Selective association of nerve fibres immunoreactive for substance P or bombesin with putative cholinergic neurons of the male rat major pelvic ganglion

The male rat major pelvic ganglion contains both sympathetic and parasympathetic neurons that supply the lower urinary and digestive tracts, and the reproductive organs. The aim of this study was to describe the distribution and identify potential targets of sensory and intestinofugal axons in this ganglion. Two putative markers of these projections were chosen, substance P for primary sensory axons and bombesin for myenteric intestinofugal projections. Varicose substance P-immunoreactive axons were associated only with non-noradrenergic (putative cholinergic) somata, and most commonly with those that contained vasoactive intestinal peptide. Immunoreactivity for substance P was also present in a small group of non-noradrenergic somata, many of which were immunoreactive for enkephalins, neuropeptide Y or vasoactive intestinal peptide. Bombesin immunoreactivity was found only in preterminal and terminal (varicose) axons, the latter of which were exclusively associated with non-noradrenergic somata that contain neuropeptide Y-immunoreactivity. Some varicose axons containing either substance P-or bombesin-immunoreactivity were intermingled with clumps of small, intensely fluorescent cells. These studies indicate that substance P-and bombesin-immunoreactive axons are likely to connect with numerically small, but discrete, populations of pelvic neurons.     

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.     

Biondi bodies in the choroid plexus epithelium of the human brain

Summary Scanning electron microscopy (SEM) was used to examine choroid plexuses in the brain of two human adults aged 44 and 46, respectively, and 12 older subjects from 67 to 98 years of age. It was possible to obtain a three-dimensional view of the ring-like structures (Biondi bodies) located in the cytoplasm of choroid plexus epithelial cells in the older-age group. The filaments forming the rings were clearly visible. No such structures were found between epithelial cells. The intracellular location of the Biondi bodies and their state of preservation compared to other cytoplasmic elements suggest that they may have a destructive effect on epithelial cells of choroid plexuses. The same material was examined by transmission electron microscopy (TEM); the results obtained were in full agreement with the evidence obtained with SEM.     

Sequential expression of Efhc1/myoclonin1 in choroid plexus and ependymal cell cilia

EFHC1 is a gene mutated in patients with idiopathic epilepsies, and encodes the myoclonin1 protein. We here report the distribution of myoclonin1 in mouse. Immunohistochemical analyses revealed that the myoclonin1 first appeared at the roof of hindbrain at embryonic day 10 (E10), and moved on to choroid plexus at E14. At E18, it moved to ventricle walls and disappeared from choroid plexus. From neonatal to adult stages, myoclonin1 was concentrated in the cilia of ependymal cells at ventricle walls. At adult stages, myoclonin1 expression was also observed at tracheal epithelial cilia in lung and at sperm flagella in testis. Specificities of these immunohistochemical signals were verified by using Efhc1-deficient mice as negative controls. Results of Efhc1 mRNA in situ hybridization were also consistent with the immunohistochemical observations. Our findings raise “choroid plexusopathy” or “ciliopathy” as intriguing candidate cascades for the molecular pathology of epilepsies caused by the EFHC1 mutations.     

Characterization of folate uptake by choroid plexus epithelial cells in a rat primary culture model

Reduced derivatives of folic acid (folates) play a critical role in the development, function and repair of the CNS. However, the molecular systems regulating folate uptake and homeostasis in the central nervous system remain incompletely defined. Choroid plexus epithelial cells express high levels of folate receptor α (FRα) suggesting that the choroid plays an important role in CNS folate trafficking and maintenance of CSF folate levels. We have characterized 5-methyltetrahydrofolate (5-MTHF) uptake and metabolism by primary rat choroid plexus epithelial cells in vitro . Two distinct processes are apparent; one that is FRα dependent and one that is independent of the receptor. FRα binds 5-MTHF with high affinity and facilitates efficient uptake of 5-MTHF at low extracellular folate concentrations; a lower affinity FRα independent system accounts for increased folate uptake at higher concentrations. Cellular metabolism of 5-MTHF depends on the route of folate entry into the cell. 5-MTHF taken up via a non-FRα -mediated process is rapidly metabolized to folylpolyglutamates, whereas 5-MTHF that accumulates via FRα remains non-metabolized, supporting the hypothesis that FRα may be part of a pathway for transcellular movement of the vitamin. The proton-coupled folate transporter, proton-coupled folate transporter (PCFT), mRNA was also shown to be expressed in choroid plexus epithelial cells. This is consistent with the role we have proposed for proton-coupled folate transporter in FRα-mediated transport as the mechanism of export of folates from the endocytic compartment containing FRα.     

Cerebral perfusion pressure in giraffe: modelling the effects of head-raising and -lowering

Loss of consciousness caused by positional changes of the head results from reduced cerebral blood flow (CBF). CBF is related to cerebral perfusion pressure (CPP). CPP is the difference between mean arterial pressure (MAP) at the head and intracranial pressure (ICP). The positional change of the giraffe head between ground level and standing upright is the largest of all animals yet loss of consciousness does not occur. We have investigated the possibility that an increase in CPP protects giraffe from fainting, using a mechanical model that functioned as an anatomical U-tube. It consisted of a rigid ascending “carotid” limb, a collapsible “brain” tube drained by a rigid, “vertebral venous plexus” (VVP) tube, and a collapsible “head” tube drained by a collapsible tube representing the “jugular vein”. The descending tubes could be rotated relative to the “carotid” tube to be horizontal, or at 30°, 45°, and 60° to the vertical to simulate changes in head position. Pressure at the top of the “carotid” tube was intracranial MAP, at the top of the “VVP” tube was ICP, and the difference CPP. In the simulated “head-up” position and a fluid flow rate of 4 L min⁻¹, CPP was ∼170 mmHg. With the VVP tube horizontal, CPP fell from ∼170 to 45 mmHg, but increased to ∼67 mmHg at 30° “down”, to ∼70 mmHg at 45° “down” and to ∼75 at 60° “down”. The fall in CPP in the head-down positions resulted from a decrease in viscous resistance in, and dissipation of pressure to, the “head” and “jugular” tubes. These data provide an estimate of cranial pressure changes in giraffe during positional changes of the head, and suggest that an increase in CPP plays a significant role in maintaining CBF during head-raising and that it may be an important mechanism for preventing fainting in giraffe.     

Depolarization-evoked GABA release from myenteric plexus is partially coupled to L-, N-, and P/Q-type calcium channels

1. There are many evidences suggesting that -aminobutyrate (GABA) is an important neurotransmitter and/or neuromodulator in the gut.2. Using the myenteric plexus-longitudinal muscle preparation from the guinea pig ileum, we investigated the evoked release of [³H] GABA from enteric neurons by electrical pulses or high KCl, which occurs in a calcium-dependent and -independent way. In addition, using selective calcium channel blockers, we report the participation of distinct subtypes of calcium channels in the evoked release, showing a minor participation of L- and Q-type calcium channels, while N- and P-type have a participation of approximately 15%, each. However, regardless of the combination of Ca²⁺ channel blockers, we did not observe an inhibition greater than 50% of the calcium-dependent component of [³H] GABA release.3. Thus, while the observed Ca²⁺-independent release mostly probable occur via reversal of the membrane GABA transporter, in our conditions, a considerable portion of the Ca²⁺-dependent evoked release of [³H] GABA is not coupled to L-, N-, or P/Q-type calcium channels, suggesting the involvement of intracellular calcium stores or other ways of getting calcium across the membrane.     

Hydroxyurea transport across the blood-brain and blood-cerebrospinal fluid barriers of the guinea-pig

Hydroxyurea is used in the treatment of HIV infection in combination with nucleoside analogues, 2'3'-didehydro-3'deoxythymidine (D4T), 2'3'-dideoxyinosine or abacavir. It is distributed into human CSF and is transported from the CSF to sub-ependymal brain sites, but its movement into the brain directly from the blood has not been studied. This study addressed this by a brain perfusion technique in anaesthetized guinea-pigs. The carotid arteries were perfused with an artificial plasma containing [14C]hydroxyurea (1.6 microm) and a vascular marker, [3H]mannitol (4.6 nm). Brain uptake of [14C]hydroxyurea (8.0 +/- 0.9%) was greater than [3H]mannitol (2.4 +/- 0.2%; 20-min perfusion, n = 8). CSF uptake of [14C]hydroxyurea (5.6 +/- 1.5%) was also greater than [3H]mannitol (0.9 +/- 0.3%; n = 4). Brain uptake of [14C]hydroxyurea was increased by 200 microm hydroxyurea, 90 microm D4T, 350 microm probenecid, 25 microm digoxin, but not by 120 microm hydroxyurea, 16.5-50 microm D4T, 90 microm 2'3'-dideoxyinosine or 90 microm abacavir. [14C]Hydroxyurea distribution to the CSF, choroid plexus and pituitary gland remained unaffected by all these drugs. The metabolic half-life of hydroxyurea was > 15 h in brain and plasma. Results indicate that intact hydroxyurea can cross the brain barriers, but is removed from the brain by probenecid- and digoxin-sensitive transport mechanisms at the blood-brain barrier, which are also affected by D4T. These sensitivities implicate an organic anion transporter (probably organic anion transporting polypeptide 2) and possibly p-glycoprotein in the brain distribution of hydroxyurea and D4T.