Glutamatergic phenotype of glucagon-like peptide 1 neurons in the caudal nucleus of the solitary tract in rats.

The expression of a vesicular glutamate transporter (VGLUT) suffices to assign a glutamatergic phenotype to neurons and other secretory cells. For example, intestinal L cells express VGLUT2 and secrete glutamate along with glucagon-like peptide 1 (GLP1). We hypothesized that GLP1-positive neurons within the caudal (visceral) nucleus of the solitary tract (cNST) also are glutamatergic. To test this, the axonal projections of GLP1 and other neurons within the cNST were labeled in rats via iontophoretic delivery of anterograde tracer. Dual immunofluorescence and confocal microscopy was used to visualize tracer-, GLP1-, and VGLUT2-positive fibers within brainstem, hypothalamic, and limbic forebrain nuclei that receive input from the cNST. Electron microscopy was used to confirm GLP1 and VGLUT2 immunolabeling within the same axon varicosities, and fluorescent in situ hybridization was used to examine VGLUT2 mRNA expression by GLP1-positive neurons. Most anterograde tracer-labeled fibers displayed VGLUT2-positive varicosities, providing new evidence that ascending axonal projections from the cNST are primarily glutamatergic. Virtually all GLP1-positive varicosities also were VGLUT2-positive. Electron microscopy confirmed the colocalization of GLP1 and VGLUT2 immunolabeling in axon terminals that formed asymmetric (excitatory-type) synapses with unlabeled dendrites in the hypothalamus. Finally, in situ hybridization confirmed that GLP1-positive cNST neurons express VGLUT2 mRNA. Thus, hindbrain GLP1 neurons in rats are equipped to store glutamate in synaptic vesicles, and likely co-release both glutamate and GLP1 from axon varicosities and terminals in the hypothalamus and other brain regions.

C1 neurons excite locus coeruleus and A5 noradrenergic neurons along with sympathetic outflow in rats.

C1 neurons activate sympathetic tone and stimulate the hypothalamic­pituitary­adrenal axis in circumstances such as pain, hypoxia or hypotension. They also innervate pontine noradrenergic cell groups, including the locus coeruleus (LC) and A5. Activation of C1 neurons reportedly inhibits LC neurons; however, because these neurons are glutamatergic and have excitatory effects elsewhere, we re-examined the effect of C1 activation on pontine noradrenergic neurons (LC and A5) using a more selective method. Using a lentivirus that expresses channelrhodopsin2 (ChR2) under the control of the artificial promoter PRSx8, we restricted ChR2 expression to C1 neurons (67%), retrotrapezoid nucleus neurons (20%) and cholinergic neurons (13%). The LC contained ChR2-positive terminals that formed asymmetric synapses and were immunoreactive for vesicular glutamate transporter type 2. Low-frequency photostimulation of ChR2-expressing neurons activated LC (38 of 65; 58%) and A5 neurons (11 of 16; 69%) and sympathetic nerve discharge. Locus coeruleus and A5 inhibition was not seen unless preceded by excitation. Locus coeruleus activation was eliminated by intracerebroventricular kynurenic acid. Stimulation of ChR2-expressing neurons at 20 Hz produced modest increases in LC and A5 neuronal discharge. In additional rats, the retrotrapezoid nucleus region was destroyed with substance P­saporin prior to lentivirus injection into the rostral ventrolateral medulla, increasing the proportion of C1 ChR2-expressing neurons (83%). Photostimulation in these rats activated the same proportion of LC and A5 neurons as in control rats but produced no effect on sympathetic nerve discharge owing to the destruction of bulbospinal C1 neurons. In conclusion, low-frequency stimulation of C1 neurons activates pontine noradrenergic neurons and sympathetic nerve discharge, possibly via the release of glutamate from monosynaptic C1 inputs.

Identification of neurotransmitters and co-localization of transmitters in brainstem respiratory neurons.

Identifying the major ionotropic neurotransmitter in a respiratory neuron is of critical importance in determining how the neuron fits into the respiratory system, whether in producing or modifying respiratory drive and rhythm. There are now several groups of respiratory neurons whose major neurotransmitters have been identified and in some of these cases, more than one transmitter has been identified in particular neurons. This review will describe the physiologically identified neurons in major respiratory areas that have been phenotyped for major ionotropic transmitters as well as those where more than one transmitter has been identified. Although the purpose of the additional transmitter has not been elucidated for any of the respiratory neurons, some examples from other systems will be discussed.

Central nervous system distribution of the transcription factor Phox2b in the adult rat.

Phox2b is required for development of the peripheral autonomic nervous system and a subset of cranial nerves and lower brainstem nuclei. Phox2b mutations in man cause diffuse autonomic dysfunction and deficits in the automatic control of breathing. Here we study the distribution of Phox2b in the adult rat hindbrain to determine whether this protein is selectively expressed by neurons involved in respiratory and autonomic control. In the medulla oblongata, Phox2b-immunoreactive nuclei were present in the dorsal vagal complex, intermediate reticular nucleus, dorsomedial spinal trigeminal nucleus, nucleus ambiguus, catecholaminergic neurons, and retrotrapezoid nucleus (RTN). Phox2b was expressed by both central excitatory relays of the sympathetic baroreflex (nucleus of the solitary tract and C1 neurons) but not by the inhibitory relay of this reflex. Phox2b was absent from the ventral respiratory column (VRC) caudal to RTN and rare within the parabrachial nuclei. In the pons, Phox2b was confined to cholinergic efferent neurons (salivary, vestibulocochlear) and noncholinergic peritrigeminal neurons. Rostral to the pons, Phox2b was detected only in the oculomotor complex. In adult rats, Phox2b is neither a comprehensive nor a selective marker of hindbrain autonomic pathways. This marker identifies a subset of hindbrain neurons that control orofacial movements (dorsomedial spinal trigeminal nucleus, pontine peritrigeminal neurons), balance and auditory function (vestibulocochlear efferents), the eyes, and both divisions of the autonomic efferent system. Phox2b is virtually absent from the respiratory rhythm and pattern generator (VRC and dorsolateral pons) but is highly expressed by neurons involved in the chemical drive and reflex regulation of this oscillator.

Opioid circuits originating from the nucleus paragigantocellularis and their potential role in opiate withdrawal.

Neurons in the rat nucleus paragigantocellularis (PGi), located in the ventrolateral medulla, send collateral projections to the locus coeruleus (LC) and to the nucleus of the solitary tract (NTS). The present study examined whether neurons in the PGi that project to both the LC and NTS contain leucine(5)-enkephalin (ENK), and also whether opioid-containing neurons in the PGi are activated following withdrawal from opiates. Retrograde transport of Fluoro-Gold (FG) from the LC and transport of a protein-gold tracer from the NTS was combined with detection of an antibody directed against ENK in the PGi. Using fluorescence and brightfield microscopy, it was established that more than half of the neurons containing both FG and the protein-gold tracer, also exhibited immunolabeling for ENK. The most frequent location of triply labeled neurons was the retrofacial portion of the PGi. In a separate series, rats were chronically implanted with morphine or placebo pellets and, on the fifth day, were subjected to an intraperitoneal injection of naltrexone. Two hours following initiation of withdrawal, rat brains were obtained and processed for detection of c-fos and in situ hybridization labeling of preproenkephalin (PPE) mRNA. Naltrexone injections into morphine-dependent rats caused a dramatic increase in c-fos as compared to control rats. Approximately 66% of the c-fos-labeled neurons exhibited labeling for PPE mRNA. These were also enriched in the retrofacial portion of the PGi. Taken together, the present data indicate that withdrawal from opiates engages opioid neurons in the PGi, some of which may coordinate activity of neurons in both the NTS and the LC.

Regulation of sympathetic tone and arterial pressure by the rostral ventrolateral medulla after depletion of C1 cells in rats.

This review describes experiments designed to determine the role of bulbospinal (BS) C1 cells in regulating the sympathetic outflow and blood pressure. This goal was achieved by analyzing the physiological consequences of destroying BS C1 cells. These cells were destroyed by suicide transport of an anti-dopamine-beta-hydroxylase antibody conjugated to saporin (anti-D beta H-SAP). Two to 3 weeks after spinal cord injection (T2-T6), the toxin destroyed 75-85% of BS C1 and C3 cells along with > 95% of BS noradrenergic neurons (A5, A6, A7). The toxin spared BS noncatecholaminergic cells. Under anesthesia, toxin-treated rats had a normal blood pressure and an apparently normal sympathetic nerve discharge (SNA, splanchnic), and intravenous clonidine caused a normal degree of sympathoinhibition. Inhibition of rostral ventrolateral medulla (RVLM) neurons by bilateral injection of muscimol caused the same hypotension and sympathoinhibition as in control rats. The baroreflex range was 41% attenuated by the toxin, but the MAP50 was unchanged. Sympathoexcitatory responses to stimulation of peripheral chemoreceptors with cyanide or to electrical stimulation of RVLM were severely depressed (60% to 80%) in toxin-treated rats. Rats in which A5 neurons were selectively destroyed had no deficit in the parameters tested. Unit recordings of BS RVLM neurons indicated that the toxin destroyed most barosensitive C1 neurons, but spared noncatecholaminergic lightly myelinated BS cells. In summary, the integrity of C1 neurons is not essential for the generation of SNA and the maintenance of BP under resting conditions, perhaps because these functions are performed primarily by noncatecholaminergic BS neurons. However, the deficits caused by treatment with anti-D beta H-SAP indicate that BS C1 neurons play a crucial role in several sympathoexcitatory responses mediated by the RVLM.

Preproenkephalin mRNA is expressed by C1 and non-C1 barosensitive bulbospinal neurons in the rostral ventrolateral medulla of the rat.

The autonomic regions of the thoracolumbar spinal cord receive a dense enkephalinergic (ENK) innervation from supraspinal sources, including the rostral ventrolateral medulla (RVLM). In the present study, we sought to determine whether the barosensitive bulbospinal (BSBS) neurons of the RVLM express preproenkephalin (PPE) mRNA. After injection of Fluoro-Gold (FG) into the upper thoracic spinal cord, neurons with PPE mRNA (PPE(+) neurons) were retrogradely labeled throughout the ventrolateral medulla. At the most rostral RVLM level, 29% of bulbospinal PPE+ cells were tyrosine hydroxylase-immunoreactive (TH-ir) and the latter constituted 19.4% of the bulbospinal TH-ir cells. We determined whether the bulbospinal PPE(+) RVLM neurons are barosensitive in two ways. First, we examined Fos production by FG-labeled RVLM neurons after 2 hours of hydralazine-induced hypotension (to 73 +/- 2 mm Hg) in conscious rats. Hydralazine (10 mg/kg i.v.) increased the number of Fos-ir neurons by two- to eightfold at all levels of the ventrolateral medulla examined. In the RVLM, 54% of bulbospinal PPE(+) neurons were Fos-ir, whereas such cells were more rarely found at caudal ventrolateral medullary levels. Second, we recorded individual BSBS RVLM units extracellularly in anesthetized rats and filled them juxtacellularly with biotinamide. Most biotinamide-filled neurons were PPE(+) (10 of 17), and the PPE(+) BSBS cells had a faster axonal conduction velocity than those without PPE mRNA (4.2 vs. 0.67 m/sec). Four of the 10 PPE(+) BSBS RVLM neurons were TH-ir. In summary, PPE mRNA is predominantly expressed by RVLM BSBS neurons with lightly myelinated spinal axons. PPE mRNA is present in most noncatecholaminergic BSBS neurons and also in approximately 20% of the bulbospinal C1 neurons. BSBS RVLM neurons most likely provide a major ENK input to sympathetic preganglionic neurons and PPE mRNA is the first identified positive phenotype of the non-C1 BSBS RVLM neurons.

Neurokinin-1 receptor-immunoreactive neurons of the ventral respiratory group in the rat.

The rostral end of the ventral respiratory group (VRG) contains neurons that are intensely neurokinin-1 receptor (NK1R) immunoreactive (ir). It has been theorized that some of these cells might be critical to respiratory rhythmogenesis (Gray et al. [1999] Science 286:1566-1568). In the present study we determined what major transmitter these NK1R-ir cells make and whether they are bulbospinal or propriomedullary. NK1R-ir neurons were found in the VRG between Bregma levels -11.7 and -13.6 mm. The highest concentration was found between Bregma -12.3 and -13.0 mm. This region overlaps with the pre-Bötzinger complex (pre-BötC) as it was found to contain many pre-inspiratory neurons, few E2-expiratory neurons, and no I-incremental neurons. VRG NK1R-ir neurons contain neither tyrosine hydroxylase (TH) nor choline acetyl-transferase (ChAT) immunoreactivity, although dual-labeled neurons were found elsewhere within the rostral medulla. GAD67 mRNA was commonly detected in the ventrolateral medulla (VLM) but rarely in the NK1R-ir neurons of the pre-BötC region (6 % of somatic profiles). GlyT2 mRNA was commonly found in the pre-BötC region but rarely within NK1R-ir neurons (1.3 %). Up to 40% of VRG NK1R-ir neurons were retrogradely labeled by Fluoro-Gold (FG) injected in the contralateral pre-BötC region. Some NK1R-ir VRG neurons located caudal to Bregma -12.6 mm were retrogradely labeled by FG injected in the spinal cord (C4-C5, T2-T4). In sum, NK1R immunoreactivity is present in many types of ventral medullary neurons. Within the VRG proper, NK1R-ir neurons are concentrated in an area that overlaps with the pre-BötC. Within this limited region of the VRG, NK1R-ir neurons are neither cholinergic nor catecholaminergic, and very few are gamma-aminobutyric acid (GABA)ergic or glycinergic. The data suggest that most NK1R-ir neurons of the pre-BötC region are excitatory. Furthermore, the more rostral NK1R-ir cells are propriomedullary, whereas some of the caudal ones project to the spinal cord.

Regulation of sympathetic tone and arterial pressure by rostral ventrolateral medulla after depletion of C1 cells in rat.

1. In this study we examined whether the rostral ventrolateral medulla (RVLM) maintains resting sympathetic vasomotor tone and activates sympathetic nerve activity (SNA) after the depletion of bulbospinal C1 adrenergic neurones. 2. Bulbospinal C1 cells were destroyed ( approximately 84% loss) by bilateral microinjections (spinal segments T2-T3) of an anti-dopamine-beta-hydroxylase antibody conjugated to the ribosomal toxin saporin (anti-DH-SAP). 3. Extracellular recording and juxtacellular labelling of bulbospinal barosensitive neurones in the RVLM revealed that treatment with anti-DH-SAP spared the lightly myelinated neurones with no tyrosine hydroxylase immunoreactivity. 4. In rats treated with anti-DH-SAP, inhibition of RVLM neurones by bilateral microinjection of muscimol eliminated splanchnic SNA and produced the same degree of hypotension as in control rats. 5. Following treatment with anti-DH-SAP the sympathoexcitatory (splanchnic nerve) and pressor responses to electrical stimulation of the RVLM were reduced. 6. Treatment with anti-DH-SAP also eliminated the majority of A5 noradrenergic neurones. However, rats with selective lesion of A5 cells by microinjection of 6-hydroxydopamine into the pons showed no deficits to stimulation of the RVLM. 7. In summary, the loss of 84% of bulbospinal adrenergic neurones does not alter the ability of RVLM to maintain SNA and arterial pressure at rest in anaesthetized rats, but this loss reduces the sympathoexcitatory and pressor responses evoked by RVLM stimulation. The data suggest sympathoexcitatory roles for both the C1 cells and non-C1 cells of the RVLM and further suggest the C1 cells are critical for the full expression of sympathoexcitatory responses generated by the RVLM.

Location and electrophysiological characterization of rostral medullary adrenergic neurons that contain neuropeptide Y mRNA in rat medulla.

The objective of this study was to characterize the projection pattern and electrophysiological properties of the rostral medullary adrenergic neurons (C(1)) that express neuropeptide Y (NPY) mRNA in rat. NPY mRNA was found in a variable fraction of tyrosine hydroxylase immunoreactive (TH-IR) neurons depending on the medullary level. By retrograde labeling (Fast Blue, FluoroGold), NPY mRNA was detected in virtually all C(1) cells (96%) and C(3) cells (100%) with hypothalamic projections but in only 9% of C(1) cells and 58% of C(3) cells projecting to thoracic segment 3 (T(3)) or T(6) of the spinal cord. To identify the electrophysiological properties of the C(1) cells that express NPY mRNA, we recorded from baroinhibited neurons within the C(1) region of the ventrolateral medulla (RVLM) and tested for projections to segment T(3), the hypothalamus, or both. By using the juxtacellular method, we labeled these cells with biotinamide and determined whether the recorded neurons were TH-IR and contained NPY mRNA. At rostral levels (Bregma -11.8 mm), barosensitive neurons had a wide range of conduction velocities (0.4-6.0 m/second) and discharge rates (2-28 spikes/second). Most projected to T(3) only (27 of 31 cells), and 4 projected to both the hypothalamus and the spinal cord. Most of the baroinhibited cells with spinal projections but with no hypothalamic projections had TH-IR but no NPY mRNA (11 of 17 cells). Only 1 cell had both (1 of 17 cells), and 5 cells had neither (5 of 17 cells). Both TH-IR and NPY mRNA were found in neurons with dual projections (2 of 2 cells). At level Bregma -12.5 mm, baroinhibited neurons had projections to the hypothalamus only (13 of 13 cells) and had unmyelinated axons and a low discharge rate. Four of five neurons contained both TH-IR and NPY mRNA, and 1 neuron contained neither. In short, NPY is expressed mostly by C(1) cells with projection to the hypothalamus. NPY-positive C(1) neurons are barosensitive, have unmyelinated axons, and have a very low rate of discharge. Most bulbospinal C(1) cells with a putative sympathoexcitatory role do not make NPY.

Distribution of glutamic acid decarboxylase mRNA-containing neurons in rat medulla projecting to thoracic spinal cord in relation to monoaminergic brainstem neurons.

Recent neurophysiological work has suggested the existence of monosynaptic gamma-aminobutyric acidergic (GABAergic) projections from the medulla oblongata to sympathetic preganglionic neurons. The purpose of the present study was to identify the possible anatomical location of these neurons. The location of GABAergic neurons with projection to the thoracic spinal cord was studied by using in situ hybridization for both 65-kD and 67-kD isoforms of glutamic acid decarboxylase (GAD) mRNA (GAD-65 and GAD-67, respectively) combined with midthoracic spinal cord injections of the tracer Fast Blue. Tyrosine hydroxylase (TH) or tryptophan hydroxylase immunohistochemistry was combined with GAD mRNA detection and Fast Blue to determine whether any bulbospinal catecholaminergic or serotonergic cell groups in the medulla also are GABAergic. GAD-67 and GAD-65 mRNA-containing neurons had similar distribution patterns in the medulla oblongata, with some areas exhibiting lighter labeling for GAD-65 mRNA. GABAergic bulbospinal neurons were located in the caudal part of the solitary nucleus, the parasolitary nucleus, the vestibular nuclei, the ventral medial medulla, the raphe nuclei, and parapyramidal areas. TH-immunoreactive neurons in the A1, A2, C1, and C2 areas or the area postrema did not contain either GAD-67 or GAD-65 mRNA. GAD mRNA-positive bulbospinal cells were present medial to theA1 and C1 catecholaminergic cell groups, with little or no overlap. Serotonergic neurons positive for GAD mRNAwere found in the parapyramidal area and just dorsal to the pyramidal tract in the raphe magnus. This population included bulbospinal neurons. In conclusion, GABAergic neurons with projections to the thoracic spinal cord exist in a restricted number of medullary nuclei from which inhibitory sympathetic control may originate.

Evidence for glycinergic respiratory neurons: Bötzinger neurons express mRNA for glycinergic transporter 2.

Bötzinger (BOTZ) neurons in the rostral ventrolateral medulla fire during the late expiratory phase of the respiratory cycle. These cells inhibit phrenic motor neurons and several types of respiratory neurons in the medulla oblongata. BOTZ cells produce a fast, chloride-mediated inhibition of their target neurons, but the neurotransmitter used by these cells has not been determined. In the present study, we examine whether gamma-aminobutyric acid (GABA) or glycine could be the inhibitory neurotransmitter of BOTZ cells. In chloralose-anesthetized rats, we individually filled 20 physiologically characterized BOTZ neurons with biotinamide by using a juxtacellular labeling method. Medullary sections containing the labeled BOTZ neurons were processed for in situ hybridization by using digoxigenin-labeled riboprobes for glutamic acid decarboxylase isoform 67 (GAD67), a marker for GABAergic neurons, or for glycine transporter 2 (GLYT2), a marker for glycinergic neurons. All BOTZ cells examined contained GLYT2 mRNA (n = 10), whereas none had detectable levels of GAD67 mRNA (n = 10). For a positive control, 12 GABAergic neurons in the substantia nigra pars reticulata also were recorded and filled with biotinamide in vivo. Most of these cells, as expected, had detectable levels of GAD67 mRNA (11 out of 12). These results demonstrate that the juxtacellular labeling method can be combined with in situ hybridization to identify physiologically characterized cells with probable GABAergic or glycinergic phenotypes. Furthermore, these data suggest that BOTZ neurons use the neurotransmitter glycine and not GABA to provide widespread inhibition of respiratory-related neurons.

Properties of C1 and other ventrolateral medullary neurones with hypothalamic projections in the rat.

1. This study compared (i) the properties of C1 cells with those of neighbouring non-C1 neurones that project to the hypothalamus and (ii) the properties of C1 cells that project to the hypothalamus with those of their medullospinal counterparts. 2. Extracellular recordings were made at three rostrocaudal levels of the ventrolateral medulla (VLM) in alpha-chloralose-anaesthetized, artificially ventilated, paralysed rats. Recorded cells were filled with biotinamide. 3. Level I (0-300 microm behind facial nucleus) contained spontaneously active neurones that were silenced by baro- and cardiopulmonary receptor activation and virtually unaffected by nociceptive stimulation (firing rate altered by < 20 %). These projected either to the cord (type I; 36/39), or to the hypothalamus (type II; 2/39) but rarely to both (1/39). 4. Level II (600-800 microm behind facial nucleus) contained (i) type I neurones (n = 3) (ii) type II neurones (n = 11), (iii) neurones that projected to the hypothalamus and were silenced by baro- and cardiopulmonary receptor activation but activated by strong nociceptive stimulation (type III, n = 2), (iv) non-barosensitive cells activated by weak nociceptive stimulation which projected only to the hypothalamus (type IV, n = 9), (v) cells that projected to the hypothalamus and responded to none of the applied stimuli (type V, n = 7) and (vi) neurones activated by elevating blood pressure which projected neither to the cord nor to the hypothalamus (type VI, n = 4). 5. Level III (1400-1600 microm behind facial motor nucleus) contained all the cell types found at level II except type I. 6. Most of type I and II (17/26) and half of type III cells (4/8) were C1 neurones. Type IV-V were rarely adrenergic (2/12) and type VI were never adrenergic (0/3). 7. All VLM baroinhibited cells project either to the cord or the hypothalamus and virtually all (21/23) C1 cells receive inhibitory inputs from arterial and cardiopulmonary receptors.

Antagonist precipitated clonidine withdrawal in rat: effects on locus coeruleus neurons, sympathetic nerves and cardiovascular parameters.

The goal of the present study was to examine the effect of clonidine withdrawal on the neural control of blood pressure. Rats were treated for 7-13 days with clonidine via osmotic minipumps (200 microg kg(-1) day(-1), s.c.). Controls received saline or were sham operated. Withdrawal was precipitated by the alpha2-adrenergic receptor (alpha2-AR) antagonist atipamezole. Most experiments were done under halothane anesthesia. Chronic treatment with clonidine did not change mean arterial pressure (MAP) or heart rate (HR) but raised femoral artery resistance and the activity of locus coeruleus neurons slightly. Atipamezole given to rats treated chronically with clonidine produced the following effects: no change in MAP, severe tachycardia, sustained increase in splanchnic sympathetic nerve discharge (SND; +75 +/- 13%), transient increase in lumbar SND (+23 +/- 7%), ON-OFF activity pattern in the locus coeruleus (LC). The ON phase of LC activity was synchronized with upswings of SND and with small changes in MAP. A second alpha2-AR antagonist, methoxyidazoxan, produced effects identical to those of atipamezole. Atipamezole given to control rats produced no effect on MAP, HR, SND or LC activity. Atipamezole reversed the hypotension, sympathoinhibition and bradycardia produced by acute administration of clonidine. In awake rats treated chronically with clonidine, atipamezole did not change MAP but produced arterial pressure lability and tachycardia. In conclusion, under anesthesia, selective alpha2-AR antagonists elicit a clonidine withdrawal syndrome that displays autonomic characteristics reminiscent of the spontaneous withdrawal syndrome found in awake rats. The most prominent features of this syndrome are tachycardia, sympathoactivation, lack of hypertension and an oscillating activity pattern of brainstem neurons leading to abrupt changes in SND and in MAP.

Atipamezole-precipitated clonidine withdrawal induces c-Fos expression in rat central nervous system.

We sought to identify a clonidine withdrawal syndrome in conscious rats by investigating the effects of a single injection of the specific alpha2-adrenergic antagonist atipamezole (1.5 mg/kg i.p.) after chronic treatment with the alpha2-adrenergic agonist clonidine (200 microg/kg per day via osmotic mini-pump for 7-10 days). Rats treated chronically with clonidine followed by atipamezole injection (clonidine-atipamezole) demonstrated dramatic behavioral effects including shaking, vigorous digging, and whole-body seizure-like movements. Control groups (saline-saline, clonidine-saline and saline-atipamezole) showed no overt unusual behavioral effects following injection. The brains of the clonidine-atipamezole group showed massive c-Fos expression (especially in di- and telencephalon) while the other groups showed either background levels of c-Fos-immunopositive cells (saline-saline and clonidine-saline groups) or a slight increase over background in selected areas (saline-atipamezole group). Maps of c-Fos-immunolabeled cells were generated at five representative coronal planes for each treatment group. C-Fos-immunopositive cells were counted in three representative brainstem structures (locus coeruleus, nucleus of the solitary tract, rostral ventrolateral medulla (RVL)) and in three regions of the thoracic spinal cord (dorsal horn, intermediate zone and ventral horn). In the three brainstem structures the number of c-Fos-positive cells was elevated 8-10-fold in the clonidine-atipamezole group compared to the other groups. No other treatment group was significantly different from the saline-saline group. An increased number of c-Fos-positive neurons was also noted in the dorsal horn and intermediate layers of the thoracic spinal cord in the clonidine-atipamezole group compared to a sham-operated atipamezole-injected group. In the RVL, 59% of c-Fos-positive cells contained alpha2A-adrenergic receptor-like immunoreactivity in clonidine-atipamezole treated (withdrawing) rats. In addition, one-third of the tyrosine hydroxylase (TH)-immunopositive cells in RVL were also c-Fos-positive in clonidine withdrawing rats where no TH-positive cells were also c-Fos-positive in RVL of control groups. Atipamezole injected 10 min after a single injection of clonidine (200 microg/kg, i.p.) produced no behavioral effect and did not increase c-Fos expression in brainstem. Injection of the opiate antagonist naltrexone (100 mg/kg, i.p.) in rats chronically treated with clonidine did not elicit behavioral effects or result in increased c-Fos expression in brainstem. In conclusion, administration of the selective alpha2-antagonist atipamezole to rats chronically treated with the alpha2-adrenergic agonist clonidine triggers a powerful withdrawal syndrome associated with massive CNS expression of c-Fos protein. The intensity of the withdrawal syndrome indicates that chronic exposure to alpha2-adrenergic receptor agonists produces strong dependence.

Distribution of alpha 2C-adrenergic receptor-like immunoreactivity in the rat central nervous system.

The distribution of alpha 2C-adrenergic receptors (ARs) in rat brain and spinal cord was examined immunohistochemically by using an affinity purified polyclonal antibody. The antibody was directed against a recombinant fusion protein consisting of a 70-amino-acid polypeptide portion of the third intracellular loop of the alpha 2C-AR fused to glutathione-S-transferase. Selectivity and subtype specificity of the antibody were demonstrated by immunoprecipitation of [125I]-photoaffinity-labeled alpha 2-AR and by immunohistochemical labeling of COS cells expressing the individual rat alpha 2-AR subtypes. In both cases the antibody recognized only the alpha 2C-AR subtype, and immunoreactivity was eliminated by preadsorption of the antibody with excess antigen. In rat brain, alpha 2C-AR-like immunoreactivity (alpha 2C-AR-LI) was found primarily in neuronal perikarya, with some labeling of proximal dendrites; analysis by confocal microscopy revealed the intracellular localization of some of the immunoreactivity. Areas of dense immunoreactivity include anterior olfactory nucleus, piriform cortex, septum, diagonal band, pallidum, preoptic areas, supraoptic nucleus, suprachiasmatic nucleus, paraventricular nucleus, amygdala, hippocampus (CA1 and dentate gyrus), substantia nigra, ventral tegmental area, raphe (pontine and medullary), motor trigeminal nucleus, facial nucleus, vestibular nucleus, dorsal motor nucleus of the vagus, and hypoglossal nucleus. Labeling was found in specific laminae throughout the cortex, and a sparse distribution of very darkly labeled cells was observed in the striatum. At all levels of the spinal cord there were small numbers of large, darkly labeled cells in layer IX and much smaller cells in layer X. In general, the pattern of alpha 2C-LI throughout the neuraxis is consistent with previously published reports of the distribution of receptor mRNA detected by hybridization histochemistry.

Effects of morphine and morphine withdrawal on adrenergic neurons of the rat rostral ventrolateral medulla.

In urethane anesthetized rats, iontophoretic application of morphine or alpha-methylnoradrenaline (alpha-MNE) inhibited (80-100%) the discharges of all putative adrenergic (C1) cells of the rostral ventrolateral medulla (RVLM). The effect of morphine was blocked selectively by naloxone while that of alpha-MNE was blocked selectively by the alpha 2-adrenergic antagonist idazoxan. Putative C1 cells were inhibited (75-100%) by low i.v. doses of clonidine (10-15 micrograms/kg). Most cells (7/10) were also inhibited by morphine i.v. (81% at 7 mg/kg). Two cells were slightly excited at doses below 2 mg/kg and inhibited at higher doses. Three cells were excited only. All effects of morphine i.v. were reversed by naloxone (1 mg/kg, i.v.). Intravenous administration of naloxone to morphine-dependent rats increased significantly the firing rate of all putative C1 adrenergic cells (from 5.8 +/- 0.9 spikes/s to 12.3 +/- 1.5 spikes/s; n = 8). During withdrawal these cells could still be inhibited (80-100%) by i.v. injection of clonidine (15 micrograms/kg). C-Fos expression induced by naltrexone-precipitated withdrawal was examined in the brainstem of freely moving morphine-dependent rats pretreated with clonidine or saline before injection of the opioid antagonist. The locus coeruleus (LC) of the same rats was examined for comparison. Morphine withdrawal without clonidine treatment significantly increased the number of Fos-like-immunoreactive (Fos-LIR) cells in the RVLM and LC. Clonidine pretreatment (1 mg/kg, i.p.) reduced the number of withdrawal-activated Fos-LIR cells in LC by 81%. In the RVLM this reduction averaged 37% for all cell types and 48% for C1 adrenergic cells. Further, a very large proportion of RVLM neurons that expressed c-Fos during morphine withdrawal (83%) were immunoreactive for alpha 2A-adrenergic receptors. This study suggests that, like noradrenergic cells of the LC, C1 adrenergic neurons of the RVLM are: (i) inhibited by both opiate and alpha 2-adrenergic receptor agonists; and (ii) activated during naloxone-precipitated morphine withdrawal. Since C1 cells are considered essential to sympathetic tone generation, their inhibition by morphine may contribute to the hypotensive effects of this opioid agonist in non-dependent individuals. Their excitation during opiate withdrawal may also contribute to the autonomic activation that characterizes this syndrome. Finally, inhibition of C1 cells by clonidine may contribute to the clinically recognized efficacy of this drug to attenuate autonomic signs of opiate withdrawal.