Fluorescently-labeled fremanezumab is distributed to sensory and autonomic ganglia and the dura but not to the brain of rats with uncompromised blood brain barrier.

BACKGROUND: The presence of calcitonin gene-related peptide and its receptors in multiple brain areas and peripheral tissues previously implicated in migraine initiation and its many associated symptoms raises the possibility that humanized monoclonal anti-calcitonin gene-related peptide antibodies (CGRP-mAbs) can prevent migraine by modulating neuronal behavior inside and outside the brain. Critical to our ability to conduct a fair discussion over the mechanisms of action of CGRP-mAbs in migraine prevention is data generation that determines which of the many possible peripheral and central sites are accessible to these antibodies - a question raised frequently due to their large size. MATERIAL AND METHODS: Rats with uncompromised and compromised blood-brain barrier (BBB) were injected with Alexa Fluor 594-conjugated fremanezumab (Frema594), sacrificed 4 h or 7 d later, and relevant tissues were examined for the presence of Frema594. RESULTS: In rats with uncompromised BBB, Frema594 was similarly observed at 4 h and 7 d in the dura, dural blood vessels, trigeminal ganglion, C2 dorsal root ganglion, the parasympathetic sphenopalatine ganglion and the sympathetic superior cervical ganglion but not in the spinal trigeminal nucleus, thalamus, hypothalamus or cortex. In rats with compromised BBB, Frema594 was detected in the cortex (100 µm surrounding the compromised BBB site) 4 h but not 7 d after injections. DISCUSSION: Our inability to detect fluorescent (CGRP-mAbs) in the brain supports the conclusion that CGRP-mAbs prevent the headache phase of migraine by acting mostly, if not exclusively, outside the brain as the amount of CGRP-mAbs that enters the brain (if any) is too small to be physiologically meaningful.

Innervation of taste buds revealed with Brainbow-labeling in mouse.

Nerve fibers that surround and innervate the taste bud were visualized with inherent fluorescence using Brainbow transgenic mice that were generated by mating the founder line L with nestin-cre mice. Multicolor fluorescence revealed perigemmal fibers as branched within the non-taste epithelium and ending in clusters of multiple rounded swellings surrounding the taste pore. Brainbow-labeling also revealed the morphology and branching pattern of single intragemmal fibers. These taste bud fibers frequently innervated both the peripheral bud, where immature gemmal cells are located, and the central bud, where mature, differentiated cells are located. The fibers typically bore preterminal and terminal swellings, growth cones with filopodia, swellings, and rounded retraction bulbs. These results establish an anatomical substrate for taste nerve fibers to contact and remodel among receptor cells at all stages of their differentiation, an interpretation that was supported by staining with GAP-43, a marker for growing fibers and growth cones.

Proteomic Profiling of Cranial (Superior) Cervical Ganglia Reveals Beta-Amyloid and Ubiquitin Proteasome System Perturbations in an Equine Multiple System Neuropathy.

Equine grass sickness (EGS) is an acute, predominantly fatal, multiple system neuropathy of grazing horses with reported incidence rates of ∼2%. An apparently identical disease occurs in multiple species, including but not limited to cats, dogs, and rabbits. Although the precise etiology remains unclear, ultrastructural findings have suggested that the primary lesion lies in the glycoprotein biosynthetic pathway of specific neuronal populations. The goal of this study was therefore to identify the molecular processes underpinning neurodegeneration in EGS. Here, we use a bottom-up approach beginning with the application of modern proteomic tools to the analysis of cranial (superior) cervical ganglion (CCG, a consistently affected tissue) from EGS-affected patients and appropriate control cases postmortem. In what appears to be the proteomic application of modern proteomic tools to equine neuronal tissues and/or to an inherent neurodegenerative disease of large animals (not a model of human disease), we identified 2,311 proteins in CCG extracts, with 320 proteins increased and 186 decreased by greater than 20% relative to controls. Further examination of selected proteomic candidates by quantitative fluorescent Western blotting (QFWB) and subcellular expression profiling by immunohistochemistry highlighted a previously unreported dysregulation in proteins commonly associated with protein misfolding/aggregation responses seen in a myriad of human neurodegenerative conditions, including but not limited to amyloid precursor protein (APP), microtubule associated protein (Tau), and multiple components of the ubiquitin proteasome system (UPS). Differentially expressed proteins eligible for in silico pathway analysis clustered predominantly into the following biofunctions: (1) diseases and disorders, including; neurological disease and skeletal and muscular disorders and (2) molecular and cellular functions, including cellular assembly and organization, cell-to-cell signaling and interaction (including epinephrine, dopamine, and adrenergic signaling and receptor function), and small molecule biochemistry. Interestingly, while the biofunctions identified in this study may represent pathways underpinning EGS-induced neurodegeneration, this is also the first demonstration of potential molecular conservation (including previously unreported dysregulation of the UPS and APP) spanning the degenerative cascades from an apparently unrelated condition of large animals, to small animal models with altered neuronal vulnerability, and human neurological conditions. Importantly, this study highlights the feasibility and benefits of applying modern proteomic techniques to veterinary investigations of neurodegenerative processes in diseases of large animals.

[Inhibitory effect of benzimidazole derivatives on cholinesterases of animals in the presence of different substrates].

Specifically synthesized group of benzimidazole derivatives possessing varying degrees of delocalization of the positive charge in the cation group of the molecule has been studied in order to search for potential cholinergically active compounds and to study the role of the Coulomb interaction in cholinesterase catalysis. These compounds were reversible inhibitors of cholinesterase (ChE) of human erythrocytes, horse serum, brain of the frog Rana temporaria and visual ganglia of the Pacific squid Todarodes pacificus in the presence of acetylthiocholine iodide and propionylthiocholine iodide as substrates. The differences in the nature of reversible inhibitory effect were observed. The effect of the inhibitor structure and substrate nature, specific for each of the studied inhibitors, on the character of the process of reversible inhibition was found.

Immunohistochemical detection of intra-neuronal VZV proteins in snap-frozen human ganglia is confounded by antibodies directed against blood group A1-associated antigens.

Varicella-zoster virus (VZV) causes chickenpox, establishes latency in trigeminal (TG) and dorsal root ganglia (DRG), and can lead to herpes zoster upon reactivation. The VZV proteome expressed during latency remains ill-defined, and previous studies have shown discordant data on the spectrum and expression pattern of VZV proteins and transcripts in latently infected human ganglia. Recently, Zerboni and colleagues have provided new insight into this discrepancy (Zerboni et al. in J Virol 86:578-583, 2012). They showed that VZV-specific ascites-derived monoclonal antibody (mAb) preparations contain endogenous antibodies directed against blood group A1 proteins, resulting in false-positive intra-neuronal VZV staining in formalin-fixed human DRG. The aim of the present study was to confirm and extend this phenomenon to snap-frozen TG (n=30) and DRG (n=9) specimens of blood group genotyped donors (n=30). The number of immunohistochemically stained neurons was higher with mAb directed to immediate early protein 62 (IE62) compared with IE63. The IE63 mAb-positive neurons always co-stained for IE62 but not vice versa. The mAb staining was confined to distinct large intra-neuronal vacuoles and restricted to A1(POS) donors. Anti-VZV mAb staining in neurons, but not in VZV-infected cell monolayers, was obliterated after mAb adsorption against blood group A1 erythrocytes. The data presented demonstrate that neuronal VZV protein expression detected by ascites-derived mAb in snap-frozen TG and DRG of blood group A1(POS) donors can be misinterpreted due to the presence of endogenous antibodies directed against blood group A1-associated antigens present in ascites-derived VZV-specific mAb preparations.

Expression of trace amine-associated receptors in the Grueneberg ganglion.

The Grueneberg ganglion (GG) in the vestibule of the anterior nasal cavity is considered as an olfactory subcompartment based on expression of the olfactory marker protein (OMP) and axonal projection to the olfactory bulb. Searching for olfactory receptors present in the GG, it has been observed recently that V2r83, a member of the V2R class of olfactory receptors, is expressed in numerous cells in the GG of mice. However, no other olfactory receptors have been found to be present in a considerable number of GG neurons so far. Here, we report that GG neurons express trace amine-associated receptors (TAARs) that have most recently been described as a novel class of olfactory receptors. It was observed that several TAAR subtypes are expressed by defined subpopulations of GG neurons distinct from the V2r83-positive cells. Analyzing the time course of TAAR expression during pre- and postnatal development revealed that TAARs are expressed by a substantial portion of GG neurons in late embryonic and neonatal stages, whereas in juveniles and adults, the number of TAAR-positive cells in the GG was significantly decreased.

Morphological evidence of sensory neurons in the root of the rat tongue.

In our previous studies, a large number of substance P (SP)-immunoreactive (IR) nerve fibers were detected in the rat tongue and their number increased after inflammation, suggesting that these fibers might be involved in the axon reflex. Therefore, in this study, we have examined the different neuropeptide-containing nerve elements by light, electron, and confocal laser microscopy. SP, vasoactive intestinal polypeptide (VIP), and neuropeptide Y (NPY) IR varicose fibers were numerous compared with other ones. Small groups of ganglia with perikarya IR for SP, VIP, NPY, galanin, and somatostatin were observed. The SP-IR nerve cell bodies were mainly located in the tunica propria just below the epithelial lining. Double-labeling immunohistochemistry showed that the intrinsic SP-IR neurons did not colocalize VIP. The SP containing nerve terminals were observed in and below the epithelium as well as in very close contact to or making real synapses with other neurons in the intralingual ganglion. Our data confirmed the possibility of intrinsic sensory neurons, which might be the afferent branch of the intralingual reflex arch, while the VIP- and NPY-IR neurons located in the salivary glands, around the blood vessels, and in the muscle layer might constitute the efferent site of this reflex.

RhoA, RhoB, RhoC, Rac1, Cdc42, and Tc10 mRNA levels in spinal cord, sensory ganglia, and corticospinal tract neurons and long-lasting specific changes following spinal cord injury.

Inhibition of RhoA has been shown to enhance axonal regeneration following spinal cord injury. Here we mapped mRNA expression patterns of RhoA, B, and C, Rac1, Cdc42, and Tc10 in spinal cord, sensory ganglia, and sensorimotor cortex in uninjured rats, and following spinal cord injury or sham laminectomy. In the intact spinal cord, neurons displayed high levels of Rac1, Cdc42, and Tc10 mRNA hybridization signal. GFAP-immunoreactive astrocytes expressed primarily RhoB and Rac1, while oligodendrocyte-like cells expressed RhoA, Rac1, and Cdc42. Injury caused profound, long-lasting upregulation of RhoA, Rac1, Cdc42, and Tc10 mRNA in the spinal cord, while RhoB was modestly increased and RhoC did not change. GFAP-immunoreactive reactive astrocytes exhibited a dramatic increase of RhoA mRNA expression along with increases of Rac1 and Cdc42. Injury also led to elevation of RhoA, Cdc42, and Tc10 in neurons and modest increases of RhoA, Rac1, and Tc10 in oligodendrocyte-like cells. Laminectomy caused similar, but less pronounced alterations of investigated mRNA species. In dorsal root ganglia neuronal RhoA, Rac1, Cdc42, and Tc10 mRNA levels were increased similarly by spinal cord injury and sham surgery. The CST pyramidal cells expressed Tc10 mRNA and the CST itself was Tc10-immunoreactive. Tc10-immunoreactivity disappeared distal to injury. We conclude that there are gene-specific patterns of expression of the six different Rho-GTPases in normal spinal cord and dorsal root ganglia, and that specific changes of temporal and spatial expression patterns occur in response to spinal cord injury, suggesting different roles of these GTPases in the cellular sequelae of CNS injury.

PML bodies in reactive sensory ganglion neurons of the Guillain-Barré syndrome.

Acute inflammatory demyelinating polyneuropathy (AIDP) is a type of Guillain-Barré syndrome (GBS) characterized by primary nerve demyelination sometimes with secondary axonal degeneration. Studies on the fine structure of dorsal root ganglia in AIDP are lacking. Our aim was to investigate the cytology and nuclear organization of primary sensory neurons in AIDP with axonal injury using ultrastructural and immunohistochemical analysis. The light cytology of the L5 dorsal ganglion showed the characteristic findings of neuronal axonal reaction. The organization of chromatin, nucleolus, Cajal bodies, and nuclear pores corresponded to transcriptionally active neurons. However, the hallmark of the nuclear response to axonal injury was the formation of numerous nuclear bodies (NBs; 6.37 +/- 0.6, in the AIDP, vs. 2.53 +/- 0.2, in the control, mean +/- SDM), identified as promyelocytic leukemia (PML) bodies by the presence of the protein PML. In addition to PML protein, nuclear bodies contained SUMO-1 and the transcriptional regulators CREB-binding protein (CBP) and glucocorticoid receptor (GR). The presence of proteasome 19S was also detected in some nuclear bodies. We suggest that neuronal PML bodies could regulate the nuclear concentration of active proteins, a process mediated by protein interactions with PML and SUMO-1 proteins. In the AIDP case, the proliferation of PML bodies may result from the overexpression of some nuclear proteins due to changes in gene expression associated with axonal injury.

Nerve plexuses in the trachea and extrapulmonary bronchi of the rat.

Intrinsic nerve plexuses of the rat trachea and extrapulmonary bronchi were examined by immunohistochemistry. Three nerve plexuses--peritracheal and peribronchial, intramuscular, and submucosal--were found in the wall of the trachea and bronchi. Nerve cell bodies were located in the peritracheal and peribronchial nerve plexuses. They occurred singly or formed ganglia in the plexus, and regional differences in cell numbers were found in the cervical and thoracic portions of the trachea and in the extrapulmonary bronchia. In total, 83.5 +/- 28.3 ganglia (mean +/- SD, 57-131, n=5) and 749.8 +/- 221.1 nerve cell bodies (540-1,080, n=5) were found in the nerve plexus. The mean densities of ganglia were 0.31, 0.97 and 1.15/mm2, and the mean densities of the nerve cell bodies were 1.82, 9.26 and 11.54/mm2 in the cervical region, thoracic region of trachea, and extrapulmonary bronchi, respectively. Almost all nerve cell bodies in ganglia were positive for choline acetyltransferase and neuropeptide Y (NPY), and a few cells were positive for vasoactive intestinal peptide (VIP). In addition, in cholinergic nerves, a few nerve fibers in the smooth muscles were positive for substance P (SP), calcitonin gene-related peptide (CGRP), and VIP, and a moderate number of fibers were positive for NPY. Tyrosine hydroxylase-immunoreactive nerve fibers were observed around blood vessels and within nerve bundles in the tunica adventitia. In the epithelium, nerve fibers were positive for SP and CGRP. Our results indicate that postganglionic neurons form three layers of cholinergic plexuses in the rat trachea and extrapulmonary bronchi, and that all of these possess intrinsic and extrinsic peptidergic innervation.

Ca2+/calmodulin-dependent protein kinase II in the rat cranial sensory ganglia.

Immunohistochemistry for Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) was performed on the rat cranial sensory ganglia. More than one half of neurons was immunoreactive for the enzyme in the trigeminal (60%), jugular (70%), petrosal (55%) and nodose ganglia (63%). These neurons were mainly small to medium-sized. The co-expression study demonstrated that one half of CaMKII-immunoreactive (ir) neurons was also immunoreactive for calcitonin gene-related peptide (CGRP) or the vanilloid receptor subtype 1 (VR1) in the trigeminal, jugular and petrosal ganglia. In the nodose ganglion, CaMKII-ir neurons were mostly devoid of CGRP-immunoreactivity (ir) (8.2%) whereas the co-expression with VR1-ir was common among such neurons (72%). In the facial skin, nasal mucosa and palate, the epithelium and taste bud were innervated by CaMKII-ir nerve fibers. In addition, the retrograde tracing study demonstrated that 39.6% and 44.8% of trigeminal neurons which were retrogradely traced with fluorogold from the facial skin and nasal mucosa exhibited CaMKII-ir. Forty-six percent of petrosal neurons which innervated the soft palate were immunoreactive for the enzyme.

Angiotensin II subtype 1A (AT1A) receptors in the rat sensory vagal complex: subcellular localization and association with endogenous angiotensin.

Angiotensin II (Ang II) type 1 (AT1) receptors are prevalent in the sensory vagal complex including the nucleus tractus solitarii (NTS) and area postrema, each of which has been implicated in the central cardiovascular effects produced by Ang II. In rodents, these actions prominently involve the AT1A receptor. Thus, we examined the electron microscopic dual immunolabeling of antisera recognizing the AT1A receptor and Ang II to determine interactive sites in the sensory vagal complex of rat brain. In both the area postrema and adjacent dorsomedial NTS, many somatodendritic profiles were dually labeled for the AT1A receptor and Ang II. In these profiles, AT1A receptor-immunoreactivity was often seen in the cytoplasm beneath labeled portions of the plasma membrane and in endosome-like granules as well as Golgi lamellae and outer nuclear membranes. In addition, AT1A receptor labeling was detected on the plasma membrane and in association with cytoplasmic membranes in many small axons and axon terminals. These terminals were morphologically heterogeneous containing multiple types of vesicles and forming either inhibitory- or excitatory-type synapses. In the area postrema, AT1A receptor labeling also was detected in many non-neuronal cells including glia, capillary endothelial cells and perivascular fibroblasts that were less prevalent in the NTS. We conclude that in the rat sensory vagal complex, AT1A receptors are strategically positioned for involvement in modulation of the postsynaptic excitability and intracrine hormone-like effects of Ang II. In addition, these receptors have distributions consistent with diverse roles in regulation of transmitter release, regional blood flow and/or vascular permeability.

The co-expression of VR1 and VRL-1 in the rat vagal sensory ganglia.

Immunohistochemistry for two nociceptive transducers, the vanilloid receptor 1 (VR1) and vanilloid receptor 1-like receptor (VRL-1), was performed on the vagal sensory ganglia. In the jugular ganglion, VR1-immunoreactive (IR) neurons were small to medium-sized (range 49.7-1,125.6 microm(2), mean+/-S.D. 407.7+/-219.7 microm(2)), whereas VRL-1-IR neurons were medium-sized to large (range 223.6-1,341.1 microm(2), mean+/-S.D. 584.3+/-253.5 microm(2)). In the nodose ganglion, VR1- and VRL-1-IR neurons were mostly small to medium-sized (VR1: range 148.5-1464.4 microm(2), mean+/-S.D. 554.3+/-207.4 microm(2); VRL-1: range 161.7-1166.2 microm(2), mean+/-S.D. 541.9+/-186.2 microm(2)). The double immunofluorescence method revealed that co-expression of VR1-immunoreactivity among VRL-1-IR neurons was more abundant in the nodose ganglion (63%) than in the jugular ganglion (4%). The present study suggests that co-expression of VR1 and VRL-1 may be more common in visceral sensory neurons than in somatic sensory neurons.

4,5-diaminofluoroscein imaging of nitric oxide synthesis in crayfish terminal ganglia.

We have analyzed the synthesis of nitric oxide in the terminal abdominal ganglion of the crayfish using the fluorescent probe 4,5-Diaminofluoroscein diacetate, DAF-2 DA. Following DAF-2 loading, ganglia showed cell-specific patterns of fluorescence in which the occurrence of strongly fluorescent cell bodies was highest in specific anterior, central, and posterior regions. We found that preincubation with the nitric oxide synthase (NOS) inhibitor L-NAME prevented much of the initial development of DAF-2 fluorescence, whereas the inactive isomer D-NAME had no effect. Washout of preincubated L-NAME caused increased cell-specific fluorescence due to endogenous NOS activity. Application of the NOS substrate L-arginine also resulted in an increase of DAF-2 fluorescence in a cell-specific manner. We bath applied the NO donor SNAP to increase exogenous NO levels which resulted in DAF-2 fluorescence increases in most cells. We therefore presume that the cell-specific pattern of DAF-2 fluorescence indicates the distribution of neurones actively synthesizing NO. The similarity between the DAF-2 staining pattern and previously published studies of NOS activity are discussed.

Intestinal inflammation and activation of sensory nerve pathways: a functional and morphological study in the nematode infected rat.

BACKGROUND: In the rat, gastric distension elicits an intensity dependent pseudoaffective bradycardia mediated via capsaicin sensitive afferent and cholinergic efferent vagal pathways. Inflammation alters visceral perception although the mediators responsible have not been identified. In the nematode infected rat, there is a substantial increase in neuronal substance P (SP) content of the gut. AIMS: To examine the effects of inflammation on perception of a noxious visceral stimulus and on SP and neurokinin 1 (NK-1) receptor immunoreactivity (IR) in visceral afferent pathways. METHODS: Immunohistochemistry was performed on sections from the jejunum, dorsal root ganglia (DRG), and spinal cord (T1-L1) using SP and NK-1 rabbit polyclonal antibodies. In the DRG, the number of SP-IR or NK-1-IR neurones per section was visually quantified. The pseudoaffective cardiac reflex response to gastric stimulation was compared in control and Trichinella spiralis infected rats. RESULTS: Intestinal inflammation induced a rightward shift in the intensity dependent bradycardic response to gastric distension. This was associated with a marked increase in SP-IR not only in the gut wall but also in the DRG and dorsal horn of the spine. In contrast, NK-1-IR was not increased in the gut wall. Moreover, inflammation evoked a decrease in NK-1-IR in the dorsal horn. No NK-1-IR was identified in the DRG of either control or infected animals. CONCLUSIONS: Intestinal inflammation modulates the capsaicin sensitive pseudoaffective autonomic response to gastric distension, increases SP-IR in afferent pathways, and downregulates dorsal horn NK-1-IR. As the pseudoaffective response is capsaicin sensitive, the rightward shift of the response is likely the consequence of the decrease in NK-1 receptors in the sensory pathways.

A comparative study of three cranial sensory ganglia projecting into the oral cavity: in situ hybridization analyses of neurotrophin receptors and thermosensitive cation channels.

Peripheral cranial sensory nerves projecting into the oral cavity receive food intake stimuli and transmit sensory signals to the central nervous system. To describe and compare the features of the cranial sensory ganglia that innervate the oral cavity, i.e., the trigeminal, petrosal, and geniculate ganglia (TG, PG, and GG, respectively), in situ hybridization was conducted using riboprobes for neurotrophin receptors (TrkA, TrkB, and TrkC), a neurotransmitter (substance P), and ion channels important for thermosensation (VR1 and TREK-1). In TG, all in six probes yielded positive signals to various extent in intensity and frequency. In addition, a strong correlation between the expression of VR1 and those of TrkA and substance P was observed as in the case of the dorsal root ganglia. In PG, positive signals to all six probes were also detected, and the correlation of expression was similar to that shown by TG. On the other hand, most cells in GG were positive to the TrkB probe, and a small number of cells were positive to the TrkC probe, but no significant signal was observed for the other four probes. These results indicate that TG and PG consist of cells that are heterogeneous in terms of neurotrophin requirement and somatosensory functions, and that GG seems to consist mainly of a homogeneous cell type, gustatory neurons. In conclusion, TG, PG, and GG, show gene expression characteristics intrinsic to the three ganglia. It is also concluded that TG and a portion of PG project several types of somatosensory nerves. This is consistent with the finding that GG and a portion of PG project gustatory nerves.

The role of basic fibroblast growth factor in peripheral nerve regeneration.

In the peripheral nervous system regeneration and gradual functional restoration occur following peripheral nerve injury. Growth of regenerating axons depends on the presence of diffusible neurotrophic factors, in addition to the substratum. Neurotrophic factors that are involved in peripheral nerve regeneration include nerve growth factor, brain-derived neurotrophic factor, ciliary neurotrophic factor, glial cell line-derived neurotrophic factor, and interleukin-6. Recent functional and expression studies of basic fibroblast growth factor and its receptors have emphasized a physiological role of these molecules in the peripheral nervous system. Basic fibroblast growth factor and its receptors are constitutively expressed in dorsal root ganglia and the peripheral nerve. These molecules display an upregulation in dorsal root ganglia and in the proximal and distal nerve stumps following peripheral nerve injury. In the ganglia these molecules show a mainly neuronal expression, whereas at the lesion site of the nerve, Schwann cells and invading macrophages represent the main cellular sources of basic fibroblast growth factor and the receptors 1-3. Exogenously applied basic fibroblast growth factor mediates rescue effects on injured sensory neurons and supports neurite outgrowth of transectioned nerves. Regarding the expression patterm and the effects after exogenous administration of basic fibroblast growth factor, this molecule seems to play a physiological role during nerve regeneration. Thus, basic fibroblast growth factor could be a promising candidate to contribute to the development of new therapeutic strategies for the treatment of peripheral nerve injuries.

Calbindin-D28k in cerebrovascular extrinsic innervation system of the rat.

Calbindin-D28k, one of the calcium-binding proteins, belongs to the EF hand family and is commonly found in neurons. It serves as a representative neuronal marker for neuroanatomical investigations. The authors' knowledge of its precise function, however, is yet very limited. In this study, we examined the existence of nerve fibers with calbindin-D28k immunoreactivity in the cerebral blood vessels and ganglia that innervate the cerebral blood vessels in the rat. Numerous nerve fibers with calbindin-D28k immunoreactivity were observed on the walls of the major extracerebral arteries forming the circle of Willis and its branches. Calbindin-D28k immunoreactivity was seen in many neurons of the trigeminal, dorsal root and jugular ganglia. A small number of neurons showed calbindin-D28k immunoreactivity in the otic and superior cervical ganglia. Calbindin-D28k immunoreactivity was not detected in the sphenopalatine or internal carotid ganglia. Pericellular basket-like formations of nerve terminals with calbindin-D28k immunoreactivity were observed in the sphenopalatine, otic, internal carotid and superior cervical ganglia. The present study demonstrated calbindin-D28k immunoreactivity in the cerebrovascular nerve fibers as well as in their origins--the cranial ganglia. These findings are significant in understanding the calcium-mediated mechanism of the neural control of the cerebral blood vessels.

5-HT5a receptors in the carotid body chemoreception pathway of rat.

By using a specific antibody, 5-HT5a receptor-like immunoreactivity was revealed in the chemoreceptive, oxygen sensitive, carotid body (CB) type I cells, and neurons of the petrosal ganglion (PG) and the superior cervical ganglion (SCG) in rat. mRNA encoding for the 5-HTa receptor was also detected in these tissues by RT-PCR, and confirmed with DNA sequencing. The present study provides direct evidence that 5-HT5a receptors are expressed in the CB, PG and SCG, which all likely play fundamental roles in arterial chemoreception.