Ventral Gray Matter Research Articles

Anatomy and motor pathways of the electric organ of skates.

The electric organ of skates is a paired structure within the tail consisting of two longitudinal columns of electrocytes contained within the lateral musculature on each side of the vertebral column. The electrocytes develop from hypaxial skeletal muscle fibers, and, depending upon the species, are generally classified as either cup-shaped or disc-shaped. The disc-shaped electrocytes are considered to be the more derived type. Regardless of the morphology of the electrocyte, the electric organ discharge of all skates is characterized as a weak asynchronous and long-lasting signal. Although recent behavioral investigations have revealed a communicative function for the electric organ, details as to which specific behaviors utilize this motor system remain uncertain. The electric organ is innervated by segmental motor nerves that branch from the ventral root of caudal spinal nerves at all levels of the electric organ. The cells of origin of the electromotor nerves, or electromotoneurons (EMNs), are large multipolar neurons with extensive dendrites located within the ventral gray matter of the spinal cord. The EMNs are uniformly distributed among the somatic motoneurons at levels corresponding to the rostrocaudal extent of the electric organ, and therefore do not form a discrete nucleus. The medullary command nucleus is comprised of neurons located within the nucleus raphe magnus, and forms a descending spinal pathway to the EMNs.

Synaptic targets of commissural interneurons in the lumbar spinal cord of neonatal rats.

There is strong evidence that commissural interneurons, neurons with axons that extend to the contralateral side of the spinal cord, play an important role in the coordination of left/right alternation during locomotion. In this study we investigated the projections of commissural interneurons to motor neurons and other commissural interneurons on the other side of the spinal cord in neonatal rats. To establish whether there are direct contacts between axons of commissural interneurons and motor neurons, we carried out two series of experiments. In the first experiment we injected biotinylated dextran amine (BDA) into the lateral motor column to retrogradely label commissural interneurons that may have direct projections to motor neurons. Stained neurons were recovered in the ventromedial areas of the contralateral gray matter in substantial numbers. In the second experiment BDA was injected into the ventromedial gray matter on one side of the lumbar spinal cord, whereas motor neurons were simultaneously labeled on the opposite side by applying biocytin onto the ventral roots. BDA injections into the ventromedial gray matter labeled a strong axon bundle that arose from the site of injection, crossed the midline in the ventral commissure, and extensively arborized in the contralateral ventral gray matter. Many of these axons made close appositions with dendrites and somata of motor neurons and also with commissural interneurons retrogradely labeled with BDA. The results suggest that commissural interneurons may establish monosynaptic contacts with motor neurons on the opposite side of the spinal cord. Our findings also indicate that direct reciprocal connections between commissural interneurons on the two sides of the spinal cord may also exist.

Neurons with distinctive firing patterns, morphology and distribution in laminae V-VII of the neonatal rat lumbar spinal cord.

It is generally accepted that neurons in the ventral spinal grey matter, a substantial proportion of which can be regarded as constituents of the spinal motor apparatus, receive and integrate synaptic inputs arising from various peripheral, spinal and supraspinal sources. Thus, a profound knowledge concerning the integrative properties of interneurons in the spinal ventral grey matter appears to be essential for a fair understanding of operational principles of spinal motor neural assemblies. Using the whole cell patch clamp configuration in a correlative physiological and morphological experimental approach, here we demonstrate that the intrinsic membrane properties of neurons vary widely in laminae V-VII of the ventral grey matter of the neonatal rat lumbar spinal cord. Based on their firing patterns in response to depolarizing current steps, we have classified the recorded neurons into four categories: 'phasic', 'repetitive', 'single' and 'slow'. Neurons with firing properties characteristic of the 'phasic', 'repetitive' and 'single' cells have previously been reported also in the superficial and deep spinal dorsal horn, but this is the first account in the literature in which 'slow' neurons have been recovered and described in the spinal cord. The physiological heterogeneity in conjunction with the morphological correlation and distribution of neurons argues that different components of motor neural assemblies in the spinal ventral grey matter possess different signal processing characteristics.

Human chorionic gonadotropin/luteinizing hormone receptor expression in the adult rat spinal cord

The adult and fetal rat brain contains human chorionic gonadotropin/luteinizing hormone (hCG/LH) receptors, that are functional in mediating the neurotropic, neuroendocrine and behavioral actions of gonadotropins. We hypothesized that the spinal cord also contains these receptors. We have now demonstrated by in situ hybridization, immunohistochemistry, and topical autoradiography that the adult rat spinal cord expresses hCG/LH receptors. Positive cells included motoneurons, interneurons and/or glia in the intermediate and ventral gray matter, interneurons and/or glia in the dorsal gray matter, and oligodendrocytes or astrocytes in the white matter. The receptors were able to bind an appropriate ligand, 125I-hCG. The functional significance of these receptors in the spinal cord is unknown, but we can speculate that they may be neurotrophic in function.

Developmental expression of EphA4-tyrosine kinase receptor in the mouse brain and spinal cord

Eph receptor tyrosine kinases and their ephrin ligands are involved in some of the most important steps during the development of the central nervous system, including cell migration, axon guidance, topographic mapping and synapse formation. Moreover, in the adult, they have been implicated in plasticity and regulation of neural stem cell function. One member of the Eph family, EphA4, can bind to both classes of ephrins and may have multiple functions in nervous system development. In order to look for potential sites of EphA4 action during central nervous system development, we conducted a spatio-temporal analysis of EphA4 protein expression. We used immunohistochemistry in preference to in situ hybridization because of the high likelihood that EphA4 protein is expressed on axon tracts, long distances from EphA4 mRNA. In the telencephalon, EphA4 was expressed in the developing cortex from embryonic day 11 (E11) and, later, on major cortical tracts including the corpus callosum and cortico-spinal tract. Robust EphA4 expression was also found in the hippocampus and fornix, and cells and tracts in the striatum. In the diencephalon, the thalamus, the hypothalamus and thalamo-cortical projection were strongly positive. In the mesencephalon, a number of different nuclei were weakly positive, most prominently the red nucleus. In the rhombencephalon, many nuclei were strongly positive including the cerebellum and one of its afferent paths, the inferior cerebellar peduncle, as well as the olivary region. In the spinal cord, there was a dynamic pattern of expression through development, with persistent expression in the dorsal funiculus and ventral grey matter.

Influence of electroacupuncture on stress-induced hypertension and its mechanism

Background: Electroacupuncture (EA) and activation of the sympathetic inhibitory system in the brain, i.e. nucleus arcuatus–ventral periaqueductal gray matter–nucleus raphe obscurus (ARC–vPAG–NRO) system, can lower blood pressure (BP). This study was to observe the effect of EA on stressed-induced hypertensive rats and whether nitric oxide in vPAG can be involved in the effect of EA. Methods: On the anesthetized stress-induced hypertensive rats blood pressure, heart rate (HR), cardiac functions and electrocardiogram were recorded synchronously. EA of “zusanli” acupoints with microinjections of saline (NS) and N ω-nitro- l-arginine ( LNNA) into vPAG in brain was applied. Results: EA+NS resulted in a depressor, bradycardic response and an attenuation in the maximum of left ventricular pressure, and diastolic pressure and ±d p/d t, as well as an increase of total variance, very low frequency (VLF), low frequency (LF) and the LF–high frequency (HF) ratio (LF/HF). In the EA+ LNNA group, the above effects of EA were abolished or reduced significantly. Conclusions: The results suggest that the depressor effect of EA on stress-induced hypertensive rats might be mediated by NO in the vPAG due to activation of the sympathetic inhibitory system in the brain and by attenuated cardiac activities.

Ontogeny of descending serotonergic innervation and evidence for intraspinal 5-HT neurons in the mouse spinal cord

Neuronal networks in the mouse spinal cord express serotonin (5-HT)-induced rhythmic motor activity at early developmental stages (embryonic day (E) 12.5). Later in development, by post-natal day (P) 10, the 5-HT-evoked rhythmic motor activity matures and acquires an adult locomotor-like pattern. With the view to establishing a relationship between the ontogeny of locomotor networks and the maturation of spinal 5-HT systems, we have traced 5-HT immunoreactivity in the mouse spinal cord from E12.5 to PN10. By E12.5, descending 5-HT immunoreactive (5-HT-ir) fibers that likely originate from raphe nuclei were detected in the ventral and lateral funiculi, at anterior cervical spinal levels, but not at more caudal levels. Descending 5-HT-ir axons reached thoracic levels at E14.5 and lumbar levels at E16.5. Some 5-HT-ir fibers could be detected in the ventral and intermediate gray matter by E16.5, whereas the dorsal gray matter was not invaded before PN0. At PN10, a dense serotonergic innervation was restricted to the gray matter with a high concentration of 5-HT-ir fibers in three areas: dorsal horn, ventral horn (where motoneurons are located) and intermediate area. Surprisingly, from E16.5 to PN10, 5-HT-ir intraspinal neurons were found, exclusively at sacral levels. Their somata lay in the gray matter around the central canal and preferentially in the ventro-median part of the ventral horn. The functional significance of these sacral 5-HT-ir neurons is discussed.

Upregulation of EphA Receptor Expression in the Injured Adult Rat Spinal Cord

After spinal cord injury (SCI), the inability of supraspinal neurons to regenerate or reform functional connections is likely due to proteins in the surrounding microenvironment restricting regeneration. EphAs are a family of receptor tyrosine kinases that are involved in axonal guidance during development. These receptors and their ligands, the Ephrins, act via repulsive mechanisms to guide growing axons towards their appropriate targets and allow for the correct developmental connections to be made. In the present study, we investigated whether EphA receptor expression changed after a thoracic contusion SCI. Our results indicate that several EphA molecules are upregulated after SCI. Using semiquantitative RT-PCR to investigate mRNA expression after SCI, we found that EphA3, A4, and A7 mRNAs were upregulated. EphA3, A4, A6, and A8 receptor immunoreactivity increased in the ventrolateral white matter (VWM) at the injury epicenter. EphA7 had the highest level of immunoreactivity in both control and injured rat spinal cord. EphA receptor expression in the white matter originated from glial cells as coexpression in both astrocytes and oligodendrocytes was observed. In contrast, gray matter expression was localized to neurons of the ventral gray matter (motor neurons) and dorsal horn. After SCI, specific EphA receptor subtypes are upregulated and these increases may create an environment that is unfavorable for neurite outgrowth and functional regeneration.

Temporal progression of angiogenesis and basal lamina deposition after contusive spinal cord injury in the adult rat.

After spinal cord injury (SCI), the absence of an adequate blood supply to injured tissues has been hypothesized to contribute to the lack of regeneration. In this study, blood vessel changes were examined in 28 adult female Fischer 344 rats at 1, 3, 7, 14, 28, and 60 days after a 12.5 g x cm NYU impactor injury at the T9 vertebral level. Laminin, collagen IV, endothelial barrier antigen (SMI71), and rat endothelial cell antigen (RECA-1) immunoreactivities were used to quantify blood vessel per area densities and diameters in ventral gray matter (VGM), ventral white matter (VWM), and dorsal columns (DC) at levels ranging 15 mm rostral and caudal to the epicenter. This study demonstrates an angiogenic response, defined as SMI71/RECA-1-immunopositive endothelial cells that colocalize with a robust deposition of basal lamina and basal lamina streamers, 7 days after injury within epicenter VGM. This angiogenesis diminishes concurrent with cystic cavity formation. GAP43- and neurofilament- (68 kDa and 210 kDa) immunopositive fiber outgrowth was associated with these new blood vessels by day 14. Between 28 and 60 days after injury, increases in SMI71-immunopositive blood vessel densities were observed in the remaining VWM and DC with a corresponding increase in vessel diameters up to 15 mm rostral and caudal to the epicenter. This second angiogenesis within VWM and DC, unlike the acute response observed in VGM, did not correspond to any previously described changes in locomotor behaviors in this model. We propose that therapies targeting angiogenic processes be directed at the interval between 3 and 7 days after SCI.

Encephalomyelitis associated with akabane virus infection in adult cows.

Between August and September 2000, five 2-7-year-old cows in Korea exhibited neurologic signs and were diagnosed as infected with Akabane virus based on the results of histopathology, immunohistochemistry, serology, and reverse transcription polymerase chain reaction (RT-PCR) analysis. Immunohistochemistry and RT-PCR were equally effective and sensitive for diagnosing Akabane virus infection during the early stage of infection. Typical lymphohistiocytic inflammation characterized by perivascular mononuclear cell infiltration, gliosis, neuronophagia, and neuronal loss was noted in the brain and the ventral horn gray matter of the spinal cord. The lesions in the brain were most prominent in the pons and medulla oblongata. Akabane virus antigen was detected in the brain and spinal cord, mainly in degenerating neurons and glial cells. RT-PCR analysis revealed a target band of expected size in four cows. This is the first report on an outbreak of natural Akabane virus infection in adult cattle.

Caspase-3 activation and caspase-like proteolytic activity in human perinatal hypoxic-ischemic brain injury.

Human perinatal hypoxic-ischemic brain injury is an important cause of death and morbidity. One relatively common pattern of perinatal injury involves selective neuronal death in the ventral gray matter of the pons and in the subiculum of the hippocampal formation, classically termed 'pontosubicular neuronal necrosis' (PSN). The vulnerable neurons undergo karyorrhectic condensation of their nuclear chromatin and exhibit in situ end labeling for DNA fragmentation, leading to the recent reclassification of cell death in PSN as apoptotic. Caspase activation plays a central role in apoptosis and caspase-3 appears to be an especially important effector enzyme in neuronal apoptosis. In this study we performed immunohistochemistry on brain sections from six postmortem cases of PSN using two polyclonal antisera; CM1, a specific marker of caspase-3 activation, and fractin, which specifically recognizes a neoepitope at a caspase cleavage site in actin, and is therefore a marker of caspase-like proteolytic activity. Numerous CM1- and fractin-immunolabeled neurons were seen in the nuclei pontis and subiculum in each case, and the great majority showed karyorrhectic morphology. The immunostaining involved the nuclei and cytoplasm of these cells and the proximal portions at least of their neuritic processes. Some neurons exhibited a more extensive pattern of dendritic fractin labeling. Frequent CM1- and fractin-immunoreactive axonal segments were also seen. The identification of caspase-3 activation and caspase-like proteolytic activity in PSN cases in this study suggests that caspase inhibitors may potentially have a therapeutic neuroprotective role in human perinatal hypoxic-ischemic brain injury.

Characterization of Gross and Histological Lesions in Balb/c Mice Experimentally Infected with Herpesvirus saimiri 1 (HVS1)

Accidental B virus (Herpesvirus simiae) infection of human beings working with macaques is frequently fatal. However, the pathogenic potential of other similar simian alphaherpesviruses, such as the squirrel monkey virusHerpesvirus saimiri (HVS1), is virtually unknown. As part of an effort to develop a murine model for infections with these agents, Balb/c mice were inoculated intramuscularly in the left hindlimb with 10 to 106plaque forming units (PFU) of HVS1. After observation for clinical signs of infection for 21 days, mice were killed and specimens collected for serology and histopathology. Mice receiving 5103PFU of HVS1 exhibited severe, pruritic, ulcerative skin lesions near the site of inoculation and developed unilateral or bilateral hindlimb paralysis with severe muscle atrophy. Histological lesions were characterized by a necrotizing dermatitis and folliculitis. Spinal cord lesions consisted of a non-suppurative myelitis affecting primarily the ipsilateral dorsal horn of the thoracolumbar spinal cord with occasional extension to ventral and contralateral spinal cord regions. Immunohistochemical labelling confirmed the presence of viral antigen within the lesions, and anti-HVS1 IgG concentrations were related to the occurrence of disease. HVS1 infection in some mice extended from the ipsilateral dorsal horn and funiculus into the ventral and contralateral grey and white matter, resulting in bilateral hindlimb paralysis. Thoracolumbar spinal cord lesions resolved without continued spread of the virus to cranial nervous system structures, i.ecervical spinal cord and brain.

Hyperbaric oxygen therapy given 30 minutes after spinal cord ischemia attenuates selective motor neuron death in rabbits.

Spinal cord ischemia sometimes causes paraplegia because the spinal motor neuron cells are vulnerable to ischemia. Although various protective remedies for spinal cord injury have been reported, there have been few established clinical methods. Although hyperbaric oxygen (HBO) has been used clinically as a treatment for ischemia, the reason for its effectiveness is still uncertain because sufficient experimental data are lacking. Prospective, randomized, controlled study. Experimental animal research laboratory in a university research center. Twenty-three Japanese white rabbits, weighing 2-3 kg. A modified rabbit spinal cord ischemia model of infrarenal aortic occlusion for 15 mins was employed. Rabbits were randomly assigned to four groups; the rabbits in group A did not undergo ischemic insults (n = 5). The rabbits in groups B and C underwent ischemic insult for 15 mins, followed by 1 hr of HBO treatment at 3 atm absolute with 100% oxygen at 30 mins (n = 6) or 6 hrs (n = 7) after reperfusion, respectively. The rabbits in group D underwent ischemic insult for 15 mins without HBO treatment (n = 5). We observed neurologic functions for 14 days. The sections of the spinal cords were stained with hematoxylin and eosin, and the number of spinal motor neurons in ventral region was counted by light microscopy. All rabbits in groups A and B could stand, whereas all rabbits in groups C and D showed irreversible paraplegia on days 2 and 14 after reperfusion. Spinal motor neurons in ventral gray matter in groups C and D decreased significantly compared with those in groups A and B. HBO therapy shortly after ischemic insult had protective effects against ischemic spinal cord damage. However, delayed treatment with HBO did not change the prognosis.

NITRIC OXIDE IN vPAG MEDIATES THE DEPRESSOR RESPONSE TO ACUPUNCTURE IN STRESS-INDUCED HYPERTENSIVE RATS

The hypertensive rat model was made by chronic stress of electric foot-shocks and noises. On such hypertensive rats, when anesthetized with urethane and chloralose, the electroacupuncture (EA) to bilateral "Zusanli (st.36)" acupoints for 20 min, could result in a depressor (including both systolic and diastolic pressure) and bradycardiac response as well as an attenuation in the maximum of left ventricular pressure, end diastolic pressure and +/-dp/dt. In power spectrum analysis of heart rate variability aspect, EA could increase all total variance, very low frequency component, low frequency component and the ratio of low frequency component and high frequency component. When EA with microinjection of N(omega) - Nitro- L-Arginine , a blocker of the formation of nitric oxide, into the ventral periaqueductal gray matter (vPAG), the above effects of EA were abolished or reduced significantly. The results suggest that the depressor effect of EA on stress-induced hypertensive rats might be mediated by nitric oxide in the vPAG due to activation of sympathetic inhibitory system and by attenuated cardiac activities.

Antiallodynic effects produced by stimulation of the periaqueductal gray matter in a rat model of neuropathic pain

It has been well documented that there is opioid resistance in neuropathic pain. This indicates that the endogenous opioid system may not be involved effectively in modulating neuropathic pain. The present study sought to determine if activation of the descending pain inhibition system might produce analgesia in the animal neuropathic model we developed. Under ketamine anesthesia, male Sprague–Dawley rats were chronically implanted with stimulating electrodes in the ventral periaqueductal gray matter (PAG) and both the tibial and sural nerves of the sciatic nerve branches were severed. Pain sensitivity was measured with a von Frey filament and acetone applied to the sensitive area for 1 week postoperatively. Rats with neuropathic pain syndrome after transection of the tibial and sural nerves were tested as to the analgesic effects of ventral PAG stimulation for an additional two weeks. Electrical stimulation of the ventral PAG turned out to be highly effective in alleviating neuropathic pain. Mechanical allodynia and cold allodynia were reduced by PAG stimulation. Naloxone reversed the antiallodynic effects of ventral PAG stimulation. These results suggest that activation of the descending pain inhibition system including the ventral PAG reduces neuropathic pain syndrome and that opiates are involved in this system.

Microinjection of opiates into the periaqueductal gray matter attenuates neuropathic pain symptoms in rats.

We have previously demonstrated that electrical stimulation of the ventral periaqueductal gray matter (PAG) produced analgesia in neuropathic pain in rats. Opioids were also shown to be involved in analgesic effects. This study sought to determine whether opiates microinjected into the ventral PAG produce analgesia. Male Sprague-Dawley rats were chronically implanted with a guide cannula in the PAG under pentobarbital anesthesia and both the tibial and sural nerves were completely cut. Pain sensitivity was postoperatively measured with a von Frey filament and acetone applied to the sensitive area for 1 week. Opioids such as [D-Ala2,N-MePhe4,Gly(ol)5]-enkephalin (DAMGO) and [D-Pen ,D-Pen5]-enkephalin (DPDPE) were injected into the PAG. DAMGO, a mu-opioid agonist, and DPDPE, a delta-opioid agonist, were highly effective in reducing neuropathic pain. These effects were reversed by naloxone. These results suggest that the neurons in the ventral PAG are activated by opioids to produce analgesia and that specific opioid receptors are involved in the descending pain inhibition system from the PAG.

Systemic Administration of Domoic Acid-Induced Spinal Cord Lesions in Neonatal Rats

Domoic acid (Dom) is a glutamate analog and a seafood toxin that has caused neurological disturbance and death in humans. Brain lesions caused by Dom have been documented in the literature, but the effect of Dom on the spinal cord has not been investigated as extensively. Systemic administration of glutamate agonists (i.e., homocysteate, kainate, and a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) caused spinal cord lesions in infant rats. In the present study, the toxic effects of Dom on the developing spinal cord are examined. Neonatal rats on Postnatal Day 7 were administered Dom subcutaneously at doses of 0.1 0, 0.17, 0.25, 0.33, 0.42, and 0.50 mg/kg, respectively. Motor seizures characterized by scratching, tail flicking, and swimming-like movement were induced by Dom at all doses. High doses of Dom (≥ 0.33 mg/kg) further induced a hindlimb paralysis, a forelimb tremor, and death that occurred in less than 2 hours. The percentages of death and paralysis induced by 0.33 mg/kg Dom were 47% and 65%, respectively (n = 17). At this dose, electrocorticogram was recorded and synchronized interrupted electrical activities in brains of these animals were detected. However, no brain damage was detected in these rats. Spinal cord lesions characterized by focal hemorrhage, neuronal swelling, and neuronal vacuolization were found in 73% of the animals that had shown the paralysis/tremor in their extremities, as examined 1 to 2 hours after Dom injection. These lesions were seen at all spinal cord levels. Neuronal degeneration was mainly found in the ventral and intermediate gray matter, whereas cells in the dorsal portion of the spinal cord were relatively spared. Data suggest that observed behavioral changes were due to spinal cord damage rather than seizures or brain lesions.

Distribution of preprovasopressin mRNA in the rat central nervous system

Vasopressin released in the central nervous system has been shown to be involved both in homeostatic mechanisms (e.g., water balance, thermoregulation, cardiovascular regulation, metabolism, and antinociception) and in higher brain functions (e.g., social recognition and communication, and learning and memory). Many nuclear groups have been proposed to synthesize vasopressin, but available data are conflicting. We have used a sensitive in situ hybridization technique to identify the distribution of the neurons that may be the origin of the vasopressin in the central nervous system of the male Sprague-Dawley rat. Vasopressin mRNA-expressing neurons were most abundant in the hypothalamus (e.g., the paraventricular, supraoptic, and suprachiasmatic nuclei) but were also seen in the medial amygdaloid nucleus, the bed nucleus of stria terminalis, and the nucleus of the horizontal diagonal band. Previously unreported vasopressinergic neurons were seen in the entorhinal and piriform cortices, the ventral lateral portion of the parabrachial nucleus, the pedunculopontine nucleus, and the rostral part of the ventral periaqueductal gray matter and the adjacent portion of the mesencephalic reticular nucleus. Vasopressin mRNA expression suggestive of neuronal labeling was seen in the pyramidal layer of the CA1–3 fields and the dentate gyrus of the hippocampus. In addition, vasopressin mRNA expression, probably representing axonal mRNA, was detected over the hypothalamopituitary tract. No or insignificant preprovasopressin mRNA expression was present in the cerebellum, locus coeruleus, subcoeruleus, or the spinal cord. These findings provide novel information on the distribution of vasopressin neurons that are important for our understanding of how vasopressin acts in the brain. J. Comp. Neurol. 411:181–200, 1999. © 1999 Wiley-Liss, Inc.

Distribution of preprovasopressin mRNA in the rat central nervous system

Vasopressin released in the central nervous system has been shown to be involved both in homeostatic mechanisms (e.g., water balance, thermoregulation, cardiovascular regulation, metabolism, and antinociception) and in higher brain functions (e.g., social recognition and communication, and learning and memory). Many nuclear groups have been proposed to synthesize vasopressin, but available data are conflicting. We have used a sensitive in situ hybridization technique to identify the distribution of the neurons that may be the origin of the vasopressin in the central nervous system of the male Sprague-Dawley rat. Vasopressin mRNA-expressing neurons were most abundant in the hypothalamus (e.g., the paraventricular, supraoptic, and suprachiasmatic nuclei) but were also seen in the medial amygdaloid nucleus, the bed nucleus of stria terminalis, and the nucleus of the horizontal diagonal band. Previously unreported vasopressinergic neurons were seen in the entorhinal and piriform cortices, the ventral lateral portion of the parabrachial nucleus, the pedunculopontine nucleus, and the rostral part of the ventral periaqueductal gray matter and the adjacent portion of the mesencephalic reticular nucleus. Vasopressin mRNA expression suggestive of neuronal labeling was seen in the pyramidal layer of the CA1-3 fields and the dentate gyrus of the hippocampus. In addition, vasopressin mRNA expression, probably representing axonal mRNA, was detected over the hypothalamopituitary tract. No or insignificant preprovasopressin mRNA expression was present in the cerebellum, locus coeruleus, subcoeruleus, or the spinal cord. These findings provide novel information on the distribution of vasopressin neurons that are important for our understanding of how vasopressin acts in the brain.

Delayed neurotoxicity of diisopropylfluorophosphate (DFP): autoradiographic localization of high-affinity [3H]DFP binding sites in the chicken spinal cord

The delayed neurotoxic organophosphate, diisopropylfluorophosphate (DFP) binds with high affinity to membrane-bound proteins from chicken nerve tissues. The autoradiographic distribution of [(3)H]DFP binding sites in spinal cord sections of chicken showed higher concentrations of binding sites in gray matter than in white matter. In the cervical region, fairly high densities of [(3)H]DFP binding sites were found in laminae X and to a lesser extent, in the ventral horn gray matter. To identify the membrane-associated DFP-binding proteins, detergent-solubilized membranes were labeled widi 5-10nM [(3)H]DFP (10pmol/mg protein) for 70 min at 37°C. Gel-exclusion chromatography of the [(3)H]DFP-radiolabeled membranes indicated at least two major radioactive proteins with apparent molecular weights of 150-670 kDa and 40-129 kDa. Although we could not identify the high affinity DFP binding proteins, the autoradiographic experiments clearly demonstrated that the DFP binding proteins localized on gray matter of chicken spinal cord.