) and which is innervated by neurons in A1 area abolished all the effects of KA. The pretreatment of the C1 area with 6-hydroxy-dopamine (6-OHDA) also abolished all the effects of KA. In contrast interruption of projections of the A1 area to the hypothalamus or nucleus tractus solitarii had no effects. The latency for response evoked in renal nerve by stimulation of A1 area was about 10 ms longer than that elicited from C1 area. Tyramine microinjected into the C1 area elicited a dose-dependent decrease in AP prevented by local application of desmethylimipramine. We conclude that neurons of the A1 area tonically inhibit sympathetic activity by inhibiting neurons of the C1 area probably by release of NA.

We have studied the responses to electrical and chemical stimulation of the ventrolateral medulla in the chloralose-anesthetized, paralyzed, artificially ventilated rat. Locations of most active pressor responses were compared to regions containing neurons labeled immunocytochemically for phenylethanolamine N-methyltransferase (PNMT), the enzyme catalyzing the synthesis of adrenaline. Elevations of arterial pressure (+81.6 +/- 2.5 mm Hg) and cardioacceleration (+73 +/- 13.6 bpm) were elicited with low current (5 times threshold of 9.5 +/- 1.1 microA) electrical stimulation in a region of rostral ventrolateral medullary reticular formation we have termed the nucleus reticularis rostroventrolateralis (RVL). Electrical stimulation of the RVL increased plasma catecholamines (16.8-fold for adrenaline, 5.3-fold for noradrenaline, and 1.9-fold for dopamine) and vasopressin (1.7-fold before spinal transection, 4.7-fold after). The location of the most active pressor region in the ventrolateral medulla corresponded closely with the location of C1 adrenaline-synthesizing (PNMT-containing) neurons. In addition, the location of the most active pressor region in the dorsomedial medulla corresponded with the location of a bundle of PNMT-containing axons. Unilateral injections into the RVL of the excitatory amino acid monosodium L-glutamate (50 pmol to 10 nmol), but not saline, caused transient dose-dependent and topographically specific elevations (maximum +71.6 +/- 4.9 mm Hg) of arterial blood pressure and tachycardia. Injections of the rigid structural analogue of glutamate, kainic acid, caused large, prolonged (at least 15 min) pressor responses and tachycardia. Unilateral injections of the inhibitory amino acid gamma-aminobutyric acid (GABA) into the RVL caused transient dose-dependent hypotension (maximum -40.8 +/- 6.6 mm Hg) and bradycardia, whereas the specific GABA antagonist bicuculline caused prolonged (10 to 20 min) elevations (+64.2 +/- 6.8 mm Hg) of arterial pressure and tachycardia. By contrast, injections of the glycine antagonist strychnine had no significant effect. Bilateral injections of the neurotoxin, tetrodotoxin, dropped arterial pressure to low levels (51.7 +/- 4.7) not changed by subsequent spinal cord transection at the first cervical segment (52.5 +/- 6.2). We propose the following. (1) Neurons within the RVL, most probably C1 adrenaline-synthesizing neurons, exert an excitatory influence on sympathetic vasomotor fibers, the adrenal medulla, and the posterior pituitary. (2) These neurons are tonically active and under tonic inhibitory control, in part via GABAergic mechanisms--perhaps via the nucleus of the solitary tract (NTS).(ABSTRACT TRUNCATED AT 400 WORDS)

In previous experiments we implicated projections from the medial geniculate body (MG) to a subcortical field, involving portions of the posterior caudate-putamen and amygdala, in the classical conditioning of emotional responses to acoustic stimuli in the rat. In the present series of experiments we examined whether intrinsic neurons in the subcortical field mediate emotional conditioning and, if so, whether the critical neurons are contained within the amygdala or the caudate-putamen. Rats were prepared with a unilateral electrolytic lesion of the MG. Contralaterally, intrinsic neurons were destroyed in the subcortical field by microinjection of ibotenic acid. This lesion combination leaves one MG and one subcortical field intact but disconnected. Controls received unilateral injection of phosphate buffer vehicle into the subcortical field contralateral to the MG lesion or were unoperated. After two weeks the animals were instrumented for continuous, computer-assisted recording of arterial pressure and heart rate and subjected to classical conditioning trials involving the presentation of a pure tone in association with foot-shock. The occurrence of the shock with respect to the tone was random for a pseudoconditioned control group. Conditioned changes in mean arterial pressure, heart rate and emotional behavior ('freezing') elicited by the tone were assessed during extinction trials. Following completion of the experiments, the rats were sacrificed and their brains were removed and sectioned using standard procedures. Lesion location and size was evaluated with the assistance of a computer-based image processing system. In unoperated conditioned rats the acoustic stimulus elicited increases in arterial pressure and heart rate, and induced freezing. The arterial pressure and freezing responses differed in conditioned and pseudoconditioned rats, but the heart rate response did not. Therefore, only the arterial pressure and freezing responses reflect the formation of an association between the tone and shock. Destruction of intrinsic neurons in the subcortical field contralateral to a unilateral MG lesion disrupted the associative conditioning of the arterial pressure and freezing responses. These were reduced in magnitude to the level observed in pseudoconditioned rats. The non-associative heart rate change was not affected by the lesions. That ibotenic acid destroyed intrinsic neurons and spared fibers in the subcortical field was demonstrated anatomically and biochemically.(ABSTRACT TRUNCATED AT 400 WORDS)

We studied the organization of projections from the C1 adrenergic and A1 noradrenergic cell groups in the ventrolateral medulla (VLM) to the hypothalamus and the spinal cord by using a combination of retrograde transport of fluorescent tracers and immunocytochemistry. Three issues were addressed. Neurons in the VLM that stain immunohistochemically for phenylethanolamine N-methyltransferase (PNMT) have been assumed to be adrenergic. However, the presence of PNMT-immunoreactive neurons in the hypothalamus that do not stain for tyrosine hydroxylase (TH) prompted us to re-evaluate the VLM by an elution-restaining immunohistochemical procedure. We confirmed that nearly all of the rostral medullary PNMT-immunoreactive neurons also stained for TH. By contrast, in the caudal medulla, very few TH-positive neurons stained for PNMT. Neurons of the C1 group in the rostral VLM project both to the thoracic spinal cord and to the hypothalamus. To determine whether individual C1 neurons send collaterals to the hypothalamus and spinal cord, we injected different-colored fluorescent dyes (diamidino yellow or fast blue) into the thoracic spinal gray matter and either the median preoptic (MnPO) or paraventricular (PVH) nuclei of the hypothalamus. Very few double-labeled neurons were found in the VLM, indicating that hypothalamic and spinal cord projections arise from almost completely independent populations of cells. Approximately half of the neurons projecting to the spinal cord from rostral VLM were not immunoreactive for TH or PNMT, indicating that a substantial part of this projection is noncatecholaminergic. The MnPO and the PVH both receive extensive catecholaminergic inputs from the VLM. We also used fluorescent retrograde tracers to determine whether individual VLM neurons send collaterals to both hypothalamic sites. Approximately 20% of neurons projecting to the MnPO in the rostral two thirds of the VLM also sent collaterials to the PVH, nearly all of these neurons being TH-positive. The collateralization of the VLM catecholaminergic projection to the hypothalamus may provide an anatomical substrate for integration of fore-brain participation in cardiovascular regulation. In contrast, the adrenergic projection from the VLM to the intermediolateral column of the spinal cord arises from a separate population of neurons.

We sought to determine whether the global increase in regional cerebral blood flow (rCBF) produced by electrical stimulation of the rostral cerebellar fastigial nucleus (FN) is a consequence of excitation of intrinsic neurons of the FN or of axons of fibers passing through or projecting into it. Studies were conducted in rats anesthetized with chloralose, paralyzed and ventilated. rCBF was measured with [14C]iodoantipyrine as tracer and regional cerebral glucose utilization (rCGU) by [14C]2-deoxyglucose in homogenates of 11 brain regions. Neuronal perikarya in FN were excited chemically by local microinjection of the glutamate analogue kainic acid (KA) (5 nmol in 100 nl). KA elicited a transient and significant fall of arterial pressure and heart rate, the fastigial depressor response (FDR). Associated was a significant and symmetrical reduction in rCBF, to 44% of control in all regions except medulla. The response was site- and agent-specific and unrelated to the hypotension. KA also significantly and proportionally reduced, to 52% of control, rCGU in the same 10 areas of brain. In all regions the magnitudes of the reductions in rCBF and rCGU elicited by KA were linearly related. Intrinsic neurons of FN were chronically destroyed by local microinjection of the excitotoxin ibotenic acid (IBO) (10 micrograms/microliters in 0.4 microliter). Destruction of intrinsic FN neurons had no effect on resting rCBF nor on the global cerebrovascular vasodilation elicited by electrical stimulation of the FN. We conclude that: (a) excitation of intrinsic neurons of FN elicits a widespread reduction of cerebral metabolism and, secondarily, blood flow; (b) FN neurons do not exert a long-term tonic influence on brain blood flow nor metabolism; (c) the global increase in rCBF elicited by electrical stimulation of the FN is a consequence of excitation of axons projecting into or through the nucleus.

(c) the Vmax for the high affinity uptake of [3H]norepinephrine in homogenates of lower brain stem;

We have investigated the effects of systemic administration of atropine sulfate on the global cerebrovascular vasodilation elicited by electrical stimulation of the cerebellar fastigial nucleus (FN) in chloralose-anesthetized rat. Atropine (0.3 mg/kg, i.v.) abolished the cerebrovasodilation elicited from FN but did not affect the concomitant elevation in arterial pressure and the EEG changes. We conclude that the cerebrovascular effect of FN stimulation, but not the peripheral cardiovascular or EEG changes, are mediated by cholinergic muscarinic receptors associated with cerebral vessels and/or intrinsic neural pathways.

The developmental morphology and synaptic associations of neurons in the nigrostriatal anlage are examined by the electron microscopic immunocytochemical localization of tyrosine hydroxylase at embryonic (E) day 13.5 and 14.5 in rat brain. At E 13.5, immunoreactivity for the enzyme is localized throughout the cytoplasm of neuronal perikarya and processes including somatic, dendritic, and axonal growth cones. The cytoplasmic organelles in perikarya include primarily ribonucleic-protein particles, mitochondria and an immature Golgi apparatus. At E 14.5, the tyrosine hydroxylase labeled processes are detected in the lateral hypothalamus and ventrolateral caudate-putamen. The axonal processes showing immunoreactivity in the ventral mesencephalon and more rostral portions of the nigrostriatal bundle are frequently attached to unlabeled neurites by puncta adherentia. In the hypothalamus and caudate-putamen presumably transient synaptic junctions are also detected between the labeled axons and unlabeled neurons. The immature morphological features of neurons showing immunoreactivity for tyrosine hydroxylase thus indicate, that the biochemical differentiation of the nigrostriatal neurons precedes complete cytological differentiation.

A polyclonal antiserum was raised in rabbit against the clonidine analog p-aminoclonidine (PAC) coupled to hemocyanin. The antiserum (anti-PAC3) exhibited high affinity for unconjugated [3H]PAC (Kd 0.32 +/- 0.07 nM) in a rapid-filtration radioimmunoassay. Competition experiments showed that PAC, clonidine, and naphazoline cross-reacted with the anti-PAC3 antiserum, whereas a number of other structurally related compounds did not. An endogenous clonidine-displacing substance (CDS) partially purified from bovine brain also inhibited specific [3H]PAC binding to anti-PAC3 in a dose-dependent manner. Thus, (a) anti-PAC3 antiserum is specific for clonidine and closely related compounds, and (b) CDS may structurally resemble clonidine since it is recognized by this highly specific antiserum.

The immunocytochemical localization of the biosynthetic enzymes--tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine-N-methyltransferase (PNMT)--was used to determine the cytological features and precise neuroanatomical location of catecholaminergic neurons in the medulla oblongata of rat. Perikarya labeled with TH were detected in two bilaterally symmetrical columns located in the ventrolateral and dorsomedial medulla. The distribution and the number of neuronal perikarya containing TH were the same as those containing DBH, except in the dorsal motor nucleus of the vagus at the level of the area postrema where the number of neurons immunocytochemically labeled for TH was considerably greater than those labeled for DBH. The detection of perikarya which show immunoreactivity for TH, used in the biosynthesis of dopamine, noradrenaline, and adrenaline, but not DBH, which converts dopamine to noradrenaline, suggests the existence of dopamine-synthesizing neurons in the medulla. Perikarya labeled with PNMT, used in the biosynthesis of adrenaline, were localized in more restricted regions corresponding to rostral subsets of the dorsal and ventral groups labeled for TH and DBH. Counts of neurons immunocytochemically labeled for TH or PNMT were obtained in order to determine the relative ratio of neurons which contain the enzymes necessary for the synthesis of dopamine, noradrenaline, or adrenaline at various levels of the medulla. At the most caudal levels no PNMT labeled neurons were detected. Further rostral, PNMT-labeled neurons were first detected in the ventrolateral medulla. At the level of the area postrema, the number of PNMT-labeled neurons in the ventrolateral medulla was approximately half of the number of cells showing immunoreactivity for TH. In contrast, few PNMT-labeled cells were detected in the dorsomedial medulla at the level of the area postrema compared to many neurons labeled for TH. At rostral medullary levels, in both the ventrolateral and the dorsomedial regions, the number of neurons labeled for TH and PNMT was essentially the same. Thus most, if not all, of the catecholaminergic neurons in the rostral medulla have PNMT, necessary for the synthesis of adrenaline.

Kainic acid (KA), an analogue of L-glutamate, was microinjected in 0.1 microliter of saline into the nucleus tractus solitarii (NTS) of adult rats. In rats anesthetized with halothane or alpha-chloralose, KA injected unilaterally elicited hypotension, bradycardia, and apnea. The threshold dose was 0.1-0.2 ng (10(-13) mol). Doses greater than 0.2 ng blocked responses to subsequent injections for at least 30 minutes. Doses of KA greater than 15 ng reduced the reflex bradycardia elicited by raising the arterial pressure with phenylephrine and produced arterial hypertension in rats anesthetized with alpha-chloralose or in other rats within 15 minutes of terminating halothane anesthesia. Bilateral injection of KA in doses greater than 15 ng completely blocked baroreflexes and resulted in a dose-dependent elevation of arterial pressure (167 +/- 9.4; P less than 0.001) both in alpha-chloralose-anesthetized rats and in awake rats after the termination of halothane anesthesia. The hypertension rapidly led to pulmonary edema and death. Procaine microinjected also elicited fulminating hypertension; vehicle did not. Doses of KA producing hypertension caused no histological or biochemical evidence of neuronal death. The cardiovascular responses to KA were restricted to sites in the intermediate one-third of NTS and could not be elicited by injection into adjacent sites in brainstem. The results indicate that, in low doses, KA injected into NTS stimulates neurons which mediate the baroreflex, whereas, in higher doses, it produces baroreflex blockade and neurogenic hypertension. The results suggest that fulminating hypertension can be produced by nondestructive perturbations of neurochemical transmission in brain. Since the cardiovascular responses of KA are similar to those produced by microinjection into NTS of the amino acid neurotransmitter glutamic acid, the study adds further support to the hypothesis that L-glutamate is the neurotransmitter released by baroreceptor afferent nerves.

The distribution and morphology of cells containing tyrosine hydroxylase (TH) were mapped by using the immunoperoxidase technique in the hypothalamus and preoptic area in two strains of mouse, CBA/J and BALB/cJ. On the basis of rostral-caudal contiguities between cell aggregates, hypothalamic preoptic neurons were subdivided into three arbitrary groups: (1) dorsal, (2) intermediate, and (3) ventral. New or more prominent collections of TH cells were observed, and in some regions, cells were more complexly organized than originally described. In the dorsal group, a rostral collection of small ovoid cells, previously not described, were located in the anterior preoptic nucleus (APN) of Loo ('31) and extended rostrally and ventrally into the preoptic periventricular gray. The next constituent occupied the paraventricular nucleus (PVN), and was composed of two classes of cells: (1) a small ovoid cell within anterior and medial parvocellular PVN in contiguity rostrally with a similar cell in APN and (2) a larger, angular cell within and adjacent to the lateral PVN in contiguity caudally with cells in the zona incerta (ZI). Further caudally, a larger and more pleomorphic collection of TH neurons was localized in the medial ZI, particularly at midtuberal levels. These cells were not scattered, as previously reported, but were differentiated into two clear-cut densities, a larger medial island and a more elongated lateral island. Cells of ZI, both large and small, extended caudally into the dorsal hypothalamic and subparafascicular nuclei and periventricular gray. In contrast to previous descriptions, no cells were seen in the nucleus reuniens. In the intermediate group, the most rostral constituent occupied the preoptic periventricular gray, extended as far as the lamina terminalis, and merged dorsocaudally with cells in APN. While the variably shaped cells of the hypothalamic periventricular gray (PVG) were still present in the retrochiasmatic region, a striking absence of these cells was noted at midtuberal levels between the dorsomedial and arcuate hypothalamic nuclei. At this level, a new group of small-round TH cells, resembling those of the arcuate nucleus, was identified in the dorsomedial hypothalamic nucleus (DMN). At caudal tuberal levels, similar neurons were found in the posterior hypothalamic nucleus (PH). These neurons overflowed medially into the PVG and caudoventrally into the arcuate nucleus. In the ventral group, the most rostral constituent, composed of both small and ovoid cells in the retrochiasmatic area, appeared to represent the rostral commissural portion of the arcuate nucleus (Arc).(ABSTRACT TRUNCATED AT 400 WORDS)

Although the auditory cortex is believed to be the principal efferent target of the medial geniculate body (MG), our recent behavioral studies indicate that in rats the conditioned coupling of emotional responses to an acoustic stimulus is mediated by subcortical projections of the MG. In the present study we have therefore used WGA-HRP as an anterograde and retrograde axonal marker to (1) define the full range of subcortical efferent projections of the MG; (2) identify the cells of origin within the MG of each projection; and (3) determine whether the subregions of the MG that project to subcortical areas receive inputs from acoustic relay nuclei of the mid-brain, particularly the inferior colliculus. The rat MG was first parcelled into three major cytoarchitectural areas: the ventral, medial, and dorsal divisions. The suprageniculate nucleus, located within the body of the MG just dorsal to the medial division, was also identified. Efferent projections of the MG were determined by combined anterograde and retrograde tracing methods. Injections of WGA-HRP in the MG produced anterograde transport to cortex and several subcortical areas, including the posterior caudate-putamen and amygdala, the ventromedial nucleus of the hypothalamus, and the subparafascicular thalamic nucleus. The cells of origin of the subcortical projections were then mapped retrogradely after injections in the anterogradely labeled areas. Injections in the caudate-putamen or amygdala retrogradely labeled the medial division of the MG and the suprageniculate nucleus, as well as several adjacent areas of the posterior thalamus surrounding the MG. In contrast, injections in the ventromedial nucleus of the hypothalamus or the subparafascicular thalamic nucleus only produced labeling in the areas surrounding MG. Afferents to MG from the inferior colliculus were then identified. The central nucleus of the inferior colliculus, the main lemniscal acoustic relay nucleus in the midbrain, was found to project to the ventral and medial divisions of the MG. In contrast, the dorsal cortex and external nucleus of the inferior colliculus project to each division of the MG and to several additional nuclei in adjacent areas of the posterior thalamus. These data demonstrate that the medial division of MG, the suprageniculate nucleus, and immediately adjacent areas of the posterior thalamus provide a direct linkage between auditory neurons in the inferior colliculus and subcortical areas of the forebrain and thereby support the view that thalamic sensory nuclei relay afferent signals to subcortical as well as cortical areas.

Changes in cyclic nucleotide metabolism similar to those characteristic of the chronic forms of hypertension were observed in an acute neurogenic form of hypertension in rats produced by electrolytic lesions of the nucleus tractus solitarii. These changes that were evident 2 hr after the lesions were made included decreased cyclic AMP levels in the heart, increased cGMP:cAMP ratio, cAMP phosphodiesterase (3':5'-cAMP 5'-nucleotidohydrolase, EC 3.1.4.17) and guanylyl cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) activities in the aorta and decreased snesitivity of adenylyl cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) in both the aorta and heart to stimulation by the beta-adrenergic stimulant isoproterenol. These changes appear to depend on catecholamine release and are not due to mechanical distortion secondary to the increased arterial pressure. These studies provide biochemical support to the concept that the sympathetic nervous system may play a critical role in the initiation of the hypertensive syndrome and that chronic hypertension could result from the fixation of the biochemical effects of increased sympathetic activity.

We have previously demonstrated that at embryonic Day (E) 8, some cells of the chick ciliary ganglion (CG) contain the catecholaminergic (CA) enzyme tyrosine hydroxylase (TH), but not phenylethanolamine-N-methyltransferase (PNMT); and that in culture essentially all cells express both enzymes. In the present study, we sought to determine, first, whether the expression of adrenergic traits in the CG in vivo is transient or permanent in the CG. To do so, CGs were removed from E5 to postnatal Day 5, fixed, and processed for the immunocytochemical localization of the CA enzymes: TH, L-amino acid decarboxylase (AADC), and PNMT. At all stages examined, some CG neurons expressed TH immunoreactivity (TH-IR) and all contained AADC-IR. However, none stained with PNMT antibodies, indicating that these cells stably express some, but not all, of the CA enzymes. Second, we examined whether CG neurons in culture expressed other CA markers. CG neurons did not contain detectable levels of TH enzyme activity nor did they transport and store exogenously supplied monoamines. These results indicate that some but not all traits necessary for adrenergic function are present in CG neurons in vitro. Third, we sought to establish whether CA expression in CG neurons is affected by modification in culture conditions. Cultures of CG neurons continued to express TH-IR even when grown in the presence of either 50% HCM or 20 mM KCl for 5 days. Finally, the expression of the cholinergic enzyme, choline acetyltransferase (CAT) was assessed in CG cultures by biochemical assay. CAT activity increased five-fold between 5 and 17 days in vitro, irrespective of the presence of TH-IR in 100% of the CG neurons of sister cultures. These data suggest that at least a subpopulation of CG neurons express both TH and CAT in culture. We conclude that the postmitotic neurons of the CG are able to express some but not all of the traits characteristic of a CA phenotype while maintaining cholinergic expression. These findings suggest that (1) the appearance of the full complement of adrenergic properties is not coordinated and may be regulated by different environmental cues and (2) parasympathetic neurons can express both adrenergic and cholinergic traits simultaneously.

The contribution of intrinsic neurons in the medial geniculate body (MG) of the rat to the classical conditioning of emotional responses to acoustic stimuli was examined. Injection of ibotenic acid, which destroys cell bodies but not axons of passage, into the MG disrupted the conditioned changes in mean arterial pressure and emotional behavior elicited by a tone previously associated with footshock. Unconditioned responses elicited by the tone and by the footshock were unaffected. Moreover, the same animals readily learned to associate a visual stimulus with footshock. Thus, destruction of intrinsic neurons in the MG selectively disrupts the conditioning of emotional responses to acoustic stimuli. The MG appears to be the afferent link in a modality specific emotional learning pathway.

Glutamic acid decarboxylase (GAD), the enzyme which synthesizes the inhibitory transmitter gamma-aminobutyric acid (GABA), was localized immunocytochemically within cells and processes distributed throughout the ventrolateral medulla. In caudal regions, GAD-stained cells were adjacent to the 'precerebellar' lateral reticular nucleus and partially overlapped the A1 area of norepinephrine synthesizing neurons. The largest number of labeled neurons filled the rostral ventrolateral medulla (RVL), coinciding with and extending beyond the C1 adrenergic area. GAD-positive cells also occupied the nucleus reticularis parvocellularis, raphe magnus (RM) and lateral wings of RM in the region of the pararaphe. Intrinsic GAD-containing cells in the ventrolateral medulla may tonically inhibit sympathoinhibitory neurons in the caudal ventrolateral medulla (CVL) and sympathoexcitatory neurons in the RVL.

In rat, anaesthetized with urethane and paralyzed, bilateral microinjections of histamine or naphazoline into a region of the rostral ventrolateral medulla (RVL) containing adrenergic neurons of the C1 group (C1 area) elicited a dose-dependent, anatomically specific and reversible decrease of arterial pressure (AP) and heart rate (HR). Similar effects were observed with the histamine agonists 4-imidazole acetic acid or the selective H2 agonist dimaprit. The selective H2 antagonist metiamide but not the H1 antagonist chlorpheniramine injected bilaterally into the C1 area reversibly blocked the hypotension and bradycardia elicited by histamine or dimaprit without affecting the responses produced by naphazoline. Neither metiamide nor chlorpheniramine change resting AP or HR. Pretreatment of the C1 area with the non-imidazole alpha-adrenoceptor blocker phenoxybenzamine while increasing resting AP and HR, antagonized the decrease of AP and HR elicited by the indirect sympathomimetic agonist tyramine but not those by histamine. We conclude, that histaminergic and tonic noradrenergic mechanisms coexist in the C1 area of the RVL mediating sympathoinhibitory responses, the effect of histamine is mediated by H2 but not by H1 receptors.

The peroxidase-antiperoxidase (PAP) technique is used to determine the cellular localization of tyrosine hydroxylase in the human fetal nervous system. Antiserum to trypsin-treated tyrosine hydroxylase from the bovine adrenal medulla can be detected immunocytochemically in peripheral sympathetic neurons in the 8-mm (5-week) fetus, but can not be detected in the central nervous system until later stages of development. The cytological features and distribution of the neuronal perikarya and processes labeled for the enzyme are similar to those of the catecholaminergic neurons previously identified by histofluorescence. These findings indicate that specific neurons in the human fetus have at least one of the enzymes necessary for the biosynthesis of catecholamines and that cross-species reactivity exists between antiserum produced to the bovine tyrosine hydroxylase and human tissues.

Tryptophan hydroxylase [EC 1.14.16.4; L-tryptophan, tetrahydropteridine:oxygen oxidoreductase (5-hydroxylating)], the enzyme catalyzing the rate-limiting step in the biosynthesis of serotonin, was purified 79-fold from the region of the raphe nucleus of rat midbrain by sequential column chromatography and disc-gel electrophoresis. In electrophoresis three bands were distinguished, A, B, and C, which, when separated and submitted individually to electrophoresis, reproduced the same three bands. Bands A and C were enzymatically active and inhibited by para-chlorohenylalanine. Antibodies produced to each of the three bands crossreacted by immuno double diffusion and electrophoresis with each other and homogenates of raphe nuclei; they completely inhibited enzyme activity only of tryptophan hydroxylase. Tryptophan hydroxylase was localized by light and electron immunohistochemistry to serotonin neutrons of the raphe. Ultrastructurally, in cell bodies, the enzyme was distributed in cytoplasm and in association with endoplasmic reticulum and Golgi apparatus. In dendrites and axons, it was associated with microtubules. Tryptophan hydroxylase in brain is only neuronal and cytoplasmic, exists in multiple forms, and is associated with microtubules, suggesting it may be transported from sites of synthesis in cell body into axons.

A population of neurons localized to a small region of the brain stem reticular formation, the rostral ventrolateral reticular nucleus (RVL), is the principal area of the brainstem regulating resting, reflex, and probably behaviorally coupled control of arterial pressure. The critical area of the RVL reticular nucleus engaged in cardiovascular control surrounds a cluster of adrenergic neurons of the C1 group, and is therefore designated the C1 area. C1 area neurons have a direct and extremely potent synaptic relationship with preganglionic sympathetic neurons of the intermediolateral (IML) nucleus of the spinal cord. Neurons in the C1 area are tonically active and fire in relationship to the cardiac cycle, a rhythm imposed by baroreceptors. They thereby provide the background of excitation to preganglionic neurons to maintain normal resting arterial pressure. Which transmitter released by C1 area neurons produces sympathetic excitation is uncertain, but it may be glutamate. C1 area neurons are critical to the integration of a wide range of cardiovascular reflexes, including the arterial pressure responses to arterial baroreceptor, chemoreceptor, and other cardiopulmonary receptor stimulation, to pain and possibly to exercise, to brain stem ischemia and distortion, and probably to the arterial pressure elevations associated with emotional behavior. The cardiovascular neurons of the C1 area are responsive to the actions of a number of neurotransmitters, many of which are confined to local circuit neurons in the region, including gamma-aminobutyric acid (GABA), catecholamines, acetylcholine, and several opiates. They also appear to be the site of action upon arterial pressure of a number of drugs--including clonidine and possibly the beta-blockers. Neurons of the C1 area of the RVL reticular nucleus, therefore, appear to function as one of the most critical output systems of the brain for regulating arterial pressure in health and sickness.

The effects of electrical stimulation of the pressor area of the fastigial nucleus (FN) of cerebellum on regional cerebral blood flow (CBF) were examined in anesthetized, paralyzed rats. CBF was measured in tissue homogenates by the Kety principle using 14C-iodoantipyrine as an indicator during FN stimulation, during stimulation of the vermal cortex, or after placement of electrodes in FN (unstimulated controls). Blood gases and systemic arterial pressures were comparable in the three groups. FN stimulation elicited a widespread and significant (P less than 0.005) increase in CBF in 12 of 13 areas. Flow was maximally increased in cerebral cortex (up to 220% of control) but also rose in selected regions of telencephalon, diencephalon, mesencephalon, lower brain stem, and in white matter of the corpus callosum (172%). Flow was not increased in cerebellum. The increase in CBF persisted after transection of the spinal cord at C1 or cervical sympathectomy. In five rats, hypercarbia (PCO2 to 63 mmHg) increased CBF in all 13 regions but in a pattern differing from FN stimulation. Excitation of neurons originating in, projecting to, or passing through FN can elicit a potent and virtually global increase of CBF. The effect appears mediated by intrinsic pathways of the central nervous system.

We demonstrated the coexistence of aromatic L-amino acid decarboxylase (AADC) and arginine-vasopressin in neurons of the hypothalamic suprachiasmatic nucleus of Sprague-Dawley rats. Neurons that lacked monoamines but expressed immunoreactivity to the enzyme AADC occupied the rostral and caudal poles of the suprachiasmatic nucleus and mediodorsal and dorsolateral positions along the entire extent of the nucleus. AADC was also localized in similar neurons of the suprachiasmatic nucleus of rats from other strains including the homozygous Brattleboro rat.

The activity of tyrosine hydroxylase (TH) was measured in the superior cervical ganglion (SCG) of the mouse during herpes simplex virus (HSV) infection. TH activity remained at control levels or actually increased during acute infection at a time when viral titers of SCG homogenates were at their peak and viral antigen was detected in from one-third to one-half of ganglionic neurons. A rapid decline in TH activity followed and coincided with falling viral titers, disappearance of viral antigen and replacement of neurons by inflammatory cells. Immunization partially prevented this reduction of TH activity. In addition, when mice were immunosuppressed by cyclophosphamide, TH activity was relatively preserved early in the course of infection despite high viral titers in the ganglion and the presence of viral antigen and histopathological alterations in nearly 100% of neurons. These results suggest that a cellular 'luxury function', in this case TH activity, can be preserved and perhaps even augmented during neuronal HSV infection. Indeed, activity of this enzyme may persist until late into the acute phase of infection, perhaps up to the point of cell death induced either by immune-mediated or direct virus-induced cell lysis.

Ricin conjugated to horseradish peroxidase was retrogradely transported by peripheral neurons of the nodose ganglion after microinjection into the nucleus tractus solitarius but was not transported by central neurons projecting to the nucleus tractus solitarius. Similarly, ricin was not transported by central neurons projecting to the caudate nucleus. Differences in labeling of central and peripheral neurons with ricin may reflect differences in surface glycoconjugates on processes of these neurons.

Peripheral deafferentation of the rodent olfactory bulb results in loss of dopamine content, tyrosine hydroxylase activity and immunocytochemical staining for tyrosine hydroxylase in juxtaglomerular dopamine neurons. Reinnervation of the bulb by afferent neurons results in the return of all parameters to control levels suggesting that the dopamine neurons did not degenerate but that the expression of tyrosine hydroxylase enzyme was transneuronally regulated in a static population of juxtaglomerular cells. To evaluate this possibility, we determined the activity and immunocytochemical localization of the second enzyme in the dopamine biosynthetic pathway, DOPA decarboxylase. At a time when tyrosine hydroxylase activity was reduced to 25% of control values, DOPA decarboxylase activity in the lesioned bulb was maintained at about 65% of that in the unlesioned bulb. Immunocytochemical staining with antibodies to both enzymes, performed sequentially in the same sections, demonstrated that in the unlesioned bulb tyrosine hydroxylase and DOPA decarboxylase are co-localized in the same population of juxtaglomerular neurons. Similar results were obtained in adjacent sections each stained with one of the two antibodies. In contrast, in the deafferented bulb, about three times as many neurons were stained with DOPA decarboxylase as with tyrosine hydroxylase antibodies. The DOPA decarboxylase activity measurements and immunocytochemistry argue for the continued presence, in the lesioned olfactory bulb, of a population of tyrosine hydroxylase deficient dopamine neurons. The data suggest that olfactory receptor cell innervation transneuronally regulates the expression of tyrosine hydroxylase by mechanisms separate from those controlling the levels of DOPA decarboxylase.