The morphology of the spinal cord efferent and afferent neurons contributing to the ventral roots of the cat.

Horseradish peroxidase was applied to proximal ventral roots of the coccygeal and sacral spinal cord of cats. Subsequent histochemical reaction resulted in extensive staining of spinal cord neurons that had processes in the ventral roots. This procedure was used to study four issues concerning ventral root neurons. (1) Extensive transverse dendritic arborizations were revealed for large and small neurons presumed to be alpha and gamma motoneurons respectively. Dendrites from these neurons were found to project heavily into the ipsilateral white matter, both laterally and ventrally. Dendrites also projected extensively through the anterior commissure, attaining the contralateral grey and white matter. (2) Medially-located efferent neurons were found to contribute the contralateral dendrites as well as some dorsally-directed dendrites. Laterally-located neurons projected dendrites extensively into the lateral and ventral white matter. (3) Stained neurons were found in the intermediolateral cell column, and were presumed to be preganglionic efferent neurons. Some of these neurons projected dendrites into the marginal zone of the dorsal horn, while others sent dendrites medially toward the central canal. (4) Stained fibers, presumed to be primary afferents, were found to enter from the ventral roots and course to the dorsal horn. Most of these fibers were small in diameter and distributed boutons predominantly to the substantia gelatinosa. A few large ventral root afferent fibers were observed that distributed boutons mostly to the nucleus proprius.

Thalamic projections to S-I in macaque monkey.

The organization of thalamic input to functionally characterized zones in primary somatosensory cerebral cortex (S-I) of macaque monkeys (Macaca mulatta) was investigated using the method of labelling by retrograde transport of horseradish peroxidase (HRP). It was found that the cell columns positioned at the posterior margin of the band of cortex representing a given body region receive thalamic input from a posterior level of the ventroposterior thalamic nucleus (VP), and that cell columns at successively more anterior positions within that band receive input from successively more anterior levels of VP. The extreme posterior and anterior margins of the S-I hand, foot and face areas receive input from neuron populations which are not as widely separated in the anteroposterior dimension of VP as the neurons projecting to the extreme anterior and posterior margins of the proximal limb and trunk representations in S-I. These characteristics of the organization of the projections from VP to S-I are consistent with the view that the body representations in VP and S-I have the same connectivity and differential submodality distribution; and with the idea that thalamocortical conncetions only exist between functionally equivalent neuron populations in VP and S-I.

Distribution of neurons expressing calcitonin gene-related peptide mRNAs in the brain stem, spinal cord and dorsal root ganglia of rat and guinea-pig.

In situ hybridization histochemistry was used to localize calcitonin gene-related peptide mRNAs in spinal cord, brain stem and dorsal root ganglion neurons of the rat and guinea-pig. A 32P-labeled 23-base-long (23mer) oligodeoxyribonucleotide (oligomer) complementary to calcitonin gene-related peptide mRNA sequences encoding residues 23-30 of calcitonin gene-related peptide was used primarily as a probe (CGRP I probe). A 32mer complementary to mRNA sequences for residues 10-20 of calcitonin gene-related peptide (CGRP II probe) was also used as a positive control for specificity of the 23mer for calcitonin gene-related peptide mRNA. In both the guinea-pig and rat calcitonin gene-related peptide mRNA was localized specifically to neurons of the dorsal root ganglion, to spinal motoneurons and to motoneurons of the hypoglossal, facial and accessory facial motor nuclei. Differences in the distribution of calcitonin gene-related peptide mRNA between the rat and guinea-pig included a higher proportion of rat dorsal root ganglion neurons containing calcitonin gene-related peptide mRNA and the localization of calcitonin gene-related peptide mRNA to motoneurons of the ambiguus motor nucleus, parabrachial and peripeduncular nucleus of the rat but not the guinea-pig. In the guinea-pig, in contrast, calcitonin gene-related peptide mRNA was localized also to motoneurons of the abducens, trigeminal, trochlear and oculomotor nerves. The neuronal groups in the intact rat found here to contain calcitonin gene-related mRNA have also been shown previously to contain calcitonin gene-related peptide immunoreactivity in colchicine-treated rats. Colchicine-treated rats, however, have been found to contain additional groups of calcitonin gene-related peptide immunoreactive neurons which, in the intact rats used in the present study, showed no detectable hybridization with the calcitonin gene-related peptide probe.

Colchicine enhances mRNAs encoding the precursor of calcitonin gene-related peptide in brainstem motoneurons.

Hybridization signals indicating mRNAs encoding the precursor of calcitonin gene-related peptide (CGRP) and CGRP immunoreactivity were detected on parallel sections containing brainstem motor nuclei using in situ hybridization histochemistry and immunohistochemistry. In untreated and saline-injected rats the motoneurons in the hypoglossal, facial motor nuclei and in the ambiguus nucleus showed weak to moderate hybridization signals. In these motoneurons CGRP immunoreactivity was restricted to the Nissl bodies of the perikarya. Twenty-four and 42 hours after intracerebroventricular colchicine injection the intensity of both the hybridization signal and the immunoreaction product increased. The distribution of CGRP immunoreactivity changed from discrete perikaryal localization to diffuse reaction in the perikarya and along the proximal dendritic tree. Motoneurons in the rest of the brainstem motor nuclei (VIth, Vth, IVth and IIIrd) of untreated and saline-injected rats showed neither hybridization signal nor CGRP immunoreactivity. After intracerebroventricular injection of colchicine these motoneurons showed both hybridization signal and CGRP immunoreactivity. In all nuclei the size of motoneurons decreased and their Nissl structure changed to an amorphous basophilic mass following colchicine treatment.

Selective suppression of endogenous peroxidase activity: application for enhancing appearance of HRP-labeled neurons in vitro.

A simple procedure to suppress selectively the endogenous peroxidase activity of red blood cells in histological sections of the mammalian nervous system is described. Pretreatment of sections with a series of ethanol solutions results in selective abolition of red blood cell staining and also leads to enhanced visualization of neurons that have been injected with horseradish peroxidase (HRP). The method is particularly useful for processing in vitro brain slices that contain HRP-labeled neurons. It can also be used for processing unperfused neural tissue from in vivo HRP labeling experiments. The ethanol pretreatment is compatible with several standard histochemical techniques for the demonstration of HRP.

Simultaneous visualization of chromatolytic neurons and neurons retrogradely labeled with horseradish peroxidase.

A method is described for visualization of neurons retrogradely labeled with horseradish peroxidase (HRP) in celloidin-embedded brain slabs. The preservation of cellular detail obtained with this technique facilitates (1) accurate delimination of nuclei or laminae in which HRP-positive neurons are found and (2) simultaneous identification of cells of origin of two central fiber systems, by combining the retrograde transport of HRP and the retrotrade degeneration technique. The latter approach has been used in adult cats and in kittens to identify cortical neurons which project to the dorsal column nuclei and to the spinal cord.

Correlation of antispermatozoal antibody with infertility in immunized female rabbits using 14C-protein A in a filter radioassay.

The meaningful detection of antisperm antibody in immunologically infertile females has been confounded by the many methods of assay that exist. With many of these methods there is poor correlation of assay results with infertility. In this report, female rabbits were rendered partially or completely infertile by immunization with sperm fractions. A filter radioassay for antisperm antibody was developed that consists of incubating 10(7) sperm with sperm from immunized rabbits and 14C-Protein A, a long-lived and versatile indirect radiolabel for many antibodies of the IgG class. The spermatozoa are washed by rapid vacuum filtration on polycarbonate membrane filters instead of by time-consuming centrifugation. The filters with the collected spermatozoa are then counted in a liquid scintillation counter. Sera from female rabbits isoimmunized with sperm antigens show a highly significant correlation (r = -0.904; p less than 0.001) between assay results and infertility as measured by the percentage of eggs that underwent cleavage after artificial insemination.

Sperm head decondensation by a high molecular weight fraction of sea urchin egg homogenate.

Sperm nucleus decondensing activity (NDA) of eggs has been investigated using the gametes of the sea urchin, Lytechinus variegatus. To assay for NDA, isolated sperm heads were exposed to homogenate from mature unfertilized ova and then examined with phase-contrast optics. NDA is retained by dialysis and requires Ca++ at the time of homogenization as well as at the time of assay. Other divalent cations (Mg++, Sr++, Ba++, Mn++) can substitute for Ca++ but are much less effective. NDA is stable to cold (-20 degrees C, -76 degrees C) but not to heat (56 degrees C). The pH activity optimum is in the range 7-9. NDA is present in the supernatant after centrifugation at 150,000 g for 105 minutes. It elutes shortly following the void volume on a Sephacryl S-200 column, with an approximate M.W. of 100,000 daltons.

Phospholipase activity of sea urchin sperm: its possible involvement in membrane fusion.

Phospholipase activity of egg-water treated Arbacia punctulata and Lytechinus variegatus sperm was shown to result from the sequential action of phospholipase A and lysophospholipase. A transient burst of phospholipase A activity followed induction of the acrosome reaction with egg water. The time of appearance suggested an acrosomal localization of the enzyme. The peak activity of phospholipase A correlated with initiation of sperm-egg fusion, suggesting a role for sea urchin sperm phospholipase A in membrane fusion and/or egg activation during fertilization.

Isolation of an arbacia sperm fertilization antigen.

Antisperm antibody fragments (IFab) block sea urchin fertilization by inhibiting the acrosome reaction and consequently sperm-egg attachment. We describe here a 68,000 dalton MW glycoprotein which neutralizes the fertilization-inhibiting action of IFab. This glycoprotein is a minor component is SDS-polyacrylamide gels of sperm membranes, but is greatly enriched in lithium diiodosalicylate extracts of the membranes. Final purification of the antigen was accomplished by elution from preparative SDS-polyacrylamide gels. This glycoprotein is evidently the sea urchin sperm receptor which interacts wih the egg jelly coat and acts as a "trigger" for the acrosome reaction.

Immunological immobilization of detached rabbit sperm tails.

Motile rabbit sperm tails that lack heads are immobilized by antibody plus complement. Accordingly, immunological immobilization may not necessarily require the extensive sperm acrosomal damage previously reported in immobilized normal sperm.

Mechanism of univalent antisperm antibody inhibition of fertilization in the sea urchin, Arbacia punctulata.

Univalent antisperm antibodies (IFab) markedly inhibited the fertilizing capacity of sperm when tested on intact, dejellied, and "demembranated" Arbacia punctulata eggs. Sperm motility and egg jelly penetration were not affected by IFab. Antifertilizin was excluded as the essential sperm antigen involved in the fertilization-inhibiting action. Sperm pretreated with IFab did not bind to the surfaces of either dejellied or demembranated eggs, whereas control globulin (CFab) and seawater-pretreated sperm bound to such eggs in high numbers. Electron microscopy showed that IFab-treated sperm failed to undergo the acrosome reaction. This excluded "bindin" as the essential antigen. Inhibition of fertilization by IFab was reversed or bypassed by artificial induction of the acrosome reaction with ionophore A23187. It is concluded that univalent antisperm antibody treatment inhibits the fertilizing capacity of sperm by preventing a sperm-egg interaction that results in the acrosome reaction; consequently, attachment of the sperm to the egg is prevented.

In situ hybridization of peptide mRNAs on vibratome sections.

In situ hybridization procedures that have been used successfully for the localization of somatostatin and cholecystokinin mRNAs in neurons on cryostat sections of rat brain, were tested for applicability to vibratome sections of rat and guinea pig brain. Somatostatin and cholecystokinin mRNAs were localized to neurons in 30 microns thick vibratome sections of brain from both species by use of 32P labelled oligodeoxyribonucleotide (oligomer) probes. Somatostatin mRNAs was localized to neurons in the periventricular region of the preoptic area of rat, and guinea pig brain. Cholecystokinin mRNAs were localized to neurons of rat hippocampus. Hybridization signal, background and resolution achieved with vibratome sections were comparable to those obtained with the more commonly used cryostate sections.

Representation of head and face in postcentral gyrus of the macaque.

The receptive field and submodality characteristics of individual neurons within the cytoarchitectural and topographic subdivisions of the head and face areas of the postcentral gyrus (SI) were determined with the technique of extracellular recording. Correlation of the single-unit data with the intracortical location of the recording electrode provided a detailed description of the functional organization within each of the several cytoarchitecturally distinct regions contributing to the representation of the head and face in SI. The data indicate that the functional organization of the SI cortex which receives its principal input from trigeminal mechanoreceptors is comparable to the organization within those SI regions which receive their input from the mechanoreceptors of the limbs, trunk, and tail. In each topographic subdivision of the SI cortex 1) a single region in the periphery is represented several times in widely separated locations, each time in a context of different submodalities and peripheral receptive fields; and 2) neurons belonging to the different submodality classes are segregated so that projections from cutaneous afferents terminate mainly in cytoarchitectural area 3 in the adjacent anterior portion of area 1, while projections from the afferents innervating deep tissues terminate mainly in cytoarchitectural area 3a, area 2, and the posterior part of each 1. Although the mechanoreceptor input to SI is segregated according to submodality and the mechanoreceptors from most body regions project to multiple widely separated regions within SI, neurons with receptive fields confined to the ophthalmic division of the trigeminal peripheral innervation field are found within a restricted region of the anterior postcentral gyral crown which is positioned symmetrically about the junction of cytoarchitectural areas 1 and 3. Neurons with receptive fields confined to the maxillary division of the trigeminal innervation field are found within a ring of cortex which a) completely surrounds the representation of the ophthalmic field, and b) includes parts of cytoarchitectural area 2, 1, 3, and 3a. SI neurons with receptive fields restricted to the mandibular division of the trigeminal innervation field occupy the largest portion of the SI face area and form a ring of cortical cell columns which completely surrounds that cortical region which receives its input from the maxillary peripheral innervation field.