Noradrenergic Α1 Research Articles

Projections of neurons in the periaqueductal gray to pontine and medullary catecholamine cell groups involved in the modulation of nociception

Stimulation of neurons in the periaqueductal gray (PAG) produces antinociception that is mediated in part by noradrenergic neurons that innervate the spinal cord dorsal horn. Because norepinephrine-containing neurons are not found in the PAG, noncatecholamine neurons in the PAG must project to, and activate, spinally projecting catecholamine neurons located in the pons or medulla. The present studies determined the projections of neurons in the ventrolateral PAG to the A5, A6 (locus coeruleus), and A7 catecholamine cell groups that are known to contain spinally projecting noradrenergic neurons. The anterograde tracer biotinylated dextran amine (BDA) was injected into the ventrolateral PAG, and labeled axon terminal profiles were identified near noradrenergic neurons that were visualized by processing tissue sections for tyrosine hydroxylase immunoreactivity. Highly varicose, anterogradely labeled terminal profiles were found apposed to the dendrites and somata of tyrosine-hydroxylase-immunoreactive neurons and non-tyrosine-hydroxylase-immunoreactive neurons in the dorsolateral and ventrolateral pontine tegmentum. These axon terminal profiles were more dense on the side ipsilateral to the BDA deposit, and both A7 and locus coeruleus neurons received a more dense innervation than did the A5 neurons. Although definitive evidence for a direct pathway from PAG neurons to spinally projecting A7 neurons requires ultrastructural studies, the results of the present studies provide presumptive evidence for direct projections from neurons in the PAG to noradrenergic A7 neurons that innervate the spinal cord dorsal horn and modulate pain perception. If neurons in the ventrolateral PAG do form synapses with noradrenergic A7 neurons, these spinally projecting catecholamine neurons may mediate part of the analgesic effect produced by systemic administration of morphine. In contrast, the projections of PAG neurons to the A5 cell group and the locus coeruleus may mediate the cardiovascular and motor effects produced by stimulation of sites in the ventrolateral PAG.

Anatomical basis for cannabinoid-induced antinociception as revealed by intracerebral microinjections

Cannabinoids suppress behavioral and neurophysiological responses to noxious stimuli in rodents when administered systemically. The purpose of this study was to extend previous studies of the site of cannabinoid analgesia. Rats were tested in the tail flick test before and after microinjections of the cannabinoid agonist WIN55,212-2 (5 μg) into one of 17 different brain regions. WIN55,212-2 significantly elevated tail-flick latencies when injected into the amygdala, the lateral posterior and submedius regions of the thalamus, the superior colliculus and the noradrenergic A5 region. By contrast, pain behavior was unaffected by microinjections of the cannabinoid into the other 11 areas examined (prefrontal cortex, nucleus accumbens, lateral hypothalamus, substantia nigra, cuneiform nucleus, anterior pretectal, intralaminar, parafasicular, posterior, thalamic nuclei, as well as the ventral medial, ventral lateral nuclei in the posterior thalamus).

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.

Bistability in spinal motoneurons in vivo: systematic variations in persistent inward currents.

Bistable behavior in spinal motoneurons consists of self-sustained firing evoked by a brief period of input. However, not all motoneurons possess an equal capacity for bistable behavior. In the companion paper, we found that self-sustained firing was persistent for long periods only in motoneurons with low rheobases and slow axonal conduction velocities. High rheobase, fast conduction velocity motoneurons tend to be only partially bistable in that self-sustained firing lasts at most 1-2 s. The mechanisms underlying these differences between fully and partially bistable motoneurons were investigated by measuring their current voltage (I-V) relationships in the decerebrate cat preparation after administration of the noradrenergic alpha1 agonist methoxamine. Slow (8 mV/s) triangular voltage commands were applied using the discontinuous single-electrode voltage-clamp technique. Both fully and partially bistable cells exhibited a region of negative I-V slope due to activation of a strong, persistent inward current. The peak amplitude of the total persistent inward current (IPIC) was equally large in fully and partially bistable cells, but there were substantial differences in how IPIC was activated and deactivated. In fully bistable cells, the offset of IPIC on the descending phase of the triangular voltage command occurred at a substantially more hyperpolarized voltage then its onset on the rising phase. Thus the I-V function of fully bistable cells exhibited marked hysteresis. Partially bistable cells had significantly less hysteresis. The lack of hysteresis in partially bistable cells was due to a greater decay of IPIC with time than that seen in fully bistable cells. Furthermore, the range over which activation and deactivation of IPIC occurred was more depolarized in partially than in fully bistable cells. The I-V functions were compared with frequency-current (F-I) functions from the same cells, the characteristics of which were presented in the companion paper. The strong onset-offset difference in IPIC in fully bistable cells corresponded to a similarly large hysteresis for the thresholds of their F-I functions. The reduced onset-offset difference for IPIC in partially bistable cells corresponded to a lack of hysteresis in F-I thresholds. Thus the properties of IPIC accounted for the main differences in the F-I behavior seen between fully and partially bistable cells.

Bistability in spinal motoneurons in vivo: systematic variations in rhythmic firing patterns.

In the presence of the monoamines serotonin and norepinephrine, spinal motoneurons can exhibit bistable behavior, in which a brief period of excitatory input evokes prolonged self-sustained firing. A brief inhibitory input returns the cell to the quiescent state. To determine whether motoneurons differ in their capacity for bistable behavior, intracellular recordings were obtained in the decerebrate cat preparation. To enhance the likelihood of encountering bistable behavior, the noradrenergic alpha1 agonist methoxamine was applied to the ventral surface of the cord. The capacity of the cells to produce bistable behavior was assessed from the duration of self-sustained firing evoked by a brief (1.5 s) excitatory synaptic input from muscle spindle Ia afferents. About 35% (17 of 49) of the cells produced steady self-sustained firing for >3 s and were considered fully bistable. The other 32 cells ( approximately 65%) were partially bistable, with self-sustained firing lasting <3 s. Fully bistable cells tended to have lower current thresholds for spike initiation and slower axonal conduction velocities than did partially bistable cells. This suggests that fully bistable motoneurons innervate fatigue resistant muscle fibers. The frequency-current (F-I) relations of the motoneurons were characterized with slow triangular current ramps. Fully bistable cells displayed an acceleration in firing rate immediately on initiation of rhythmic firing. The F-I gain after completion of the acceleration was positive. Fully bistable cells also displayed a hysteresis in the current level for firing threshold with the ascending threshold occurring at substantially higher current level than the descending one. Additionally, these current thresholds usually were centered about zero current, so that the ascending current threshold was positive while the descending current threshold was negative. This negative offset meant that fully bistable cells could exhibit tonic firing without depolarizing injected current. Partially bistable cells exhibited very different F-I characteristics. Firing rate acceleration was just as large as in fully bistable cells but did not occur until well above the current level needed to initiate rhythmic firing. F-I gain after acceleration was negative, there was little to no hysteresis between the ascending and descending firing thresholds, and both thresholds were above the zero current level. These properties of partially bistable cells suggest their functional role is in tasks requiring relatively brief, high forces. The low thresholds of fully bistable cells mean they will be readily recruited in low force tasks like posture, where their prolonged self-sustained firing would be advantageous.

Neurotropin induces antinociceptive effect by enhancing descending pain inhibitory systems involving 5-HT 3 and noradrenergic α2 receptors in spinal dorsal horn

Neurotropin, a non-protein extract from the inflamed skin of rabbits inoculated with vaccinia virus, has been clinically used as an analgesic drug in Japan. Its analgesic effect has been demonstrated by reduced mechano-nociception in hyperalgesic rats exposed to SART-stress (a repeated cold stress) for 5 days. In order to clarify the mechanism of the analgesic effect of neurotropin at the spinal cord level, we examined the effects of several neurotransmitter receptor antagonists given by intrathecal (i.t.) injection on the antinociceptive effect of intraperitoneally (i.p.) injected neurotropin [100 and 200 Neurotropin Unit (NU)/kg]. The analgesic effect of neurotropin was significantly inhibited not only by methysergide (100 nmol Rat , i.t.), a non-selective antagonist against serotonin (5-HT), but also MDL 72222 (30 nmol Rat , i.t.), a selective 5-HT 3 antagonist, but not influenced by ketanserin (100 nmol Rat , i.t.), a 5-HT 2A antagonist. The antinociceptive effect of neurotropin (200 NU Kg , i.p.) was significantly inhibited also by yohimbine (30 nmol Rat , i.t.), a noradrenergic α 2 antagonist. However, the analgesic effect of neurotropin (100 and 200 NU Kg , i.p.) was not influenced by naloxone (30 nmol Rat , i.t.), an opioid antagonist. These results suggest that the mechanism of the antinociceptive effect of neurotropin is via enhancement of endogenous descending pain inhibitory pathways of the serotonergic and noradrenergic systems, especially involving 5-HT 3 and noradrenergic α 2 receptors in spinal dorsal horn in which these neurons terminate. No influence of opioid receptors at the spinal cord level is indicated.

Enkephalin neurons that project to the A7 catecholamine cell group are located in nuclei that modulate nociception: ventromedial medulla

The location of methionine enkephalin neurons in the medulla oblongata that project to the dorsolateral pontine tegmentum was investigated using anterograde and retrograde tract tracing combined with immunocytochemical neurotransmitter identification. The results of these experiments demonstrate that enkephalinergic neurons from areas known to modulate nociception project to the region of the A7 catecholamine cell group in the dorsolateral pontine tegmentum. The medullary nuclei that contain these enkephalinergic neurons include the nucleus raphe magnus and the nucleus reticularis gigantocellularis pars α in the ventromedial medulla. While some of these enkephalinergic axons appose the somata and dendrites of A7 neurons, the majority of these axons appear to contact non-catecholamine neurons in the dorsolateral pontine tegmentum. Unidentified neurons located in the nucleus raphe magnus, the nucleus reticularis gigantocellularis pars α, and the nucleus reticularis gigantocellularis also project to the A7 area. Many of the neurons in the nucleus reticularis gigantocellularis pars α appear to contact both noradrenergic A7 neurons and non-catecholamine neurons in the dorsolateral pontine tegmentum, whereas most of those in the nucleus raphe magnus appear to contact non-catecholamine neurons. The anatomical findings described in this report and the results of preliminary behavioral studies provide evidence to support a model in which activation of the enkephalin-containing neurons in the ventromedial medulla facilitates nociception, while the non-enkephalin neurons mediate part of the antinociception produced by stimulating sites in the ventromedial medulla.

Regionally different noradrenergic innervation and receptor distribution in submucous plexus of guinea-pig intestine.

Regional differences of noradrenergic innervation and noradrenergic α2 receptor distribution in the submucous plexus of guinea-pig caecum, large intestine, and rectum were investigated with immunofluorescence confocal microscopy. In the caecal submucous plexus, three dimensional basket-like arrangements of small and bright dots of tyrosine hydroxylase-like immunoreactivity (TH-LIR) were observed, whereas noradrenergic α2A receptor-like immunoreactivity (NAR-LIR) was detected in somata. Both TH-LIR and NAR-LIR were localized in neuronal somata of the transverse colon. A few TH-LIR positive cell somata and many TH-LIR positive spots forming chains passing through ganglia and interganglionic nerve strands were observed, but no NAR-LIR were seen in the submucous plexus of the descending colon. There were a few TH-LIR positive somata and puncta, but no NAR-LIR were observed in the ascending colon and rectum. All of these observations show the distinct regional differences in noradrenergic innervation and noradrenergic α2A receptor distribution in the guinea-pig submucous plexus.

Expression ofc-fos in the rat brainstem after exposure to hypoxia and to normoxic and hyperoxic hypercapnia

In this study, Fos immunohistochemistry was used to map brainstem neuronal pathways activated during hypercapnia and hypoxia. Conscious rats were exposed to six different gas mixtures: (a) air; (b) 8% CO2 in air; (c) 10% CO2 in air; (d) 15% CO2 in air; (e) 15% CO2 + 60% O2, balance N2; (f) 9% O2, balance N2. Double-staining was performed to show the presence of tyrosine hydroxylase. Hypercapnia, in a dose-dependent way caused Fos expression in the following areas: caudal nucleus tractus solitarius (NTS), with few labeled A2 noradrenergic neurons; noradrenergic A1 cells and noncatecholaminergic neurons in the caudal ventrolateral medulla; raphe magnus and gigantocellular nucleus pars alpha (GiA); many noncatecholaminergic (and relatively few C1) neurons in the lateral paragigantocellular nucleus (PGCl), and in the retrotrapezoid nucleus (RTN); locus coeruleus (LC), external lateral parabrachial and Kölliker-Fuse nuclei, and A5 noradrenergic neurons at pontine level; and in caudal mesencephalon, the ventrolateral column of the periaqueductal gray (vlPAG). In most of these nuclei, hypoxia also induced Fos expression, albeit generally less than after hypercapnia. However, hypoxia did not cause labeling in RTN, juxtafacial PGCl, GiA, LC, or vlPAG. After normoxic hypercapnia, more labeled cells were present in NTS and PGCl than after hyperoxic hypercapnia. Part of the observed labeling could be caused by stress- or cardiovascular-related sequelae of hypoxia and hypercapnia. Possible implications for the neural control of breathing are also discussed, particularly with regard to the finding that several nuclei, not belonging to the classical brainstem respiratory centres, contained labeled cells.

Monoamine transporter gene expression in the central nervous system in diabetes mellitus.

Diabetic animals exhibit altered neurotransmission in brain monoaminergic systems. By means of in situ hybridization, we have investigated the expression of dopamine, noradrenaline, and serotonin transporter (DA-T, NA-T, and 5-HT-T, respectively) mRNAs in the brains of alloxan- and streptozotocin-diabetic rats. The expression of DA-T mRNA is decreased in 1- (-11%) and 4- (-17%) week alloxan-diabetic and 4- (-9%) and 8- (-20%) week streptozotocin-diabetic rats in the ventral medial bundle. The expression of NA-T mRNA is decreased in the locus coeruleus of 8- (-26%) week streptozotocin-diabetic rats, in the noradrenergic A1 cell group of 4- (-27%) week alloxan- and 8- (-25%) week streptozotocin-diabetic rats, and in the noradrenergic A2 cell group of 1- (-21%) and 4- (-28%) week alloxan-diabetic and 4- (-27%) and 8- (-25%) week streptozotocin-diabetic animals. The expression of 5-HT-T mRNA in the dorsal raphe nucleus is increased in 1- (+14%) and 4- (+44%) week alloxan- and 4- (+28%) and 8- (+44%) week streptozotocin-diabetic rats. The expression of each of the three monoamine transporter genes may be differentially regulated in diabetes and dependent on the duration of diabetes. Altered monoamine transporter gene expression may possibly contribute to the observed dysfunctions in brain monoamine transmission in chronic diabetes.

Clonidine Antagonizes Pressor Effect of N-Methyl-D-Aspartate in the Rostral Ventrolateral Medulla of Rats

The purpose of the present study was to investigate the modulatory actions of adrenoreceptor agonists on N-methyl-D-aspartate (NMDA)-induced pressor effect in rostral ventrolateral medulla (RVLM). These drugs were locally applied into RVLM of urethane-anesthetized Sprague-Dawley rats through multibarrel pipettes. Microinjection of NMDA increased the arterial pressure, an effect which was abolished by pretreatment with clonidine, whereas neither the β-adrenergic agonist isoproterenol nor the α1-adrenergic agonist phenylephrine did alter this pressor response. Previous experiments demonstrated that clonidine binds to noradrenergic α2 and imidazoline receptors in the RVLM. Norepinephrine, which has high affinity for the α2 receptor and low affinity to the imidazoline receptor, partially antagonized NMDA-induced hypertension. On the other hand, administration of selective imidazoline receptor antagonist idazoxan partially reversed clonidine-mediated antagonism of NMDA. Taken together, these results suggest that clonidine may modulate the excitatory amino acid -induced pressor response through noradrenergic α2 and imidazoline receptors in the RVLM.

Neurones in the ventrolateral pons are required for post‐hypoxic frequency decline in rats.

1. The breathing pattern following acute hypoxia (arterial O2 pressure (Pa,O2), 27.4 +/- 7.7 mmHg) was measured in intact, anaesthetized and spontaneously breathing adult rats (n = 4) and in anaesthetized, vagotomized, paralysed and ventilated animals (n = 14). Measurements were made both before and after bilateral lesions or chemical inactivation of neurones in the lateral pons. Respiratory motor activity was recorded as an index of the respiratory cycle. We tested the hypothesis that the ventrolateral pons is required for expression of post-hypoxic frequency decline, defined as a decrease in respiratory frequency below steady-state baseline levels following brief exposures to hypoxia. 2. We identified an area in the ventrolateral pons where brief (1 ms) low current (< or = 20 microA) pulses evoked a short-latency inhibitor of phrenic nerve activity. At this site, bilateral electrical or chemical lesions (n = 3) were performed, or neural activity was inhibited by focal injections of 10 mM muscimol (n = 9). In six control animals, neural activity was inhibited by muscimol injections into the lateral pons, dorsal to the target site. 3. Prior to pontine intervention, respiratory frequency decreased below baseline levels following 20-110 s of 8% O2. The decrease in frequency resulted from a prolongation of expiration (up to 276%), which gradually returned to baseline levels (tau = 45 s). 4. Following lesions or inhibition of neural activity in the ventrolateral pons, baseline inspiratory (TI) and expiratory (TE) durations were altered, albeit minimally, in the animals with intact vagus nerves. Expiratory duration following hypoxia was not different from baseline levels either in vagotomized (P = 0.18) or intact (P > 0.05) animals. In contrast, injections of muscimol at more dorsal sites did not alter the decrease in frequency normally seen following hypoxia. 5. Histological examination revealed that effective lesion or injection sites were within the lateral pontine tegmental field and included portions of the noradrenergic A5 cell group. 6. We conclude that the mechanism responsible for post-hypoxic frequency decline involves an active neural process that depends on the integrity of the ventrolateral pons.

Localization of neuropeptide Y Y1 receptor-like immunoreactivity in catecholaminergic neurons of the rat medulla oblongata.

Neuropeptide Y receptors in the medulla oblongata participate in central cardiovascular control. The neuropeptide Y1 receptor subtype gene and amino acid sequence have been identified by molecular cloning studies. In this study, a C-terminal peptide representing amino acids 355-382 of the neuropeptide Y1 receptor was synthesized and cross-linked to thyroglobulin to produce an antibody against a partial sequence of the neuropeptide Y1 receptor, used to localize neuropeptide Y1 receptor-like immunoreactivity in the catecholaminergic neurons of the medulla oblongata. The double colour immunofluorescence technique with a polyclonal antibody against the neuropeptide Y1 receptor and a monoclonal antibody against tyrosine hydroxylase revealed that in the rat medulla oblongata, a weak (the C3 cell group) to moderately intense (the A1, A2, C1 and C2 cell groups), diffuse cytoplasmic neuropeptide Y1 receptor-like immunoreactivity was distributed primarily in the noradrenergic and adrenergic cell bodies and occasionally seen in the noradrenergic and adrenergic cell processes. Almost all tyrosine hydroxylase-like immunoreactive cell bodies in the A1, A2, C1, C2 and C3 cell groups showed neuropeptide Y1 receptor-like immunoreactivity. The neuropeptide Y1 receptor-like immunoreactivity in the A2 cell group was somewhat stronger. The present findings show localization of specific neuropeptide Y1 receptor-like immunoreactivity in the vast majority of the noradrenergic and adrenergic cell bodies of the A1, A2, C1, C2 and C3 cell groups, which are putative cardiovascular regions. The results support the view that neuropeptide Y1 receptors in the medulla oblongata are involved in central cardiovascular control and may coexist with another important receptor, the alpha 2A-adrenoceptor, also involved in central, cardiovascular regulation, since the alpha 2A-adrenoceptor-like immunoreactivity has been shown to exist in almost all noradrenergic and adrenergic cell bodies in the brainstem. In conclusion, centrally administered neuropeptide Y may act in part via neuropeptide Y1 receptors located on the soma and dendrites of noradrenergic and adrenergic neurons, where it may interact with alpha 2-adrenoceptors at least in the noradrenergic A2 neurons. This noradrenaline system may be involved in at least part of the vasodepressor actions of neuropeptide Y, noradrenaline and adrenaline in the nucleus tractus solitarii in view of the present findings.

Afferent projections to the rat locus coeruleus demonstrated by retrograde and anterograde tracing with cholera-toxin B subunit and Phaseolus vulgaris leucoagglutinin

The aim of this study was to examine the afferents to the rat locus coeruleus by means of retrograde and anterograde tracing experiments using cholera-toxin B subunit and phaseolus leucoagglutinin. To obtain reliable injections of cholera-toxin B in the locus coeruleus, electrophysiological recordings were made through glass micropipettes containing the tracer and the noradrenergic neurons of the locus coeruleus were identified by their characteristic discharge properties. After iontophoretic injections of cholera-toxin B into the nuclear core of the locus coeruleus, we observed a substantial number of retrogradely labeled cells in the lateral paragigantocellular nucleus and the dorsomedial rostral medulla (ventromedial prepositus hypoglossi and dorsal paragigantocellular nuclei) as previously described. We also saw a substantial number of retrogradely labeled neurons in (1) the preoptic area dorsal to the supraoptic nucleus, (2) areas of the posterior hypothalamus, (3) the Kölliker-Fuse nucleus, (4) mesencephalic reticular formation. Fewer labeled cells were also observed in other regions including the hypothalamic paraventricular nucleus, dorsal raphe nucleus, median raphe nucleus, dorsal part of the periaqueductal gray, the area of the noradrenergic A5 group, the lateral parabrachial nucleus and the caudoventrolateral reticular nucleus. No or only occasional cells were found in the cortex, the central nucleus of the amygdala, the lateral part of the bed nucleus of the stria terminalis, the vestibular nuclei, the nucleus of the solitary tract or the spinal cord, structures which were previously reported as inputs to the locus coeruleus. Control injections of cholera-toxin B were made in areas surrounding the locus coeruleus, including (1) Barrington's nucleus, (2) the mesencephalic trigeminal nucleus, (3) a previously undefined area immediately rostral to the locus coeruleus and medial to the mesencephalic trigeminal nucleus that we named the peri-mesencephalic trigeminal nucleus, and (4) the medial vestibular nucleus lateral to the caudal tip of the locus coeruleus. These injections yielded patterns of retrograde labeling that differed from one another and also from that obtained with cholera-toxin B injection sites in the locus coeruleus. These results indicate that the area surrounding the locus coeruleus is divided into individual nuclei with distinct afferents. These results were confirmed and extended with anterograde transport of cholera-toxin B or phaseolus leucoagglutinin. Injections of these tracers in the lateral paragigantocellular nucleus, preoptic area dorsal to the supraoptic nucleus, the ventrolateral part of the periaqueductal gray, the Kölliker-Fuse nucleus yielded a substantial to large number of labeled fibers in the nuclear core of the locus coeruleus.(ABSTRACT TRUNCATED AT 400 WORDS)

Changes in corticotropin releasing factor neurotransmission induced by sleep deprivation in rat brain

The aim of the present study was to identify cytochrome P450 (CYP) isoenzymes involved in the 5-sulfoxidation, mono-N-demethylation and di-N-demethylation of the aliphatic-type phenothiazine neuroleptic chlorpromazine in human liver. Experiments were performed in vitro using cDNA-expressed human CYP isoforms (Supersomes 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4), liver microsomes from different donors and CYP-selective inhibitors. The obtained results indicate that CYP1A2 is the only CYP isoform that catalyzes the mono-N-demethylation and di-N-demethylation of chlorpromazine (100%) and is the main isoform responsible for chlorpromazine 5-sulfoxidation (64%) at a therapeutic concentration of the drug (10 μM). CYP3A4 contributes to a lesser degree to chlorpromazine 5-sulfoxidation (34%). The role of CYP2B6, CYP2C19 and CYP2D6 in catalyzing of the latter reaction is negligible (0.1–2%). Similar results were obtained at a higher, non-therapeutic concentration of the drug (100 μM); however, the contribution of CYP1A2 to chlorpromazine mono-N-demethylation was noticeably lower (75%), mostly in favour of CYP2C19 and CYP3A4 (about 12% each). The obtained results indicate that the catalysis of chlorpromazine N-demethylation and 5-sulfoxidation in humans exhibits a stricter CYP1A2 preference compared to the previously tested phenothiazines (promazine, perazine, and thioridazine). Hence pharmacokinetic interactions involving chlorpromazine and CYP1A2 substrates and inhibitors are likely to occur. Considering strong dopaminergic D2, noradrenergic α1 and cholinergic M1 receptor blocking properties of chlorpromazine and some of its metabolites, as well as their serious side effects, the obtained results may be of pharmacological and clinical importance.

Evidence that activation of the hypothalamo-pituitary-adrenal axis by electrical stimulation of the noradrenergic A1 group is not mediated by noradrenaline.

The paraventricular nucleus (PVN) of the hypothalamus, where the CRF-containing neurosecretory cells controlling the hypothalamo-pituitary-adrenal (HPA) axis are located, receives a dense noradrenergic innervation from the A1 group of the caudal ventrolateral medulla. In the present study we studied the relationship between release of noradrenaline (NA) in the PVN and activation of the HPA axis in response to electrical stimulation of the A1 region. In the urethane-anesthetized male rat, extracellular NA in the PVN was monitored on line by electrochemical recording while the activity of the HPA axis was estimated by measurement of ACTH in blood samples. A 1 min, 10 Hz stimulation evoked a significant increase of extracellular NA in the PVN as well as an ACTH surge in blood. The NA and ACTH response evoked by stimulation in the 3- to 14-Hz range were found to be frequency dependent. However, whilst the NA response increased in an exponential manner with respect to frequency, the ACTH response appeared to plateau between 10 and 14 Hz. Specific lesions of the noradrenergic terminals in the PVN, by bilateral local administration of 6-hydroxydopamine, markedly reduced the ACTH response to stimulation. Intracerebroventricular injection of desmethylimipramine, a NA uptake inhibitor, enhanced the increase in extracellular NA evoked by submaximal stimulation about 2.5-fold but did not modify the corresponding ACTH response. Combined intracerebroventricular injection of alpha- and beta-adrenergic antagonists, phentolamine and propanolol respectively, did not prevent the ACTH response evoked by stimulation. Following stimulation of the caudal ventrolateral medulla, the ACTH response thus appears to result from the stimulation of the A1 noradrenergic group projecting to the PVN. However, the inability of pharmacological manipulations which enhance or block central noradrenergic transmission to influence the ACTH response suggests that the noradrenergic endings in the PVN originating from the A1 group use a transmitter other than NA to activate the HPA axis at the PVN level.

Placebo‐controlled study of ABT‐200 versus fluoxetine in the treatment of major depressive disorder

AbstractABT‐200 is a novel potential antidepressant which antagonizes both norepi‐nephrine uptake and noradrenergic a2 receptors. This single‐site study conducted over 5 months compared the safely and efficacy of ABT‐200 (titrated up to 280 mg/day) to a fixed dose offluoxetine (20 mg/day) and placebo in 216 patients meeting DSM‐III‐R criteria for major depressive disorder. Following a 1‐week placebo lead‐in phase, patients were randomized to receive ABT‐200 (n = 72), fluoxetine (n = 72), or placebo (n = 72) for 9 weeks of double‐blind treatment. At the end of the study there were no differences (P &gt; 0.05) in HAM‐D total score changes from baseline between ABT‐200‐treated and placebo‐treated patients, whose mean scores were reduced by 4.6 points (16.8%) and 6.4 points (23.3%), respectively. Patients on fluoxetine, however, showed statistically significant improvement over placebo on total HAM‐D scores with a decrease of 8.6 points (30.5%,P &lt; 0.05). While there were no serious adverse events thought to be related to the study drug, 36.1% of ABT‐200 patients discontinued the study due to adverse events, compared to only 5.6% of placebo patients and 4.2% offluoxetine patients. If ABT‐200 produced the spectrum of biochemical effects in humans predicted from preclinical studies, our results suggest that these actions do not lead to antidepressant effects in general depressed outpatient populations. Depression 3:199–203 (1995). © 1995 Wiley‐Liss, Inc.

Afferents to the midline thalamus issue collaterals to the nucleus tractus solitarii: an anatomical basis for thalamic and visceral reflex integration

The goal of this study was to establish a structural basis for thalamic and visceral integration. We sought to define neural networks that convey visceral or integrated environmental stimuli to the diffuse thalamocortical relay system and that link periodic changes in forebrain and visceral reflex function. Our experiments were designed to determine whether afferents to the midline-intralaminar thalamic nuclei (MIT) issue collaterals to the general viscerosensory division of the nucleus tractus solitarii (NTS). Experiments were performed on anesthetized male Sprague-Dawley rats. Two tracers, FluoroGold and rhodamine latex microbeads, were stereotaxically centered on the MIT and NTS, respectively, in each animal. Subsets of midline thalamic afferents were identified that issue collaterals to the solitary complex. In the cerebral cortex, dually labeled soma were detected in layer V of the insular and infralimbic areas. In the subcortical forebrain, the lateral septal nucleus, anterolateral area of the bed nuclei of stria terminalis, medial preoptic nucleus, medial and central amygdaloid nuclei, caudal lateral hypothalamic area, supramammillary nucleus, and parvicellular division of the paraventricular hypothalamic nucleus constitute other newly identified sources of collateral projection. In the midbrain and pons, collateral projection cells were observed in the periaqueductal gray, dorsal raphe nucleus, mesencephalic reticular formation, laterodorsal tegmental nucleus, lateral and medial parabrachial nuclei, and noradrenergic A5 area. In the lateral parabrachial nucleus, dually labeled neurons were detected in the dorsal-lateral division. In the medulla, collaterals are derived from cells in the rostral and caudal ventrolateral reticular formation and parapyramidal area. Dually labeled cells were also found in the cerebellar fastigial nucleus. Collaterals may coordinate changes in visceral reflex excitability and thalamocortical rhythms during phases of sleep-wake cycle and behavioral expression.

Alpha 2A-adrenergic receptors are present in lower brainstem catecholaminergic and serotonergic neurons innervating spinal cord

A subtype-specific polyclonal antibody was used for the immunohistochemical detection of alpha 2A-adrenergic receptors (α 2A-ARs) in the rat lower brainstem (medulla and pons). Using dual-label fluorescence histochemistry, punctate α 2A-AR-like immunoreactivity (α 2A-AR-LIR) was identified in noradrenergic, adrenergic, and serotonergic neurons of the pontomedullary region. Confocal microscopic examination of material simulataneously labeled for TH-LIR and α 2A-LIR revealed that the clusters of α 2A-LIR were located intracellularly. Lower medullary neurons with spinal protections to segment T 3 were retrogradely labeled using FITC-conjugated microbeads and the material was processed for simulatneous detection of α 2A-LIR and either TH-LIR or 5-HT-LIR. Using this triple-label approach, we found that virtually all medullary serotonergic cells (raphe pallidus, raphe obscurus and parapyramidal area) including those with identified spinal projections contain α 2A-AR-LIR. In contrast, fewer than 10% of dorsal raphe serotonergic cells examined for comparison were immunoreactive. The triple labeling approach also indicated that more than 95% of the TH-immunoreactive cells of the dorsal and ventrolateral medulla, including those with demonstrable spinal projections (A5 noradrenergic and C1/C3 adrenergic) had detectable amounts of α 2A-AR-LIR. The presence of α 2A-ARs in a large fraction of bulbospinal pre-sympathetic neurons (noradrenergic A5, adrenergic C1 and C3 and serotonergic raphe cells) could explain the powerful and relatively selective effect of clonidine and other centrally acting α 2A-AR agonists on sympathetic efferent activity and hypertension.

Efferent connections of the hypothalamic “grooming area” in the rat

The efferent connections of the hypothalamic area, where grooming can be elicited by local electrical stimulation or injection of various substances, were studied using iontophoretic injections of Phaseolus vulgaris leucoagglutinin. This hypothalamic “grooming area” consists of parts of the hypothalamic paraventricular nucleus and of the dorsal hypothalamic area. The specificity of these efferents for the hypothalamic “grooming area” was investigated by comparison with efferents of hypothalamic sites adjacent to this area. In addition, the distribution of oxytocinergic fibres was studied, since oxytocinergic neurons are present in the hypothalamic “grooming area” and oxytocin is possibly involved in grooming behaviour. The efferents of the hypothalamic “grooming area” as well as of hypothalamic sites surrounding this area and the oxytocinergic fibres studied do not form well determined bundles, but rather spread out throughout the hypothalamus. Clusters of fibres could be traced rostrally and caudally, forming diffuse fibre “streams”. Three rostral, two thalamic and three caudal fibre “streams” have been distinguished along which efferent fibres innervate different brain areas. The many varicosities on labelled fibres “en passant” suggest that hypothalamic fibres are able to influence many parts of the brain along their way. The anterior periventricular area, the median preoptic nucleus, the ventral tegmental area and nucleus of the solitary tract were found to be more or less specifically innervated by hypothalamic “grooming area” fibres and oxytocinergic fibres. Other brain areas, like the septum, the medial amygdaloid nucleus, the central gray and the paraventricular nucleus of the thalamus were found to receive efferent projections from the hypothalamic “grooming area” and hypothalamic loci outside this area, as well as from the oxytocinergie system. Within the septum and the mesencephalic central gray, differences in the spatial organization of terminating fibres from the hypothalamic “grooming area” and hypothalamic “non-grooming” sites have been found. Fibres from the grooming area clustered in the ventral part of the lateral septal nucleus, while fibres from surrounding hypothalamic loci innervated other parts of that brain area. In the central gray, fibres from the hypothalamic “grooming area” clustered in rostrodorsal and caudoventral parts. A number of brain areas, that are innervated by hypothalamic “grooming area” fibres and oxytocinergic fibres, like central gray, ventral tegmental area and the noradrenergic A5 area, have been reported previously to be involved in grooming behaviour. It is concluded from the present findings, that the hypothalamic “grooming area” has preferential connections with a number of brain sites, not shared with hypothalamic projections from outside the “grooming area”. Some of these destinations are known to be involved in grooming behaviour. The unravelling of these behaviourally relevant circuitries, together with the characterization of its connections and the description of the behavioural responses elicited by stimulation of parts of this circuitry, is important in the study on brain mechanisms that are involved in the regulation of behaviour.