Intracellular Iontophoresis Of Horseradish Peroxidase Research Articles

Androgenic regulation of dendritic trees of motoneurons in the spinal nucleus of the bulbocavernosus: Reconstruction after intracellular iontophoresis of horseradish peroxidase

Androgen-sensitive motoneurons in the spinal nucleus of the bulbocavernosus (SNB) in adult male rats were labeled after intracellular iontophoresis of horseradish peroxidase, after which they were fully reconstructed in three dimensions in order to measure their dendritic trees. Three groups of rats were compared: intact adult male rats and male rats castrated as adults and given Silastic tube implants containing either testosterone or nothing. In the high-androgen groups (intact males and testosterone-treated castrates), soma size and the diameter of the first-order dendrites were larger than in blank-treated castrates. Moreover, the terminal dendrites in all groups possessed growth cones, implying that the dendrites of these motoneurons are capable of growth in adulthood. However, there were no statistically significant group differences in the length, membrane surface area, or volume of the dendritic trees, or in the orientation or branching symmetry of dendrites. In general, there were positive correlations between the size of the motoneuronal soma and various measures of the size of the dendritic tree and between the diameter of individual stem (first-order) dendritic branches and the size of remainder of that dendrite. These data suggest that there may be a modest effect of androgen on the size of the dendritic trees of SNB motoneurons in adulthood, although the effect is much smaller than has previously been reported.

Intracellular labeling of neurons in the medial accessory olive of the cat: I. Physiology and light microscopy

This study is the first of three reports on the detailed morphology of horseradish peroxidase injected neurons in the medial accessory olive of the cat. Intracellular, in vivo recordings of olivary cells were made and their response to mesodiencephalic stimulation was tested. In 44 units a short latency action potential could be recorded, which was very suggestive of a monosynaptic excitatory pathway. The short latency response was frequently followed by a long latency (mean 188 msec) or rebound action potential. Recordings were followed by intracellular iontophoresis of horseradish peroxidase. A total of 21 neurons, all located within the medial accessory olive were chosen for morphological analysis. Cells could be divided into two categories on the basis of their overall morphological appearance. Type I cells (n = 5) had sparsely branching dendrites that radiated away from the soma and were usually found in the caudal part of the medial accessory olive. The axon usually originated from the soma. Type II cells (n = 16) were located more rostrally. They had larger cell bodies with dendrites that ramified extensively, forming a globular structure (mean diam. 338 microns). The axon usually originated from a first order dendrite. No recurrent axon collaterals were observed on either type I or II cells. Both cell types carried long and complex spiny appendages; however, they were most numerous on the second and higher order dendrites of type II cells. Since the soma of these cells is usually not found in the centre of its dendritic field, even if the cell is located in the center area of the neuropil, it is suggested that the dendritic trees of up to 100 neurons may be intricately interwoven, establishing clusters with intensive intercommunication by means of dendritic gap junctions. The abundance, length and complexity of the spiny appendages suggest an important role in this process, but may also be relevant instruments in enhancing the computational capabilities of these neurons, especially in time sensitive processes. When relating the physiological and the morphological results, it was noted that both type I and type II cells could respond to mesodiencephalic stimulation and were both able to trigger a rebound action potential. No significant correlations were found between cell size and the latency of the rebound.

Morphology of corticotectal cells in the primary visual cortex of hooded rats.

In primary visual cortex of hooded rats, pyramidal cells in layer V may be classified as long, medium, or short, on the basis of the layer in which the apical dendrite terminates. The present study determines which of these types of pyramidal cells project to the superior colliculus. Two different strategies were used to label corticotectal cells with horseradish peroxidase (HRP). In the first set of experiments, a large number of corticotectal cells were labeled by retrograde transport following injection of HRP into the superior colliculus. In the second set of experiments, single unit recording was used to identify corticotectal cells physiologically by antidromic activation from the superior colliculus. These cells were then impaled and labeled by intracellular iontophoresis of HRP. The results from both techniques suggest that only long pyramidal cells send an axon to the superior colliculus. These cells are distinguished by an apical dendrite that extends into layer I. We conclude that in hooded rats corticotectal cells in primary visual cortex are the long pyramids in layer V.

Morphology of cortically projecting basal forebrain neurons in the rat as revealed by intracellular iontophoresis of horseradish peroxidase

The intracellular horseradish peroxidase technique was employed to study the morphology of basal forebrain neurons that were identified as cortically projecting by antidromic invasion from the cerebral cortex. Four neurons were examined in detail; they were located at different rostrocaudal levels within the basal forebrain. Their somata were large, 30–50 μm in longest dimension, and gave rise to three to eight primary dendrites, which ramified into third- to fifth-order dendrites. The longest observed dendrite in each neuron terminated at a distance of 600–900 μm from the soma. The sizes of soma and dendritic field of the two most rostrally located cells were smaller than those of the other two cells located more caudally. Dendritic spines were seen in all four cortically projecting basal forebrain neurons. Spines had shafts of variable lengths and usually had spherical or elongated heads. The density of spines varied among the four neurons; one neuron, a type II cortically projecting basal forebrain neuron as defined physiologically by Reiner et al., 37 had a much greater number of dendritic spines than the other three neurons, which were type I neurons. No somatic spines were observed. Presumptive axons were identified in three of the four cortically projecting basal forebrain neurons. These axons originated from either the soma or a primary dendrite, and two of them gave off local collaterals, which displayed occasional bouton-like swellings. The above observations confirm and extend previous findings that cortically projecting neurons in the basal forebrain are large multipolar cells, and provide evidence to support the conclusion that these cells, although somewhat variable in size, generally have extensive dendrites which display frequent spines.

Cerebellar macroneurons in microexplant cell culture. Methodology, basic electrophysiology, and morphology after horseradish peroxidase injection.

Cerebellar macroneurons, including Purkinje cells, survive and differentiate in long-term monolayer cultures, which are prepared by a partial dissociation procedure we refer to as a microexplant technique. Intracellular recording demonstrated that these neurons were functional, showing spontaneous spiking activity and electrical excitability, and both spontaneous and evoked synaptic activity. In order to further characterize cell types, light and electron microscopic studies were performed after intracellular iontophoresis of horseradish peroxidase. Purkinje neurons were identified by their form of dendritic arborization and numerous dendritic spines. Cortical granule cells and macroneurons derived from the deep nuclei could also be demonstrated.

Effects of monocular deprivation on the structure-function relationship of individual neurons in the cat's lateral geniculate nucleus

The retino-geniculo-cortical pathways of the cat composed of at least three parallel channels. The neurons in these channels (W-, X-, and Y-cells) have different physiological properties. In earlier studies, we used a direct structure-function for analysis for individual geniculate neurons in normal cats. This was accomplished by electrophysiological characterization of the neuron followed by intracellular iontophoresis of horseradish peroxidase into the same neuron. By this technique, we demonstrated that W-, X-, and Y-cells have distinctive morphological features in the lateral geniculate nucleus of the act. We have extended this analysis in cats reared with monocular eyelid suture to 24 geniculate neurons innervated by the sutured eye in lamina A or A1. Ten of the 24 neurons (42%) had abnormal structure-function relationships. All but 1 of the normal cells were X-cells. Abnormalities included: (1) Y-cells with normal physiology but abnormally small somata; (2) Y-cells with abnormal physiology and morphology, including poor responsiveness and unusually thin, tortuous, and beaded dendrites; (3) visually unresponsive cells with morphology similar to that of the physiologically abnormal Y-cells; and (4) cells with the physiology of normal X-cells but the morphology of normal Y-cells. Furthermore, soma size measurements indicate that the abnormally small somata found in deprived geniculate laminae result from a selective effect on Y-cells.

Optical sectioning of HRP-stained molluscan neurons

The use of high-resolution differential interference contrast (DIC) microscopy on cleared whole-mounts of the circumesophageal nervous system from Hermissenda crassicornis permits visualization of neuronal morphology in detail without the need for physical sectioning. Such optical sectioning, when preceded by intracellular iontophoresis of horseradish peroxidase (HRP) permits rapid and accurate examination of the arborization of electrically characterized neurons. Details such as varicosities and terminal swellings can readily be resolved. This method has revealed new morphological features of neurons implicated in training-specific behavioral modification in Hermissenda, and promises to be of further general use for the quantitative morphometry of electrically identified neurons.

Intracellular marking of identified neurones in the superficial dorsal horn of the cat spinal cord.

Intracellular iontophoresis of horseradish peroxidase has been used to mark identified neurones in the superficial dorsal horn of cats. The cats were anaesthetized with alpha-chloralose and paralysed with gallamine triethodide. Four specific nocireceptive neurones in the marginal zone and seven neurones in the substantia gelatinosa were recovered. One of the former and four of the latter are described in detail and are illustrated as reconstructions from serial sections. The results confirm previous conclusions that the substantia gelatinosa contains neurones classified as 'inverse' and that the nocireceptive neurones of lamina I are Waldeyer Cells.

A light and electron microscopic study of the rat superior cervical ganglion cells by intracellular HRP-labeling

Horseradish peroxidase (HRP) was injected intracellularly into single neurons of the isolated rat superior cervical ganglia. Intracellular iontophoresis of HRP did not seem to damage the sympathetic neurons or to affect synaptic transmission. Under the light microscope, 9 of the 27 HRP-labeled sympathetic neurons exhibited varicosites in their dendrites, but not in their axons; the varicose dendrites came into close contacts with adjacent non-labeled neurons. With the electron microscope, the varicose dendrites of 3 separate neurons lightly stained with HRP, morphological features of synapses could be identified at the contact site: clusters of vesicles in the varicose dendrites, intercellular space of about 20 nm separating the apposed membranes, and an intermediate density on the postjunctional membrane. These findings suggest at the ultrastructural level the occurrence of dendro-dendritic and dendro-somatic synapses in mammalian sympathetic ganglia.

Ultrastructure of photoreceptors in the eye of Hermissenda labelled with intracellular injections of horseradish peroxidase.

The terminal processes of single and of pairs of identified photoreceptors in the eyes of the nudibranch mollusc Hermissenda crassicornis were studied by light and transmission electron microscopy after their somata were labelled by intracellular iontophoresis of horseradish peroxidase (HRP). The HRP spread from the somata into the axons and fine terminal processes within the neuropil of the cerebropleural ganglia. The photoreceptors ended in extensive secondary branches in the neuropil where previous electrophysiological studies had indicated the probable site of synaptic interactions between photoreceptors. Clear round vesicles (54-126 nm diameter) within labelled processes were similar to the vesicles found in the somata and axon hillocks. The terminal processes of pairs of type B photoreceptors contained different intensities of the HRP label. Uniform intensities of the HRP label were found in the terminal processes of single type B photoreceptors. These differences in intensity suggested that the terminal processes were from different type B photoreceptors. This finding suggests that the connections between type B photoreceptors are probably monosynaptic.