Donald J. Reis

Clonidine binds to imidazole binding sites as well as α2-adrenoceptors in the ventrolateral medulla

Binding sites labeled by [3H]p-aminoclonidine ([3H]PAC) were characterized in bovine brain membranes prepared from the ventrolateral medulla, the probable site of the antihypertensive action of clonidine and analogs. Comparison was made with [3H]PAC binding to membranes prepared from frontal cortex, which has been studied extensively. Saturation binding isotherms for [3H]PAC were similar in the two regions, although Bmax values were approximately two-fold lower in ventrolateral medulla relative to frontal cortex. Norepinephrine and other phenylethylamines displaced [3H]PAC from a maximum of 70% of the total sites in the ventrolateral medulla. The remaining 30% were norepinephrine-insensitive, non-adrenoceptor sites which displayed high affinity for imidazole compounds. Ligand selectivity differed markedly between ventrolateral medulla and frontal cortex, since some imidazole compounds which potently inhibited [3H]PAC binding in the ventrolateral medulla had no effect in frontal cortex. Imidazole binding sites may mediate, in part, the hypotensive action of clonidine and other imidazole compounds in the ventrolateral medulla. These sites may also participate in the functions of a putative endogenous clonidine-like substance.

Is agmatine a novel neurotransmitter in brain

Recent evidence suggests that agmatine, which is an intermediate in polyamine biosynthesis, might be an important neurotransmitter in mammals. Agmatine is synthesized in the brain, stored in synaptic vesicles in regionally selective neurons, accumulated by uptake, released by depolarization, and inactivated by agmatinase. Agmatine binds to alpha2-adrenoceptors and imidazoline binding sites, and blocks NMDA receptor channels and other ligand-gated cationic channels. Furthermore, agmatine inhibits nitric oxide synthase, and induces the release of some peptide hormones. As a result of its ability to inhibit both hyperalgesia and tolerance to, and withdrawal from, morphine, and its neuroprotective activity, agmatine has potential as a treatment of chronic pain, addictive states and brain injury.

Ultrastructural immunocytochemical localization of tyrosine hydroxylase in the neostriatum

The morphology and synaptic associations of dopaminergic axons in the n. caudate-putamen (neostriatum) of the adult rat brain are examined. Identification of dopaminergic axons is based upon the electron microscopic immunocytochemical localization of the catecholamine synthesizing enzyme, tyrosine hydroxylase. Immunoreactivity for the enzyme is detected in unmyelinated axons and axon terminals in serial sections collected throughout the neostriatum. The labeled terminals range from 0.1 to 1.5 micron in diameter and have peroxidase reaction product located around closely packed, round vesicles with a diameter of 40-60 nm. The tyrosine hydroxylase containing axon terminals constitute approximately 21% of the total number of terminals in the n. caudatus-putamen and include 3 types which differ in size and synaptic specializations. The most prevalent (82% of total), type I, is small (0.15-0.39 micron in diameter) and forms symmetric junctions with dendrites and dendritic spines. The other two terminal types (II and III) have a medium to large diameter (0.4-1.5 micron) and show either no membrane specializations or asymmetric junctions with dendrites. The axon terminals without observable membrane densities are occasionally oriented so as to suggest an association with dopaminergic and non-dopaminergic axon terminals. These findings indicate that while the dopaminergic terminals may form axoaxonic connections, the primary synaptic contacts are with dendrites of intrinsic neurons in all regions of n. caudatus-putamen.

Acute Fulminating Neurogenic Hypertension Produced by Brainstem Lesions in the Rat

Bilateral electrolytic lesions of the nucleus tractus solitarii in the rat at the level of the obex abolished baroreceptor reflexes and resulted in an immediate, marked elevation in systemic blood pressure without a change in heart rate. In unanesthetized rats the hypertension was associated with a marked increase in total peripheral resistance, a reduction in blood flow in the abdominal aorta, and an increase in central venous pressure. The cardiac output was reduced to 62% of control as a consequence of reduced stroke volume, which was reflected, in turn, by increased end-diastolic pressure. The hypertension was abolished and the end-diastolic pressure lowered by blockade of alpha receptors with phentolamine. The hypertension was not due to changes in blood gases or to release of agents from the kidneys or the adrenal glands; it was very sensitive to anesthetics and was abolished or aborted by midcollicular decerebration. Within hours after lesioning, the rats developed progressive congestive heart failure and died in shock, often in association with pulmonary edema. We concluded that the fulminating hypertension evoked by lesions of the nucleus tractus solitarii was due to the increased vasoconstriction caused by the augmented discharge of sympathetic nerves in response to central deafferentation of baroreceptor reflexes; the hypertension was mediated by alpha receptors and depended on the integrity of structures lying above the midbrain.

Electron microscopic localization of substance P and enkephalin in axon terminals related to dendrites of catecholaminergic neurons

Morphological and pharmacological data suggest that catecholaminergic neurons receive afferent axons positively labeled for the peptides, substance P and [Met5]-enkephalin. In the present study, electron microscopic immunocytochemistry was used to determine whether a positive reaction for these peptides could be localized to axon terminals forming synapses with catecholaminergic neurons in the locus coeruleus and A2 regions of rat brain. Adjacent sections through these areas were incubated with antiserum to either substance P, [Met5]-enkephalin, or tyrosine hydroxylase, a specific marker for catecholaminergic neurons. The sections were subsequently processes by the peroxidase-antiperoxidase immunocytochemical technique. In both the locus coeruleus and A2 region, tyrosine hydroxylase was localized primarily to perikarya and dendrites of intrinsic neurons; whereas substance P and enkephalin-like immunoreactivity was localized to axons and axon terminals. The axon terminals showing positive reactions for substance P and [Met5]-enkephalin were morphologically similar to each other and to one type of axon terminal which formed synapses with dendrites labeled for tyrosine hydroxylase. This type of axon terminal always formed asymmetric synaptic junctions and contained 3-4 large (75-100 nm) dense vesicles (LDVs) and many small (40-60 nm) clear vesicles (SCVs). The reaction product for substance P and [Met5]-enkephalin was distributed throughout the lumen of the LDVs and formed a rim of labeling around the outer boundaries of the SCVs. These findings demonstrate that substance P and [Met5]-enkephalin-positive reactions are selectively localized to subcellular organelles in axon terminals in the locus coeruleus and A2 region of rat brain. They further suggest that the labeled axon terminals form synapses with dendrites of the catecholaminergic neurons.

Heat Shock Protein 70 Suppresses Astroglial-inducible Nitric-oxide Synthase Expression by Decreasing NFκB Activation

In brain glial cells, expression of calcium independent nitric-oxide synthase (NOS-2) is induced following stimulation with bacterial endotoxin (lipopolysaccharide (LPS)) and/or pro-inflammatory cytokines. We have investigated the effects of heat shock (HS), which can reduce inflammatory responses in several cell types, on the induction of glial NOS-2 expression. Preincubation of cells for 20-60 min at 43°C decreased subsequent levels of NOS-2 induction, with a maximal 80% reduction after 60 min of HS. Following HS, cells were refractory to NOS inducers for up to 4 h, after which time little or no suppression was observed. HS reduced cytosolic NOS-2 enzymatic activity (3-fold), steady state mRNA levels (2-3-fold), and gene promoter activity (by 50%). HS also reduced LPS-induced nuclear accumulation of transcription factor NFκB p65 subunit, suggesting perturbation of NFκB activation. A role for HS protein (HSP) 70 in NOS-2 suppression by HS is supported by the demonstration that 1) transfection with human HSP70 cDNA partially replicated HS effects; 2) antisense, but not sense, oligonucleotides directed against rat HSP70 partially blocked HS effects; and 3) rat fibroblasts stably expressing human HSP70 did not express NOS-2 in response to LPS plus cytokines. As with heat-shocked cells, HSP70-expressing cells also exhibited decreased NFκB p65 subunit nuclear accumulation. These results demonstrate that in glial cells, as well as other cell types, NOS-2 induction can be modulated by the HS response, mediated at least in part by HSP70 expression.

A serotonergic innervation of noradrenergic neurons in nucleus locus coeruleus: Demonstration by immunocytochemical localization of the transmitter specific enzymes tyrosine and tryptophan hydroxylase

Immunocytochemical localization of the neurotransmitter synthesizing enzymes, tyrosine and tryptophan hydroxylase, was used to determine whether the noradrenergic neurons in the nucleus locus coeruleus of the rat are innervated by serotonergic (5-HT) neurons. Specific antibodies were prepared to tyrosine hydroxylase, purified from the bovine adrenal medulla, and tryptophan hydroxylase, purified from rat midbrain. These were localized by both light and electron microscopy by the use of the peroxidase-antiperoxidase method. In the nucleus locus coeruleus, tyrosine hydroxylase was contained in the cytoplasm, proximal axons, and dendrites of intrinsic neurons. Tryptophan hydroxylase, on the other hand, was only contained within processes surrounding the perikarya and dendrites of the catecholaminergic neurons. The processes labeled with tryptophan hydroxylase were unmyelinated, ranged in size from 0.1 to 1.4 micron, and consisted of terminal varicosities separated by intervaricose segments. Although in close approximation to noradrenergic neurons, these processes, presumably axons, rarely formed synatic contacts with thickened membrane specializations. In processes, tryptophan hydroxylase was associated with subcellular organelles which had size and distribution of microtubules, and small and large synaptic vesicles. These observations provide a morphological basis to support the hypothesis that the activity of noradrenergic neurons may be modulated by a direct action of 5-HT neurons.

Role of the nucleus parabrachialis in cardiovascular regulation in cat

Electrical stimulation of the nucleus parabrachialis (NPB) and surrounding areas of the dorsolateral pons in anesthetized immobilized cats elicits a rise of arterial pressure (AP) and tachycardia: the parabrachial pressor response (PBPR). The most excitable sites were concentrated within the intermediate one-third of the NPB in its medial and lateral subdivisions. The magnitude of pressor responses and their stimulus sensitivity were substantially greater in NPB than in adjacent areas of the dorsal pons including nucleus locus coeruleus and brachium conjunctivum, suggesting that cardiovascular responses heretofore attributed to locus coeruleus may have been due to excitation of the NPB. The PBPR persisted after chronic cerebellectomy, acute transection of the brain stem at the lower midbrain, or acute bilateral lesions of the nucleus tractus solitarii (NTS), the latter abolishing baroreceptor reflexes. Thus the PBPR cannot be attributed to antidromic or orthodromic stimulation or from NTS. Change in blood flow and regional vascular resistances during the PBPR were measured by electromagnetic flow meters placed on the thoracic aorta, superior mesenteric, renal and femoral arteries. When elicited with stimuli 5 times threshold, the PBPR was associated with an 80% increase in AP, 14+ increase in heart rate, 25% increase in cardiac output, and a 42% increase in total peripheral resistance. There was a differentiated vasoconstriction in the order of superior mesenteric greater than renal greater than femoral arteries. The baroreflex elicited by electrical stimulation of the carotid sinus nerve was reduced during stimulation of the NPB. The tachycardia was abolished by bilateral vagotomy, combined with beta-adrenergic blockade. Such treatment attenuated but did not abolish the hypertension which was only eliminated by subsequent alpha-adrenergic blockade. Thus the hypertension caused by stimulation of NPB is a result both of an increase of total peripheral resistance and of cardiac output. The cardiovascular pattern of the PBPR differ from other responses elicited from the dorsal pons, including the defense response, and the response to cerebral ischemia. We conclude that a powerful cardiovascular response pattern is organized within intrinsic neurons of the NPB. This nucleus may play an important role in organization of cardiovascular control by brain.

The role of the solitary and paramedian reticular nuclei in mediating cardiovascular reflex responses from carotid baro- and chemoreceptors

1. With dye‐filled micro‐electrodes single neurones in the medulla of anaesthetized paralysed cats were identified which: (a) fired rhythmically in synchrony with or were modulated by the cardiac cycle, and which ceased firing with occlusion of the ipsilateral common carotid artery (carotid sinus baroreceptor neurones); (b) were excited by stimulation of carotid body chemoreceptors by close intra‐arterial injection of lobeline into the thyroid artery (carotid body chemoreceptor neurones).

Adrenaline neurons in the rostral ventrolateral medulla innervate thoracic spinal cord: A combined immunocytochemical and retrograde transport demonstration

Adrenaline-containing neurons in the C1 group of the ventrolateral rostral medullary reticular formation which project to the thoracic spinal cord were identified by a combined retrograde transport immunocytochemical technique. No other medullary catecholamine neurons, including the A1 and A2 noradrenaline cells, project to thoracic spinal cord. These data, taken with results of other studies of spinal catecholamine innervation, suggest a segmental segregation of projections to spinal cord by dopaminergic, noradrenergic, and adrenergic neurons.