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The immune system plays a key role in the dissemination of prion infections from the periphery to the central nervous system (CNS). While follicular dendritic cells are critical for prion replication in lymphoid tissue and subsequent neuroinvasion, myeloid dendritic cells (DCs) have been implicated in both the clearance and propagation of pathological prion protein. Since nothing is known on the ability of DCs to migrate to the CNS during prion diseases, we investigated the immunohistochemical localization of CD205(+) DCs in the brain of C57BL/6 mice intraperitoneally infected with the mouse-adapted KFu strain of Gerstmann-Sträussler-Scheinker syndrome, a human genetic prion disorder. In normal brain, CD205(+) cells were present in the meninges and choroid plexus, whereas in the majority of mice sacrificed between 120 and 300 days post infection, CD205(+) DCs were also detected in the cerebral cortex, subcortical white matter, thalamus and medulla oblongata. These findings demonstrate that DCs can enter the CNS of prion-infected mice, suggesting a possible role for these cells in the pathogenesis of prion disorders.

Elucidation of the neuronal mechanisms underlying respiratory rhythmogenesis is a major focal point in respiratory physiology. An area of the ventrolateral medulla, the pre-Bötzinger complex (preBotC), is a critical site. Attention is now focused on understanding the cellular and network properties within the preBotC that underlie this critical function. The inability to clearly identify key "rhythm-generating" neurons within the heterogeneous population of preBotC neurons has been a significant limitation. Here we report an advancement allowing precise targeting of neurons expressing neurokinin-1 receptors (NK1Rs), which are hypothesized to be essential for respiratory rhythmogenesis. The internalization of tetramethylrhodamine conjugated substance P in rhythmically active medullary slice preparations provided clear visualization of NK1R-expressing neurons for subsequent whole-cell patch-clamp recordings. Among labeled neurons, 82% were inspiratory modulated, and 25% had pacemaker properties. We propose that this approach can be used to greatly expedite progress toward understanding the neuronal processes underlying the control of breathing.

1. The effect of hemisection of the cervical spinal cord on NADPH-diaphorase staining in the reticular nuclei of the rabbit medulla was investigated using histochemical technique. 2. A quantitative assessment of somal and neuropil NADPH-diaphorase staining was made by an image analyzer in a selected area of each reticular nucleus of the rabbit medulla. 3. On the 7th postsurgery day, the highest up-regulation of somatic NADPH-diaphorase staining was observed in regions regulating cardiorespiratory processes; however, the highest increase of neuropil NADPH-diaphorase staining was found in the reticular nuclei modulating the tonus of postural muscles. 4. The degeneration of non-NADPH-diaphorase-stained neurons was detected throughout the reticular formation of the medulla, but the extent of neuronal death did not correlate with the up-regulation of the NADPH-diaphorase staining in the reticular nuclei of the medulla. 5. The findings provide evidence that NADPH-diaphorase-exhibiting neurons are refractory to the hemisection of the cervical spinal cord and that the neuronal up-regulation of NADPH-diaphorase at the medullar level is probably not a causative factor leading to the death of the reticulospinal neurons.

In the hypoglossal nucleus, GABA and glycine mediate inhibition at separate or mixed synapses containing glycine receptors (GlyRs) and/or GABA(A) receptors (GABA(A)Rs). The functional development of mixed inhibitory synapses depends on the brain area studied, but their relative proportion to total synapses generally decreases with time. We have determined the sequential process of inhibitory synapse maturation in the hypoglossal nucleus in vivo. Immunocytochemistry and confocal microscopy were used for codetection of VIAAT, the common presynaptic vesicular transporter of glycine and GABA, GlyRs, GABA(A)R alpha1 and gamma2 subunits, and gephyrin, the scaffold protein implicated in the synaptic localization of inhibitory receptors. In E17 embryos, GlyRs were already clustered while GABA(A)R alpha1 and gamma2 subunit immunoreactivity (IR) displayed both diffuse and clustered patterns. Quantitative analysis at this stage revealed that the majority of GlyR clusters were apposed to VIAAT-IR accumulation and that 30% of them colocalized with gamma2GABA(A)R clusters. This proportion increased with age to 50% at P30. GlyR clusters that did not colocalize with gamma2GABA(A)R clusters were associated with GABA(A)R gamma2 diffuse IR. Interestingly, the percentage of GlyR clusters surrounded by GABA(A)R gamma2 diffuse IR decreased with age, while GlyR clusters colocalized with gamma2GABA(A)R clusters increased. The developmental coclustered pattern of gephyrin and GABA(A)R alpha1 and gamma2 subunits paralleled the coclustered pattern of GlyRs and GABA(A)R alpha1 and gamma2 subunits. Our results indicate that the proportion of GlyR-GABA(A)R coclusters increases until adulthood. A developmental sequence of the postsynaptic events is proposed in which diffuse extrasynaptic GABA(A)Rs accumulate at inhibitory synapses to form postsynaptic clusters, most of them being colocalized with GlyR clusters in the adult.

The synuclein family includes three isoforms, termed alpha, beta and gamma. alpha-Synuclein accumulates in various pathological lesions resulting from neurodegenerative disorders including Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy. However, neither beta- nor gamma-synuclein has been detected in Lewy bodies, and thus it is unclear whether these isoforms contribute to neurological pathology. In the present study, we used immunohistochemistry to demonstrate accelerated accumulation of beta- and gamma-synucleins in axonal spheroids in gracile axonal dystrophy (gad) mice, which do not express ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1). gamma-Synuclein immunoreactivity in the spheroids appeared in the gracile nucleus at 3 weeks of age and was maintained until 32 weeks. beta-Synuclein immunoreactivity appeared in spheroids around 12 weeks of age. In contrast, alpha-synuclein immunoreactivity was barely detectable in spheroids. Immunoreactivity for synaptophysin and ubiquitin were either faint or undetectable in spheroids. Given that UCH-L1 deficiency results in axonal degeneration and spheroid formation, our findings suggest that beta- and gamma-synuclein participate in the pathogenesis of axonal swelling in gad mice.

The midbrain periaqueductal gray matter (PAG) integrates the basic responses necessary for survival of individuals and species. Examples are defense behaviors such as fight, flight, and freezing, but also sexual behavior, vocalization, and micturition. To control these behaviors the PAG depends on strong input from more rostrally located limbic structures, as well as from afferent input from the lower brainstem and spinal cord. Mouton and Holstege (2000, J Comp Neurol 428:389-410) showed that there exist at least five different groups of spino-PAG neurons, each of which is thought to subserve a specific function. The lateral cervical nucleus (LCN) in the upper cervical cord is not among these five groups. The LCN relays information from hair receptors and noxious information and projects strongly to the contralateral ventroposterior and posterior regions of thalamus and to intermediate and deep tectal layers. The question is whether the LCN also projects to the PAG. The present study in cat, using retrograde and anterograde tracing techniques, showed that neurons located in the lateral two-thirds of the LCN send fibers to the lateral part of the PAG, predominantly at rostrocaudal levels A0.6-P0.2. This part of the PAG is known to be involved in flight behavior. A concept is put forward according to which the LCN-PAG pathway alerts the animal about the presence of cutaneous stimuli that might represent danger, necessitating flight. J. Comp. Neurol. 471:434-445, 2004., (Copyright 2004 Wiley-Liss, Inc.)

Tumor necrosis factor alpha (TNF(alpha)) is a potent modulator of autonomic reflex mechanisms that control the stomach. Evidence suggests that TNF(alpha) action directly on vago-vagal reflex control circuits causes the autonomic misregulation of digestion manifested as gastrointestinal stasis, nausea, and emesis associated with illness. Neurophysiological studies indicated that TNF(alpha) may have effects on vagal afferents in the solitary nucleus, as well as neurons of the solitary nucleus (NST) and dorsal motor nucleus (DMN) of the vagus. The aim of this study was to determine the location of the TNFR1 receptor (p55) in the medulla using immunocytochemical methods. We devised a technique for localizing the p55 receptor using heat-induced antigen recovery in fixed tissue sections. This protocol allowed us to demonstrate that dense p55-immunoreactivity (p55-ir) is constitutively present on central (but not peripheral) vagal afferents in the solitary tract (ST) and nucleus; p55-ir is also present on afferents entering the spinal trigeminal nucleus. Unilateral supra-nodose vagotomy eliminated p55-ir from ipsilateral central vagal afferents. Virtually all neurons in the brainstem appeared to express p55-ir at a low level, i.e., just above background. However, vagotomy caused a dramatic up-regulation of p55-ir in vagal motor neurons. This increase in p55-ir in axotomized neurons may play a pivotal role in the connection between the occurrence of the injury and the initiation of apoptotic processes resulting in elimination of damaged neurons.

Experiments were performed in the male Wistar rat to investigate the projections from cardiovascular responsive sites in the ventrolateral medulla (VLM) to the subfornical organ (SFO). Unilateral iontophoretic injections of Phaseolus vulgaris leucoagglutinin (PHA-L) were made into either caudal VLM (CVLM) sites at which microinjection of l-glutamate (10 nl; 0.25 M) elicited decreases in mean arterial pressure or into rostral VLM (RVLM) sites at which l-glutamate microinjection elicited increases in arterial pressure. After a survival period of 7-10 days, transverse sections of the forebrain and brainstem were processed for PHA-L immunoreactivity. After injections of PHA-L into the CVLM, axonal and presumptive terminal labeling was found bilaterally throughout the rostrocaudal extent of the SFO, although most of the projections were observed within the rostral half of the nucleus. Within the SFO, labeling was found primarily in the lateral aspects of the nucleus, often in close proximity to blood vessels. In addition, CVLM injections resulted in labeling within the organum vasculosum of the laminae terminalis (OVLT) and within the ventral and dorsal components of the median preoptic nucleus (MnPO) bilaterally, but with an ipsilateral predominance. In contrast, PHA-L injections into the RVLM did not result in axonal labeling in the SFO or OVLT, although a few labeled axons were found to course through the region of the ventral component of MnPO. These data have demonstrated that neurons within the cardiovascular responsive region of the CVLM send direct axonal projections to the SFO and other structures of the laminae terminalis, and suggest that the CVLM may function in the modulation of the activity of neurons of circumventricular organs to intra- and extracellular signals of body fluid balance.

The study was undertaken to examine the involvement of the renin-angiotensin system in the reversal by endogenous central histamine of critical haemorrhagic hypotension in anaesthetised Wistar rats. Histamine N-methyltransferase inhibitor metoprine (20 microg) administered intracerebroventricularly at 5 min of critical hypotension 20-25 mmHg produced increases in histamine concentrations as measured 20 min after treatment in the hypothalamus (581.33 +/- 63.23 vs. 488.26 +/- 56.34 ng/g of wet tissue; P < 0.01) and medulla oblongata (53.42 +/- 14.65 vs. 34.68 +/- 13.52 ng/g of wet tissue; P < 0.05). That was accompanied by 34.7% higher plasma angiotensin II concentration in comparison to the control group. Metoprine produced dose-dependent (5-20 microg) rises in mean arterial pressure (MAP) and heart rate, which were significantly higher than those in normotensive animals. The resuscitating action of metoprine (20 microg) was associated with rises in renal, mesenteric and hindquarters blood flows, and a 100% survival at 2 h after treatment, while in the saline-treated group, all the animals died within 30 min. Angiotensin type 1 (AT(1)) receptor antagonist ZD 7155 (0.5 mg/kg; iv) decreased regional vascular resistance and inhibited metoprine-induced increase in MAP, whereas AT(2) receptor blocker PD 123319 (10 mg/kg; i.v.) had no effect. Angiotensin-converting enzyme inhibitor captopril (30 mg/kg; i.v.) reduced the increase in plasma angiotensin II level and the haemodynamic effects of metoprine. Neither capropril, nor angiotensin receptor antagonists influence the survival at 2 h after treatment. In conclusion, the renin-angiotensin system is involved in central histamine-induced resuscitating action in rats.

In Diptera, subsets of small retinotopic neurons provide a discrete channel from achromatic photoreceptors to large motion-sensitive neurons in the lobula complex. This pathway is distinguished by specific affinities of its neurons to antisera raised against glutamate, aspartate, gamma-aminobutyric acid (GABA), choline acetyltransferase (ChAT), and a N-methyl-D-aspartate type 1 receptor protein (NMDAR1). Large type 2 monopolar cells (L2) and type 1 amacrine cells, which in the external plexiform layer are postsynaptic to the achromatic photoreceptors R1-R6, express glutamate immunoreactivity as do directionally selective motion-sensitive tangential neurons of the lobula plate. L2 monopolar cells ending in the medulla are accompanied by terminals of a second efferent neuron T1, the dendrites of which match NMDAR1-immunoreactive profiles in the lamina. L2 and T1 endings visit ChAT and GABA-immunoreactive relays (transmedullary neurons) that terminate from the medulla in a special layer of the lobula containing the dendrites of directionally selective retinotopic T5 cells. T5 cells supply directionally selective wide-field neurons in the lobula plate. The present results suggest a circuit in which initial motion detection relies on interactions among amacrines and T1, and the subsequent convergence of T1 and L2 at transmedullary cell dendrites. Convergence of ChAT-immunoreactive and GABA-immunoreactive transmedullary neurons at T5 dendrites in the lobula, and the presence there of local GABA-immunoreactive interneurons, are suggested to provide excitatory and inhibitory elements for the computation of motion direction. A comparable immunocytological organization of aspartate- and glutamate-immunoreactive neurons in honeybees and cockroaches further suggests that neural arrangements providing directional motion vision in flies may have early evolutionary origins., (Copyright 2003 Wiley-Liss, Inc.)

An in vitro brain stem preparation from turtles exhibits a neural correlate of eyeblink classical conditioning during pairing of auditory (CS) and trigeminal (US) nerve stimulation while recording from the abducens nerve. The premotor neuronal circuits controlling abducens nerve-mediated eyeblinks in turtles have not been previously described, which is a necessary step for understanding cellular mechanisms of conditioning in this preparation. The purpose of the present study was to neuroanatomically define the premotor pathways that underlie the trigeminal and auditory nerve-evoked abducens eyeblink responses. The results show that the principal sensory trigeminal nucleus forms a disynaptic pathway from both the trigeminal and auditory nerves to the principal and accessory abducens motor nuclei. Additionally, the principal abducens nucleus receives vestibular inputs, whereas the accessory nucleus receives input from the cochlear nucleus. The late R2-like component of abducens nerve responses is mediated by the spinal trigeminal nucleus in the medulla. Both the principal sensory trigeminal nucleus and the abducens motor nuclei receive CS-US convergence and therefore both, or either, might be considered potential sites of synapse modification during in vitro abducens conditioning. Further data are required to determine the role of the principal sensory trigeminal nucleus in in vitro conditioning., (Copyright 2004 S. Karger AG, Basel.)

The effect of chronic aluminum intake has been investigated in the brain of aged male Wistar rats to assess the potential role of the accumulation of this metal ion on the development of neurodegenerative features observed in Alzheimer's disease. AlCl3 x 6 H2O (2g/L) was administered to experimental animals for 6 months in the drinking water. The total content of Al (microg/g fresh tissue) was measured by inductively coupled plasma atomic emission spectrometry (ICP-AES), while the content of Cu, Zn and Mn was determined by flame AAS in the prosencephalon + mesencephalon, pons-medulla and cerebellum of control and Al(III)-treated animals. The area occupied by mossy fibres in the CA3 field of the hippocampus was estimated by a computer-assisted morphometric method following Timm's preferential staining. In Al(III)-treated rats the concentration of Cu, Zn and Mn did not increase significantly (p < 0.5) in prosencephalon + mesencephalon, nor in pons-medulla (p < 0.5) except for Cu (p < 0.05) in pons-medulla. In the cerebellum the only significant increase was seen for Zn (p < 0.01) while no change was observed for Cu and Mn. The area occupied by the mossy fibres in the hippocampal CA3 field was significantly increased (+32%) in aged Al(III)-treated rats. Since Cu, Zn and Mn are essential components of the cytosolic and mitochondrial superoxide dismutases, it is possible that the increased content of these ions in aged Al(III)-treated rats represents an increased amount of genetic expression of these antioxidant enzymes. Considering that the positivity to Timm's reaction is based on the presence of free or loosely bound Zn2+ ions within synaptic terminals and that Zn2+ ions are reported to be accumulated by hippocampal neurons when tissue injury occurs, the increased area of the mossy fibres in CA3 field of Al(III)-treated rats could indicate increased hippocampal damage in these animals. Taken together, the present findings indicate that the aging CNS is particularly susceptible to Al(III) toxic effects which may increase the cell load of oxidative stress and may contribute, as an aggravating factor, to the development of neurodegenerative events as observed in Alzheimer's disease.

We studied the distribution of medullary glycinergic neurons in relation to GABAergic neurons, by using in situ hybridization method for mRNA encoding either glycine transporter 2 (GLYT2) or glutamic acid decarboxylase isoform 67 (GAD67). GLYT2 mRNA-positive (GLYT2+) neurons were distributed widely and clustered in (1). the respiration-related area of the ventrolateral medulla called the Bötzinger complex, (2). the nucleus retroambiguus caudal to the obex or the caudal ventral respiratory group, (3). the spinal trigeminal nucleus, (4). a small area immediately dorsal to the inferior olivary nucleus, and (5). the border zone between the hypoglossal nucleus and the surrounding reticular formation. It was characteristic that in the dorsomedial medulla, GLYT2+ neurons were distributed only sparsely in contrast to dense GAD67+ neurons. Only few GLYT2+ neurons were distributed in the medial and interstitial subnuclei of the nucleus tractus solitarii. In particular virtually no GLYT2+ neurons were found in the area postrema. Furthermore, in the reticular formation and the spinal trigeminal nucleus, GAG67+ neurons tended to be distributed in the area where GLYT2+ neurons were sparse, and vice versa. These results provide useful information for the effort of determining neurotransmitters involved in the medullary neurons.

Glutamatergic signal transduction occurs in CNS white matter, but quantitative data on glutamate uptake and metabolism are lacking. We report that the level of the astrocytic glutamate transporter GLT in rat fimbria and corpus callosum was approximately 35% of that in parietal cortex; uptake of [3H]glutamate was 24 and 43%, respectively, of the cortical value. In fimbria and corpus callosum levels of synaptic proteins, synapsin I and synaptophysin were 15-20% of those in cortex; the activities of glutamine synthetase and phosphate-activated glutaminase, enzymes involved in metabolism of transmitter glutamate, were 11-25% of cortical values, and activities of aspartate and alanine aminotransferases were 50-70% of cortical values. The glutamate level in fimbria and corpus callosum was 5-6 nmol/mg tissue, half the cortical value. These data suggest a certain capacity for glutamatergic neurotransmission. In optic and trigeminal nerves, [3H]glutamate uptake was < 10% of the cortical uptake. Formation of [14C]glutamate from [U-14C]glucose in fimbria and corpus callosum of awake rats was 30% of cortical values, in optic nerve it was 13%, illustrating extensive glutamate metabolism in white matter in vivo. Glutamate transporters in brain white matter may be important both physiologically and during energy failure when reversal of glutamate uptake may contribute to excitotoxicity.

Distribution and maturation of catecholaminergic (CA) neurons have been studied by tyrosine hydroxylase immunohistochemistry in the medulla oblongata of human fetuses aged 14.5-25 weeks of gestation. Already at 14.5 weeks, CA neurons were observed in two longitudinally oriented cell clusters, one located ventrolaterally in the area of the lateral reticular and ambiguous nuclei, the other one dorsomedially forming 4 groups related to the dorsal vagal nucleus, the commissural nucleus of the vagus, the nucleus of the tractus solitarius and the area postrema. CA neurons in the area postrema were often found close to blood vessels. Scattered intermediate CA neurons were seen between these two larger clusters. CA neurons still appeared immature exhibiting bipolar morphology with only one or two short stout processes, which hardly branched. At 21 weeks, CA neurons occupied essentially the same location, but had a more mature morphology. Though still bipolar in shape, they had thinner and much longer processes which frequently branched. Both in the ventrolateral and the dorsomedial cell clusters, these processes were frequently lying close to blood vessels. At 25 weeks, CA cells had matured into multipolar neurons with long thin processes forming fine fiber networks in the ventrolateral medulla as well as around and within the dorsal vagal and solitarius nuclei. Only at this stage, a distinct CA fiber tract was seen located in the region of the tractus solitarius. Our results indicate that CA neurons in the human medulla, which are presumably involved in the control of ventilation and blood pressure, though generated rather early during development, mature relatively late.

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease characterized by demyelination and inflammatory infiltrates in the CNS, and it is an animal model of multiple sclerosis. Piperonyl butoxide (PBO) suppresses disease in EAE mice, and it exhibits a dual effect on cytochrome P450s that manifests in a transient inhibitory phase followed by induction. In order to identify the expression of proteins associated with EAE, a proteomic screening was performed on hindbrain microsomes from control + vehicle, control + PBO, EAE + vehicle, and EAE + PBO female mice. Glucose regulated protein 94 (Grp94) and coagulation factor VIII were among the proteins identified in EAE + vehicle and EAE + PBO mice. Immunohistochemical staining of Grp94 was present in some neurons and oligodendrocytes in hindbrain sections from control animals, and in some cells within inflammatory infiltrates in EAE animals. Since Grp94 (also known as Gp96) can partake in antigen presentation and induction of proinflammatory cytokine expression, its presence in these cells suggests that it may play a role in the pathogenesis of EAE. Coagulation factor VIII is carried and protected by von Willebrand factor. Immunohistochemical staining of von Willebrand factor revealed its presence in some vessels within hindbrain sections from control animals. In EAE animals, the number of labeled vessels was significantly increased, and extracellular granular deposits were observed around labeled vessels indicating that the breakdown of the blood-brain barrier that occurs in EAE permitted its extravasation into the CNS. Additional proteins were identified in the different groups of mice by proteomic screening, but confirmation of their expression profile awaits investigations by independent measures.

Globoid cell leukodystrophy (Krabbe disease) is caused by mutations in galactosylceramidase, a lysosomal enzyme that acts to digest galactosylceramide, a glycolipid concentrated in myelin, and psychosine (galactosylsphingosine). Globoid cell leukodystrophy has been identified in many species including humans and twitcher mice. Several studies on human tissue have examined the lipid profile in this disease by gas, liquid or thin layer chromatography. Electrospray ionization tandem mass spectrometry combined with reverse phase HPLC has become a powerful alternative strategy, used here to compare the sphingolipid profile of pons/medulla tissue from twitcher mice with control tissue. In this lipidomics LC-MS approach, we scanned for precursors of m/z 264 to obtain a semi-quantitative profile of ceramides and galactosylceramides. Sphingosine-1-phosphate, C18:0 ceramide, C22:0 ceramide and C24:0 ceramide levels were reduced in the pons/medulla of twitcher mice compared to levels in control mice at 31 and 35-37 days of age. The levels of C22:0 and C24:0 galactosylceramide were similar between twitcher and control specimens and there was a trend toward reduced levels of C24:1 galactosylceramide and C24:1 hydroxy-galactosylceramide in twitcher specimens. Psychosine, C 16:0 ceramide and C 18:0 galactosylceramide levels were increased in the CNS of twitcher mice compared to levels in control mice. These data indicate that there is a trend toward decreased levels of long chain fatty acids and increased levels of shorter chain fatty acids in galactosylceramides and ceramides from twitcher mice compared with control mice, and such changes may be due to demyelination characteristic of acute pathology.

The major goal of this study was to determine whether there is increased activation of medullary neurons that participate in the central baroreceptor reflex pathway in dogs with obesity-induced hypertension, a model of hypertension that is associated with increased sympathetic activity. We used Fos-like (Fos-Li) protein immunohistochemical methods to determine activation of neurons in the nucleus tractus solitarius (NTS), caudal ventrolateral medulla (CVLM), and rostral ventrolateral medulla (RVLM). Dogs were fed either a regular diet or an identical diet with the addition of 0.5 to 0.9 kg of cooked beef fat. After approximately 6 weeks of the high fat diet, body weight (36.3+/-0.4 vs 21.5+/-0.5 kg), mean arterial pressure (105+/-4 vs 91+/-3 mm Hg), and heart rate (97+/-4 vs 70+/-3 bpm) were significantly greater in obese than in control dogs, respectively. There was little Fos-Li immunoreactivity in medullary neurons of control dogs but marked reactivity in obese dogs. Specifically, the number of Fos-Li-positive cells in the NTS and CVLM was 3 to 5 times greater in obese than in control dogs. Furthermore, despite sustained activation of these baroreceptor-sensitive neurons, there was a significantly greater number of Fos-Li positive cells in the RVLM of dogs fed the high fat diet. As baroreceptor suppression of sympathoexcitatory cells in the RVLM is mediated by activation of neurons in the NTS and CVLM, these results support recent findings indicating that baroreflex suppression of sympathetic activity is a long-term compensatory response in hypertension. However, sympathoexcitatory inputs onto RVLM neurons would appear to predominate over the inhibitory effects of the baroreflex in obesity hypertension.

The present study was designed to investigate the effect of repeated administration of a selective kappa-opioid receptor agonist (1S-trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide hydrochloride [(-)U-50,488H] on antinociception and G-protein activation induced by mu-opioid receptor agonists in mice. A single s.c. injection of (-)U-50,488H produced a dose-dependent antinociception, and this effect was reversed by a selective kappa-opioid receptor antagonist nor-binaltorphimine (nor-BNI). Furthermore, a single s.c. pre-treatment with (-)U-50,488H had no effect on the mu-opioid receptor agonist-induced antinociception. In contrast, repeated s.c. administration of (-)U-50,488H resulted in the development of tolerance to (-)U-50,488H-induced antinociception. Under these conditions, we demonstrated here that repeated s.c. injection of (-)U-50,488H significantly enhanced the antinociceptive effect of selective mu-opioid receptor agonists endomorphin-1, endomorphin-2 and [d-Ala2,N-MePhe4,Gly-ol5] enkephalin (DAMGO). Using the guanosine-5'-o-(3-[35S]thio) triphosphate ([35S]GTP gamma S) binding assay, we found that (-)U-50,488H was able to produce a nor-BNI-reversible increase in [35S]GTP gamma S binding to membranes of the mouse thalamus, which has a high level of kappa-opioid receptors. Repeated administration of (-)U-50,488H caused a significant reduction in the (-)U-50,488H-stimulated [35S]GTP gamma S binding in this region, whereas chronic treatment with (-)U-50,488H exhibited the increase in the endomorphin-1-, endomorphin-2- and DAMGO-stimulated [35S]GTP gamma S bindings in membranes of the thalamus and periaqueductal gray. These results suggest that repeated stimulation of kappa-opioid receptors leads to the heterologous up-regulation of mu-opioid receptor functions in the thalamus and periaqueductal gray regions, which may be associated with the supersensitivity of mu-opioid receptor-mediated antinociception.

We evaluated the distribution of nitric oxide synthase (NOS) isoforms in the rostral ventrolateral medulla (RVLM), the medullary origin of sympathetic neurogenic vasomotor tone, and the contribution of NOS III to the cardiovascular actions of endogenous NO in the RVLM. Adult Sprague-Dawley rats were used. Reverse transcription-polymerase chain reaction or Western blot analysis revealed that NOS I, II or III was expressed in the ventrolateral medulla at the mRNA or protein level under basal conditions. However, laser scanning confocal microscopic analysis of double-immunofluorescence images showed that whereas NOS I or II immunoreactivity colocalized with cells within the confines of the RVLM that stained positively with the neuronal marker, NeuN, NOS III immunoreactivity was associated primarily with blood vessels. Furthermore, bilateral microinjection into the RVLM of the selective NOS III inhibitor, N(5)-(1-iminoethyl)-L-ornithine, elicited minimal alterations in baseline systemic arterial pressure, heart rate or sympathetic vasomotor outflow in rats anesthetized with propofol. We conclude that whereas NOS I and II are present in neurons within the confines of the RVLM, NOS III is associated primarily with blood vessels. Our results further indicate that NOS III does not appear to contribute to the maintenance of basal sympathetic vasomotor outflows and arterial pressure by the endogenous NO at the RVLM., (Copyright 2003 National Science Council, ROC and S. Karger AG, Basel)

Elucidating the central sensory projection pathways of extra- and intracranial vessels appears to be of fundamental importance for understanding the pathogenetic mechanisms of primary headaches. In this paper, two kinds of tracers, choleragenoid (cholera toxin subunit b, CTb) and wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP), were used to transganglionically label the central sensory projections of the innervation of the superficial temporal artery (STA). Following either of the tracers applied on the adventitia of the STA, labelled terminations were found mainly in the ipsilateral C1-C3 spinal dorsal horns. Sparse labelling was also found in the interpolar and caudal parts of the spinal trigeminal nucleus. In the spinal cord, CTb labelled profiles were mainly located in laminae III and IV, whereas WGA-HRP labelled profiles were mainly located in laminae I and II. In the medulla, CTb but not WGA-HRP labelled terminals were found in a small dorsolateral extension of the cuneate nucleus. The present results indicate that the primary sensory nervous center of the STA is located in the rostral cervical spinal dorsal horn. The caudal parts of the spinal trigeminal nucleus, which has been demonstrated as a center of pain and temperature sensations of the head and face, transmits limited information from the STA to higher nervous centers.

We examined immunohistochemically whether the vesicular glutamate transporters (VGluTs), VGluT1 and VGluT2, might be expressed in synaptic terminals of nociceptive primary afferent fibers within laminae I and II of the medullary and spinal dorsal horns of the rat. VGluT1 immunoreactivity (IR) was intense in the inner part of lamina II but weak in lamina I and the outer part of lamina II. VGluT2-IR was most intense in lamina I and the outer part of lamina II. Expression of VGluTs in synaptic terminals was confirmed by dual immunofluorescence histochemistry for VGluTs and synaptophysin. Expression of VGluTs in axon terminals of primary afferent fibers terminating in laminae I and II was also confirmed immunohistochemically after unilateral dorsal rhizotomy. The dual immunofluorescence histochemistry indicated expression of VGluTs in substance P (SP)-containing axon terminals in lamina I and the outer part of lamina II. Electron microscopy confirmed the coexpression of VGluTs and SP in axon terminals within laminae I and II; VGluTs was associated with round synaptic vesicles at the asymmetric synapses. It was further observed that isolectin IB4, a marker for unmyelinated axons, often bound with VGluT2-immunopositive structures but rarely with VGluT1-immunopositive structures in lamina II. Thus, the results indicated in laminae I and II of the medullary and spinal dorsal horns that both VGluT1 and VGluT2 were expressed in axon terminals of primary afferent fibers, including SP-containing nociceptive fibers and that VGluT in unmyelinated primary afferent fibers terminating in lamina II was primarily VGluT2., (Copyright 2003 Wiley-Liss, Inc.)

In the present study, we have investigated the effects of a novel prolyl endopeptidase (EC 3.4.21.26, PEP) inhibitor, compound S 17092, on substance P (SP) and alpha-melanocyte-stimulating hormone (alpha-MSH) metabolism in the rat brain. In vitro experiments revealed that S 17092 inhibits in a dose-dependent manner PEP activity in rat cortical extracts (IC50 = 8.3 nm). In addition, S 17092 totally abolished the degradation of SP and alpha-MSH induced by bacterial PEP. In vivo, a significant decrease in PEP activity was observed in the medulla oblongata after a single oral administration of S 17092 at doses of 10 and 30 mg/kg (-78% and -82%, respectively) and after chronic oral treatment with S 17092 at doses of 10 and 30 mg/kg per day (-75% and -88%, respectively). Concurrently, a single administration of S 17092 (30 mg/kg) caused a significant increase in SP- and alpha-MSH-like immunoreactivity (LI) in the frontal cortex (+41% and +122%, respectively) and hypothalamus (+84% and +49%, respectively). In contrast, chronic treatment with S 17092 did not significantly modify SP- and alpha-MSH-LI in the frontal cortex and hypothalamus. Collectively, the present results show that S 17092 elevates SP and alpha-MSH concentrations in the rat brain by inhibiting PEP activity. These data suggest that the effect of S 17092 on memory impairment can be accounted for, at least in part, by inhibition of catabolism of promnesic neuropeptides such as SP and alpha-MSH.

Several aspects of the central regulation of respiratory control have been investigated on brainstem-spinal cord preparations isolated from newborn rats whose dam was given 0.02% caffeine in water as drinking fluid during the whole period of pregnancy. Analysis of the central respiratory drive estimated by the recording of C4 ventral root activity was correlated to Fos ponto-medullary expression. Under normoxic conditions, preparations obtained from the caffeine-treated group of animals displayed a higher respiratory frequency than observed in the control group (9.2 +/- 0.5 versus 7.2 +/- 0.6 burst/min). A parallel Fos detection tends to indicate that the changes of the respiratory rhythm may be due to a decrease in neuronal activity of medullary structures such as the ventrolateral subdivision of the solitary tract, the area postrema, and the nucleus raphe obscurus. Under hypoxic conditions, the preparations displayed a typical hypoxic respiratory depression associated with changes in the medullary Fos expression pattern. In addition, the hypoxic respiratory depression is clearly emphasized after in utero exposure to caffeine and coincides with an increased Fos expression in the area postrema and nucleus raphe obscurus, two structures in which it is not increased in the absence of caffeine. Taken together, these results support the idea that in utero caffeine exposure could affect central respiratory control.

The ventrolateral cell column of the midbrain periaqueductal gray matter (vl-PAG) plays a major role in the attenuation of pain behaviour. It is established that this effect is exerted via modulation of neuronal activities in the rostral ventromedial medulla (RVM). Until recently it has been generally accepted that the vl-PAG exerts its modulatory effects upon RVM neurons through a direct monosynaptic pathway. However, recent data suggest that an additional indirect, di- or polysynaptic pathway may also exist. Using in vivo intracellular recordings we tested this hypothesis, by studying synaptic responses of somatosensory receptive RVM neurons evoked by electric stimulation of the vl-PAG in rats. RVM neurons were regarded as somatosensory receptive if they responded to electrical stimulation of the sciatic nerve. Most of the recorded RVM cells were excited by vl-PAG stimulation. Some of them responded with a short onset latency (3.6+/-0.9 ms) corresponding to monosynaptic excitation. All of these neurons were also excited by sciatic nerve stimulation at nociceptive intensities. In contrast to this, another proportion of the recorded RVM neurons responded with a four times longer (14.8+/-3 ms) onset latency to the vl-PAG stimulation, corresponding to polysynaptic modulation. All of these neurons were inhibited by sciatic nerve stimulation. The findings show that RVM neurons receive heterogeneous monosynaptic and polysynaptic inputs from the vl-PAG. The results also suggest that the monosynaptic and polysynaptic pathways modulate the activity of functionally distinct groups of RVM neurons.

Metastin, the product of metastasis suppressor gene KiSS-1, is proposed to be the natural ligand for the G-protein-coupled receptor GPR54, known also as AXOR12. This immunohistochemical study, using a rabbit polyclonal antiserum against the human metastin fragment (45-54)-NH(2), showed that in rats metastin-like immunoreactivity (MTS-LI) was present in neurons of the nucleus of the solitary tract and caudoventrolateral reticular nucleus, and in cell processes of the spinal trigeminal tract and lateral reticular nucleus. MTS-LI was confined mainly to neurons and fibers at or caudal to the area postrema. In the spinal cord, MTS-LI cell processes formed a dense plexus in superficial layers I and II of the dorsal horn. The pattern of distribution of MTS-LI in the medulla and spinal cord suggests that this novel peptide may participate in autonomic and sensory neural signaling.

A great variety of alterations have been described in the nervous system of diabetic animals. They are named as diabetic neuropathy and affect the brain, spinal cord and peripheral nerves. In diabetic animals, plasma and tissue catecholamine levels have been reported to be increased, decreased or unchanged, and these disparities have been explained by differences in the tissues selected, severity or duration of diabetes. Dopamine, norepinephrine and epinephrine from different tissues were extracted by absorption onto alumina, and measured by high performance liquid chromatography with electrochemical detection. We found that diabetes alters catecholaminergic systems in a highly specific manner. The dopamine content is reduced in the dopaminergic nigrostriatal system only. Norepinephrine is differently altered in several areas of the sympathetic nervous system. It is increased in cardiac ventricles, and decreased in stellate ganglia and the blood serum. However, it is not altered in the central nervous system. Finally, epinephrine is only altered in the adrenal gland where it is increased, and in the serum where it is reduced. Our results suggest that diabetes reduces the activity of the nigrostriatal dopaminergic system. Changes found at the sympathoadrenal level could be explained by reduced norepinephrine and epinephrine synthesis, with increased storage due to a reduced release from synaptic vesicles.

In lampreys as in other vertebrates, brainstem centres play a key role in the initiation and control of locomotion. One such centre, the mesencephalic locomotor region (MLR), was identified physiologically at the mesopontine border. Descending inputs from the MLR are relayed by reticulospinal neurons in the pons and medulla, but the mechanisms by which this is carried out remain unknown. Because previous studies in higher vertebrates and lampreys described cholinergic cells within the MLR region, we investigated the putative role of cholinergic agonists in the MLR-controlled locomotion. The local application of either acetylcholine or nicotine exerted a direct dose-dependent excitation on reticulospinal neurons as well as induced active or fictive locomotion. It also accelerated ongoing fictive locomotion. Choline acetyltransferase-immunoreactive cells were found in the region identified as the MLR of lampreys and nicotinic antagonists depressed, whereas physostigmine enhanced the compound EPSP evoked in reticulospinal neurons by electrical stimulation of this region. In addition, cholinergic inputs from the MLR to reticulospinal neurons were found to be monosynaptic. When the brainstem was perfused with d-tubocurarine, the induction of swimming by MLR stimulation was depressed, but not prevented, in a semi-intact preparation. Altogether, the results support the hypothesis that cholinergic inputs from the MLR to reticulospinal cells play a substantial role in the initiation and the control of locomotion.

In rat fetuses, neurons generated in the lower rhombic lip on embryonic day 14 (E14) specifically express the transcription factor Pax-6 and migrate circumferentially in the subpial region along axons which are immunoreactive for TAG-1, a neural cell adhesion molecule. These neurons ultimately settle from E17 in the contralateral medulla oblongata to form the precerebellar nuclei, the lateral reticular and external cuneate nuclei. We have examined this migratory process in rat small eye (rSey2) which has a mutation in the Pax-6 gene. Both the migration and settlement of neurons labelled with 5-bromo-2'-deoxyuridine (BrdU) at E14 delayed in the mutant. Furthermore, in the ventrolateral part of the E16 mutant medulla oblongata, cells both expressing Pax-6 mRNA and prelabelled with BrdU at E14 were ectopically localized in the deep zone of the medullary parenchyma in addition to the normal subpial location. These ectopic neurons remained in close contact with ectopic TAG-1-immunoreactive axons. These results indicate that Pax-6 plays a role in the migration of medullary precerebellar neurons, although neurons generated in the lower rhombic lip can nevertheless migrate and settle to form the external cuneate nucleus in the absence of Pax-6.

Metabotropic glutamate receptors (mGluRs) in the medulla oblongata have been suggested to have a functional role in the regulation of cardiovascular baroreflexes. The present study examines the localization of mGluR8 autonomic nuclei of the medulla of the rat. mGluR8 immunoreactivity was observed in the cell bodies and/or processes of the dorsolateral, interstitial, medial, intermediate, ventral, ventrolateral, subpostremal, commissural, parvicellular and gelatinosus subnuclei of the nucleus tractus solitarius (NTS). The intensity of mGluR8 staining was highest in the commissural and interstitial subnuclei at the level of the area postrema. Commissural NTS is involved in regulation of baro-, and chemo-reflexes whereas the interstitial nucleus mediates respiratory reflexes. In the area postrema, diffuse staining was observed in the cell bodies, dendrites and fibers of the dorsal and central regions. In vagal outflow nuclei, mGluR8 immunoreactivity was observed in: (1). the cell bodies and processes of the dorsal motor nucleus of the vagus (DMN) throughout the rostro-caudal extent; and (2). the cell bodies and fibers throughout the rostro-caudal extent of the dorsal and ventral division the nucleus ambiguus (NA). Staining in the ventrolateral medulla was restricted to regions ventral to the nucleus ambiguus and dorsal to the lateral reticulate nucleus. The present study is the first to provide a detailed mapping of mGluR8 within the autonomic nuclei of the medulla and suggests that this subtype may be involved in shaping synaptic transmission in these central nuclei.

Mechanisms involved in the cephalic phase of gastric acid secretion were studied in awake fasted rats with chronic gastric fistula and exposed to the sight and smell of chow for 30 min. Acid secretion was monitored using constant intragastric perfusion and automatic titration. Sham feeding induced a peak acid response reaching 82 +/- 7 micromol/10 min within 20 min compared with the average 22 +/- 2 micromol/10 min in controls. The sham-feeding response was abolished by intracisternal pretreatment with the TRH(1)-receptor antisense oligodeoxynucleotides or subcutaneous injection of atropine, whereas TRH(1) mismatch oligodeoxynucleotides had no effect. Serum gastrin was not altered by the sham feeding and increased by refeeding. Gastrin antibody did not block the rise in acid during sham feeding, although the net acid response was reduced by 47% compared with the control group. Glycine-gastrin antibody, indomethacin and nitro-l-arginine methyl ester had no effect. Atropine and gastrin antibody decreased basal acid secretion by 98 and 75%, respectively, whereas all other pretreatments did not. These results indicate that the cholinergic-dependent acid response to sham feeding is mediated by brain medullary TRH(1) receptors in rats.

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.

Purpose: To evaluate the feasibility of single voxel 1H-MRS of the CNS structures contained in the posterior cranial fossa and to determine the distribution of the normal metabolite ratios, concentrations, and T2 relaxation times in the midbrain, pons, medulla, dentate nucleus and cerebellar vermis., Materials and Methods: A total of 147 single voxel 1H-MR spectra with a point-resolved proton spectroscopy sequence (PRESS) sequence and echo time (TE) of 136 or 272 msec were obtained in the midbrain, pons, medulla, dentate, and vermis of 31 healthy volunteers. In seven additional patients; the concentrations and T2 relaxation times of metabolites were obtained in the same locations (except the medulla) with an external phantom calibration method and a four TE PRESS technique., Results: Ten (27%) of 36 spectra acquired in the medulla were of poor quality. A similar ranking of the N-acetyl aspartate (NAA)/creatine (Cr) ratio and choline(Cho)/Cr ratios in the five locations for the two TEs was observed, with the highest values in the pons (mean NAA/Cr = 4.16 +/- 0.6 and Cho/Cr =2.66 +/- 0.6 at TE 272) and the lowest values in the dentate and vermis (mean NAA/Cr = 1.66 +/- 0.2 and Cho/Cr = 1.20 +/- 0.2 at TE 272). The analysis of variance showed significant regional differences of the NAA and Cr concentrations, which had the highest values in the dentate. Non-significant regional differences were observed for the concentration of Cho and for the T2 of the metabolites., Conclusion: With the exception of the medulla, single voxel 1H-MRS enables an in vivo biochemical analysis of the CNS structures contained in the posterior cranial fossa. Regional differences in the metabolite ratios and concentrations must be considered when employing 1H-MRS for evaluation of diseases of the brainstem and cerebellum., (Copyright 2002 Wiley-Liss, Inc.)

Recent studies have linked fetal exposure to a suboptimal intrauterine environment with adult hypertension. The aims of the present study were to see whether prenatal dexamethasone administered intravenously to the ewe between 26 to 28 days of gestation (1) resulted in high blood pressure in male and female offspring and whether hypertension in males was modulated by testosterone status, and (2) altered gene expression for angiotensinogen and angiotensin type 1 (AT1) receptors in the brain in late gestation and in the adult. Basal mean arterial pressure (MAP) at 2 years of age was significantly higher in wethers exposed to prenatal dexamethasone (group D; 106+/-5 mm Hg, n=9) compared with the control group (group S; 91+/-3 mm Hg, n=8; P<0.01). Infusion of testosterone for 3 weeks had no effect on MAP in either treatment group. At 130 days of gestation, dexamethasone administered between 26 to 28 days of gestation (group DF; n=8), resulted in an increased expression of angiotensinogen in hypothalamus (in arbitrary units: 2.5+/-0.3 versus 1.3+/-0.3 in the saline group [group SF], n=10; P<0.05). In addition, there was higher expression of the AT1 receptors in medulla oblongata in group DF (2.6+/-0.6 versus 1.1+/-0.2 in group SF; P<0.01). This effect of prenatal dexamethasone treatment was still evident in females at 7 years of age (group DA; n=5; 2.6+/-0.5 versus 1.1+/-0.2 in group SA; n=6, P<0.05). In conclusion, brief prenatal exposure of the pregnant ewe to dexamethasone leads to hypertension in adult animals of both sexes. Most interestingly, the mechanism leading to programming of hypertension might be linked with the brain angiotensin system.

Pressor responses elicited by stimulation of the nucleus raphe obscurus (NRO) depend on the integrity of the rostral ventrolateral medulla (RVLM). Therefore, to test the participation of excitatory amino acid (EAA) receptors in the cardiovascular responses evoked by NRO stimulation (1 ms, 100 Hz, 40-70 microA, for 10 s), the EAA antagonist kynurenic acid (Kyn) was microinjected at different sites in the ventrolateral medullar surface (2.7 nmol/200 nl) of male Wistar rats (270-320 g, N = 39) and NRO stimulation was repeated. The effects of NRO stimulation were: hypertension (deltaMAP = +43 +/- 1 mmHg, P<0.01), bradycardia (deltaHR = -30 +/- 7 bpm, P<0.01) and apnea. Bilateral microinjection of Kyn into the RVLM, which did not change baseline parameters, almost abolished the bradycardia induced by NRO stimulation (deltaHR = -61 +/- 3 before vs -2 +/- 3 bpm after Kyn, P<0.01, N = 7). Unilateral microinjection of Kyn into the CVLM did not change baseline parameters or reduce the pressor response to NRO stimulation (deltaMAP = +46 +/- 5 before vs +48 +/- 5 mmHg after Kyn, N = 6). Kyn bilaterally microinjected into the caudal pressor area reduced blood pressure and heart rate and almost abolished the pressor response to NRO stimulation (deltaMAP = +46 +/- 4 mmHg before vs +4 +/- 2 mmHg after Kyn, P<0.01, N = 7). These results indicate that EAA receptors on the medullary ventrolateral surface play a role in the modulation of the cardiovascular responses induced by NRO stimulation, and also suggest that the RVLM participates in the modulation of heart rate responses and that the caudal pressor area modulates the pressor response following NRO stimulation.

The effects on myelin gene expression of prenatal diets with a different lipid content and fatty acid composition were investigated in undernourished pups. Three groups of rats were fed ad libitum during gestation diets containing either 10% of margarine or 10% of microbial lipids or a standard diet containing 3.5% of lipids. After birth, all groups were switched to a reduced intake (60%) of the standard diet. A control group received ad libitum, the standard diet during pregnancy and throughout lactation. At birth no difference in body and brain weight was observed among the groups and the only significant difference in the brain fatty acid composition was the presence of odd-chain fatty acids in the group fed microbial lipids. Milk was removed from the stomach of pups at 1, 5 and 9 days of lactation for fatty acid analysis. During undernourishment, the monoenoic fatty acid and polyenoic fatty acid percentage was always higher and lower, respectively, in the groups fed 10% than in the group fed 3.5% of lipids during pregnancy. The expression of myelin genes at 11 days of undernutrition was determined in different brain regions by Northern analysis. In the standard group, the proteolipid protein and myelin oligodendrocyte glycoprotein transcripts were well detected only in the medulla whereas in groups fed 10% of lipids the transcripts were also visible in the cerebellum. These data suggest that the high lipid content of the prenatal diet independently from its fatty acid composition affects the myelin gene expression decreasing myelin susceptibility to postnatal undernutrition.

Previous investigations from our laboratory have documented that the neuropil of the phrenic nucleus contains a dense accumulation of punctate nicotinamide adenine dinucleotide phosphate diaphorase staining. In this study we investigated the occurrence and origin of punctate nitric oxide synthase immunoreactivity in the neuropil of the phrenic nucleus in C3-C5 segments, supposed to be the terminal field of the premotor bulbospinal respiratory nitric oxide synthase-immunoreactive pathway in the dog. As the first step, nitric oxide synthase immunohistochemistry was used to characterize nitric oxide synthase-immunoreactive staining of the phrenic nucleus and nitric oxide synthase-containing neurons in the dorsal and rostral ventral respiratory group and in the Bötzinger complex of the medulla. Dense punctate nitric oxide synthase immunoreactivity was found on control sections in the neuropil of the phrenic nucleus. Several thin bundles of nitric oxide synthase-immunoreactive fibers were found to enter the phrenic nucleus from the lateral and ventral column. Nitric oxide synthase-containing neurons were revealed in the dorsal respiratory group of medulla corresponding to the ventrolateral nucleus of the solitary tract and in the rostral ventral respiratory group beginning approximately 1 mm caudal to the obex and reaching to 650 microm rostral to the obex. Axotomy-induced retrograde changes, consisting in a strong upregulation of nitric oxide synthase-containing neurons, were found in the dorsal and rostral ventral respiratory group contralateral to the hemisection performed at the C2-C3 level. Concurrently, a strong depletion of the punctate nitric oxide synthase immunopositivity in the neuropil of the phrenic nucleus ipsilaterally with the hemisection was detected, thus revealing that a crossed premotor bulbospinal respiratory pathway contains a fairly high number of nitric oxide synthase-immunopositive fibers terminating in the phrenic nucleus. The use of the retrograde fluorescent tracer Fluorogold injected into the phrenic nucleus and an analysis of sections cut through the dorsal and rostral ventral respiratory group and Bötzinger complex of medulla and processed for nitric oxide synthase immunocytochemistry revealed that approximately 73.8% of crossed premotor bulbospinal respiratory nitric oxide synthase-immunoreactive axons originate in the rostral ventral respiratory group and 26.2% is given by nitric oxide synthase-containing neurons of the dorsal respiratory group. A few premotor nitric oxide synthase-immunoreactive axons originating from the Bötzinger complex were found. In summary, the present study provides evidence for a hitherto unknown premotor bulbospinal respiratory nitric oxide synthase-immunoreactive pathway connecting the bulbar respiratory centers with the motor neurons of the phrenic nucleus in the dog.

Protein kinase C gamma isoform (PKCgamma) is present at high levels in the spinal and medullary dorsal horns and is thought to play a role in the sensitization of dorsal horn neurons in certain pain states. Calbindin-D28k (CB), calretinin (CR) and parvalbumin (PV) are the most commonly expressed calcium-binding proteins and are located abundantly in the medullary dorsal horn (also called the caudal subnucleus of the spinal trigeminal nucleus). In the present study, immunofluorescence histochemical double staining for PKCgamma and CB, CR or PV was performed in the rat medullary dorsal horn. Most of the PKCgamma-, CB-, CR- and PV-immunoreactive neurons were observed in lamina II; some were also encountered in lamina I and lamina III of the medullary dorsal horn. Neurons co-expressing CB/PKCgamma, CR/PKCgamma and PV/PKCgamma were also mainly found in lamina II, while in lamina I and lamina III, only a few neurons co-expressing CB/PKCgamma, CR/PKCgamma and PV/PKCgamma were encountered. The percentages of neurons co-expressing CB/PKCgamma in the total numbers of CB- and PKCgamma-immunoreactive neurons were 6.7 and 5.9%, respectively. Of the total numbers of CR- and PKCgamma-immunoreactive neurons, 5.0 and 5.6%, respectively, showed both CR and PKCgamma immunoreactivities. The percentages of neurons co-expressing PV/PKCgamma in the total numbers of PV- and PKCgamma-immunoreactive neurons were 25.7 and 4.1%, respectively. Most of these neurons co-expressing CB/PKCgamma, CR/PKCgamma and PV/PKCgamma were small (/=36 microm) multipolar neurons were infrequently seen. The present results indicate that there are some neurons co-expressing CB/PKCgamma, CR/PKCgamma and PV/PKCgamma in the medullary dorsal horn. These neurons might play important roles in the nociceptive modulation from the oro-facial region., (Copyright 2002 S. Karger AG, Basel)

Cocaine- and amphetamine-regulated transcript (CART) peptide-immunoreactivity was detected in neurons of the rostral ventrolateral medulla (RVLM), but few in the caudal ventrolateral medulla (CVLM). Double-labeling the medullary sections with sheep polyclonal phenylethanolamine N-methyltransferase-antiserum (PNMT) or monoclonal tyrosine hydroxylase-antibody and rabbit polyclonal CART peptide-antiserum revealed that nearly all adrenergic cells in the C1 area were CART peptide-positive and vice versa; tyrosine hydroxylase-positive cells in the A1 area were not. In the thoracolumbar spinal cord, neurons in the intermediolateral cell column (IML) and other sympathetic autonomic nuclei were CART peptide-positive; some of these were contacted by immunoreactive fibers arising from the lateral funiculus. By immuno-electron microscopy, axon terminals containing closely packed agranular CART peptide-immunoreactive vesicles appeared to make synaptic contacts with immunoreactive dendrites and soma in the IML, albeit the incidence of such contacts was low. Microinjection of the retrograde tracer Fluorogold into the lateral horn area of the T1-T3 spinal segments labeled a population of neurons in the C1 area, many of which were also CART peptide-positive. The results indicate that CART peptide-immunoreactivity is expressed in C1 adrenergic neurons, some of which project to the thoracolumbar spinal cord. The presence of this novel peptide in C1 adrenergic neurons underscores the multiplicity of putative transmitters that may be involved in signaling between putative cardiovascular neurons in the medulla oblongata and sympathetic preganglionic neurons (SPNs) in the spinal cord.

Brain-derived neurotrophic factor (BDNF) is a unique neurotrophin which not only supports the development of neurons but also modulates the synaptic activity in a number of neuronal systems. BDNF is synthesized in neurons, anterogradely transported and released from nerve terminals and exerts acute effects on synaptic transmission in both peripheral and central nervous systems. Previous studies have shown that BDNF is distributed in several groups of neurons in the brain stem which regulate cardiovascular functions. Here we showed that injection of BDNF (40-400 ng/100 nl) into the rostral ventrolateral medulla resulted in a significant increase in arterial blood pressure (Delta35.5+/-3.5 mmHg) in rats. The duration of change in blood pressure was 145+/-40 s with a latency of 3-5 s. There was no significant effect on the heart rate. The injection of glutamate as a positive control also triggered an increase in blood pressure. Injection of phosphate-buffered saline as a control or the same amount of nerve growth factor did not cause significant changes in blood pressure in different preparations. Immunohistochemistry showed that the nerve terminals immunoreactive for BDNF were localized in several brain stem regions and terminate around spinal projection neurons in the rostral ventrolateral medulla. Neurons in the rostral ventrolateral medulla can uptake exogenous BDNF and express the high affinity receptor trkB. From these results we suggest that BNDF in the medulla may play a role in the regulation of blood pressure.

We subcutaneously injected 0.5 mg/kg veratrine into the musk shrew (Suncus murinus), observed the presence or absence, latency, and the incidence of vomiting in each animal for 90 min, and selected animals that frequently vomited (FV group) and those that did not vomit (NV group). Subsequently, animal brains were removed, and the induction of c-fos protein (Fos) was immunohistochemically examined to evaluate neuronal activity in the medulla oblongata. The distribution of Fos-positive neurons in the medulla oblongata was similar between FV and NV groups, with numerous neurons along the entire length of the nucleus of the solitary tract and in the ventrolateral reticular formation. Both veratrine-injected groups showed higher numbers of positive neurons than the saline administered group. However, while the FV group showed a high concentration of positive neurons in the dorsal-dorsomedial reticular formation of the nucleus ambiguus in the rostral medulla, the NV group showed few positive neurons in this area. Fos activity in neurons in this area appeared to be higher in animals with a higher incidence of vomiting.

Alpha-neoendorphin (alpha-NE) is an opiate decapeptide derived from the prodynorphin protein. Its anatomical distribution in the brain of mammals other than the rat, particularly in carnivores, is less well known than for other opiate peptides. In the present work, we have charted the distribution of alpha-NE immunoreactive fibers and perikarya in the diencephalon and the brainstem of the dog. The highest densities of labeled fibers were found in the substantia nigra and in patches within the nucleus of the solitary tract. Moderate densities appeared in the arcuate nucleus (Ar), median eminence, entopeduncular nucleus, ventral tegmental area, retrorubral area, periaqueductal central gray, interpeduncular nucleus and lateral parabrachial nucleus. Groups of numerous labeled perikarya were localized in the magnocellular hypothalamic nuclei, Ar and in the central superior and incertus nuclei in the metencephalon. Moreover, less densely packed fibers and cells appeared widely distributed throughout many nuclei in the region studied. These results are discussed with regard to the pattern described in other species. In addition, the present results were compared with the distribution of met-enkephalin immunoreactivity in the diencephalon and the brainstem of the dog that we have recently described. Although the distributions of these two peptides overlap in many areas, the existence of numerous differences suggest that they form separate opiate systems in the dog.

We evaluated the relationship between the toxicity induced by the organophosphate mevinphos (Mev) and inducible nitric oxide synthase (iNOS) in the rostral ventrolateral medulla (RVLM), the medullary origin of sympathetic neurogenic vasomotor tone. Adult Sprague-Dawley rats that were anesthetized and maintained with propofol were used. Laser scanning confocal microscopic analysis revealed colocalization of the M2 subtype of muscarinic receptors (M(2)R) and iNOS immunoreactivity in RVLM neurons. Comicroinjection bilaterally of Mev (10 nmol) and artificial cerebrospinal fluid (aCSF) into the RVLM elicited a progressive decline in systemic arterial pressure (SAP) and heart rate. This was accompanied during phase 1 Mev intoxication by an increase in the power density of the very high-frequency (VHF; 5-9 Hz), high-frequency (HF; 0.8-2.4 Hz), low-frequency (LF; 0.25- 0.8 Hz) and very low-frequency (VLF; 0-0.25 Hz) components of SAP signals. Phase 2 exhibited a reversal of the VHF and VLF power to control levels and a further reduction in the power density of both HF and LF components to below baseline. Hypotension and bradycardia promoted by Mev were significantly blunted on coadministration into the RVLM of the selective iNOS inhibitors S-methylisothiourea (250 pmol) or aminoguanidine (250 pmol). Not only was the augmented power density of HF and LF components during phase 1 Mev intoxication further enhanced, the reduced power of these two spectral components during phase 2 was appreciably antagonized. On the other hand, the temporal changes in VHF and VLF power were essentially the same as with coadministration of Mev and aCSF. We conclude that, as a cholinesterase inhibitor, Mev may induce toxicity via nitric oxide produced by iNOS on activation of the M(2)R by the accumulated acetylcholine in the RVLM., (Copyright 2001 National Science Council, ROC and S. Karger AG, Basel)

The ionotropic ATP receptor subunits P2X(1-6) receptors play important roles in synaptic transmission, yet the P2X(7) receptor has been reported as absent from neurons in the normal adult brain. Here we use RT-PCR to demonstrate that transcripts for the P2X(7) receptor are present in extracts from the medulla oblongata, spinal cord, and nodose ganglion. Using in situ hybridization mRNA encoding, the P2X(7) receptor was detected in numerous neurons throughout the medulla oblongata and spinal cord. Localizing the P2X(7) receptor protein with immunohistochemistry and electron microscopy revealed that it is targeted to presynaptic terminals in the CNS. Anterograde labeling of vagal afferent terminals before immunohistochemistry confirmed the presence of the receptor in excitatory terminals. Pharmacological activation of the receptor in spinal cord slices by addition of 2'- and 3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP; 30 microm) resulted in glutamate mediated excitation of recorded neurons, blocked by P2X(7) receptor antagonists oxidized ATP (100 microm) and Brilliant Blue G (2 microm). At the neuromuscular junction (NMJ) immunohistochemistry revealed that the P2X(7) receptor was present in motor nerve terminals. Furthermore, motor nerve terminals loaded with the vital dye FM1-43 in isolated NMJ preparations destained after application of BzATP (30 microm). This BzATP evoked destaining is blocked by oxidized ATP (100 microm) and Brilliant Blue G (1 microm). This indicates that activation of the P2X(7) receptor promotes release of vesicular contents from presynaptic terminals. Such a widespread distribution and functional role suggests that the receptor may be involved in the fundamental regulation of synaptic transmission at the presynaptic site.

The pre-Bötzinger complex (PBC), thought to be the center of respiratory rhythm generation, is a cell column ventrolateral to the nucleus ambiguus. The present study analyzed its cellular and neurochemical composition in adult rats. PBC neurons were mainly oval, fusiform, or multipolar in shape and small to medium in size. Neurokinin-1 receptor, a marker of the PBC, was present in the plasma membrane of mostly medium and small neurons and their associated processes and boutons. Among neurons immunoreactive for different neurotransmitter or receptor candidates, various numbers were colocalized with neurokinin-1 receptor. The highest ratio was with nitric oxide synthase (52.72%), and the lowest was with glycine receptors (31.93%). Glutamic acid decarboxylase- and glycine transporter 2-immunoreactive boutons, as well as GABA(A) receptor-immunoreactive plasma membrane processes and boutons, were also identified in the PBC. PBC neurons exhibited different levels of cytochrome oxidase activity, indicating their various energy demands. Our results suggest that synaptic interactions within the PBC of adult rats involve a variety of neurotransmitter and receptor types and that nitric oxide may play an important role in addition to glutamate, GABA, glycine, and neurokinin.

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., (Copyright 2001 Wiley-Liss, Inc.)

Agonists of the mu-opioid receptor (MOR) have profound effects on blood pressure, heart rate, and respiration that may be mediated by C1 adrenergic neurons in the rostral ventrolateral medulla (RVL). C1 neurons are sympathoexcitatory and are involved in both tonic and reflex regulation of sympathetic outflow. This study was designed to determine whether C1 neurons, or their afferents, contain MOR. C1 neurons were identified by using an antibody against the epinephrine synthesizing enzyme phenylethanolamine-N-methyl transferase (PNMT), whereas MOR was localized by using an antipeptide antibody that recognizes the cloned MOR, MOR1. Combined immunoperoxidase and immunogold methods were used to examine the cellular distribution of MOR1 relative to PNMT-containing neurons in the RVL. MOR1 was found in 22% of PNMT-containing dendrites (n = 392), whereas MOR1-containing axons or axon terminals contacted 14% of PNMT-containing dendrites. This distribution was heterogenous with regard to dendritic size: PNMT-labeled dendrites containing MOR1 were usually large (60% were >1.2 microm), whereas PNMT-containing dendrites that received MOR1-labeled afferents were usually small (79% were <1.2 microm). Individual dendrites rarely contained MOR1 at both pre- and postsynaptic sites. Together these results suggest that MOR agonists may directly influence the activity of C1 neurons, as well as the activity of select afferents to these cells. Plasmalemmal membrane labeling for MOR1 was more frequent in smaller PNMT-containing dendrites, suggesting that postsynaptic receptors are more readily available for ligand binding in small dendrites, although the receptor was more frequently detected in larger PNMT dendrites. The selective distribution of MORs to specific pre- and postsynaptic sites suggests the receptor may be selectively trafficked to positions where it may regulate afferent activity that is heterogeneously distributed along the dendritic tree of C1 neurons., (Copyright 2001 Wiley-Liss, Inc.)

It is known that 5-HT receptors have significant roles in nociceptive and motor functions. We have compared the cellular localization of the mRNAs encoding serotonin 5-HT(2A,) 5-HT(2C,) 5-HT(3) receptor subtypes within different levels of the rat spinal cord and medulla. In the spinal cord, 5-HT(2C) receptor mRNA is expressed at high levels in most of the gray matter, except for lamina II. In contrast, 5-HT(2A) receptor mRNA is expressed exclusively in lamina IX. 5-HT(3) receptor mRNA has a low level and diffuse pattern of expression increasing towards the ventral horn. In both gray and white matter, there is a characteristic presence of a few highly stained cells. For each subtype, the expression pattern is similar in all four levels of the spinal cord. In the medulla, 5-HT(2C) receptor mRNA is at high levels in many nuclei including the hypoglossal nucleus, the gigantocellular reticular nucleus alpha and the parvocellular reticular nucleus alpha, the spinal nucleus of the trigeminal tract, the facial, and the dorsal medullary reticular field. Moderate to low levels of expression are seen in the spinal vestibular nucleus, the vagus, the solitary nuclei and the raphe. 5-HT(2A) receptor is expressed at high levels in some nuclei such as the hypoglossal nucleus, the intercalate nucleus, the inferior olive and the lateral reticular nucleus. Moderate to low levels of expression are seen in the facial, the medial vestibular nuclei, the nucleus ambiguous, the vagus, and the gigantocellular reticular nucleus. 5-HT(3) receptor mRNA is present at low levels in most of the nuclei examined, with a few scattered strongly labeled cells. The results show a distinct distribution of the three subtypes of receptors supporting their physiological roles and will help to understand the mechanisms of nociception and motor function.

Here we examine hypothesis that short-term peripheral ZnSO(4)-induced anosmia can produce effects on c-fos expression within spinal cord and caudal medulla in male Wistar rats (n=4). Fos-like-immunoreactive cells revealed by avidin-biotin-peroxidase method show a significant bilateral increase in the nucleus proprius (layers 3 and 4) and medial part of layers 5 and 6. In substantia gelatinosa (layer 2(i)) and area 10 Fos-positive neurons were intermixed together with nicotin-amide adenine dineucleotide phosphate-diaphorase (NADPH-d)-reactive cells. Short-term anosmia enhanced c-fos expression in ventral horn (layers 7 and 8), ventrolateral segment and dorsal part of the spinal trigeminal nuclei. In anosmic rats varicose fibres and numerous NADPH-d-stained neurons were present in the gelatinous layer of the spinal trigeminal nucleus caudalis, and a separate population of Fos-positive cells was detected within this layer. Nucleus tractus solitaris also contained a few NADPH-d-reactive, medium sized neurons intermixed with Fos-immunoreactive cells.