Pubertal exposure to anabolic androgenic steroids increases spine densities on neurons in the limbic system of male rats.

Human studies show that the number of teenagers abusing anabolic androgenic steroids (AAS) is increasing. During adolescence, brain development is altered by androgen exposure, which suggests that AAS may potentially alter central nervous system (CNS) development. The goal of the present study was to determine whether pubertal AAS exposure increased dendritic spine densities on neurons within the medial amygdala and the dorsal hippocampus. Pubertal gonadally intact male rats received the AAS testosterone propionate (5 mg/kg) or vehicle for 5 days/week for 4 weeks. To determine the long-term implications of pubertal AAS use, another set of males received the same AAS treatment and was then withdrawn from AAS exposure for 4 weeks. Results showed that pubertal AAS exposure significantly increased spine densities on neurons in the anterior medial amygdala, posterodorsal medial amygdala, and the cornu ammonis region 1 (CA1) of the hippocampus compared with gonadally intact control males. Spine densities returned to control levels within the anterior medial amygdala and the posterodorsal medial amygdala 4 weeks after withdrawal. However, spine densities remained significantly elevated after AAS withdrawal in the CA1 region of the hippocampus, suggesting that pubertal AAS exposure may have a long-lasting impact on CA1 hippocampal neuroanatomy. Since pubertal AAS exposure increased spine densities and most excitatory synapses in the CNS occur on dendritic spines, AAS may increase neuronal excitation. It is proposed that this increase in excitation may underlie the behavioral responses seen in pubertal AAS-treated male rats.

Morphology of intracellularly labeled interneurons in the dentate gyrus of the immature rat.

Although many aspects of the morphological development of interneurons in the dentate gyrus have been described, the full extent of their dendrites and local axon projections in immature rodents has not been examined. Here intracellular labeling was used to assess the branching patterns of interneurons in the dentate gyrus of rat pups between 7 and 9 days of age. Labeled neurons were located within or just below the granule cell layer, and most were classified as GABAergic basket neurons on the basis of their dendritic morphologies. All labeled interneurons exhibited immature characteristics. Spines were present on cell bodies and dendrites, and growth cones were visible on some dendrites and axons. In spite of these immature features, the dendrites and axon arbors of the labeled neurons were extensive. Many apical dendrites reached the top of the molecular layer, and a number of basal dendrites extended to the CA3 pyramidal cell layer of the hippocampus. Elaborate axon plexuses were present within the dentate gyrus itself, and axon collaterals of several neurons extended beyond the dentate gyrus to branch within regions CA3 and CA1 of the hippocampus. These results indicate that the dendrites and axon collaterals of dentate interneurons are extensive at a time when the principal neurons, the granule cells, are still proliferating. These data are consistent with the idea that GABAergic interneurons may influence granule cell development in the dentate gyrus, as well as pyramidal cell maturation in the hippocampus proper.

Distribution of modulatory inputs to the stomatogastric ganglion of the crab, Cancer borealis.

The pyloric and gastric mill neural networks in the crustacean stomatogastric ganglion receive modulatory inputs from more anteriorly located ganglia via the stomatogastric nerve. In this study we employed biocytin backfilling and immunostaining, as well as electron microscopy, to determine the origin of these inputs in the crab, Cancer borealis. Fiber counts from electron micrographs of sections through the stomatogastric nerve showed that this nerve contains 55-60 medium to large diameter fibers (1-13 microns). These fibers were individually wrapped by several layers of membrane, presumably glial in origin. There was also a single cluster of jointly wrapped, small diameter (< 1 micron) fibers that may originate from peripheral sensory somata. Biocytin backfills revealed that approximately two thirds of the individually wrapped fibers in this nerve originate from somata in the other three ganglia of the stomatogastric nervous system, including the paired commissural ganglia and the single oesophageal ganglion. There were approximately 20 biocytin-labeled somata in each commissural ganglion and 3 somata in the oesophageal ganglion. An additional ten somata were localized to the stomatogastric ganglion itself. This accounts for nearly all of the medium to large diameter fibers in the stomatogastric nerve. We used double-labeling with backfills and immunocytochemistry to determine that there are two proctolin-immunoreactive neurons and four FMRFamide-like immunoreactive neurons among the biocytin-labeled neurons in each commissural ganglion. Both peptides modulate neural network activity in the stomatogastric ganglion.(ABSTRACT TRUNCATED AT 250 WORDS)

A light and electron microscopic analysis of the mossy fibers of the rat dentate gyrus.

The axon collaterals of dentate granule cells have been analyzed with the aid of a computerized microscope, following intracellular injections of horseradish peroxidase in hippocampal slice preparations. The axon of each granule cell gives rise to approximately seven primary collaterals; these collaterals usually divide into secondary and tertiary branches, which form an extensive plexus within the hilar region of the dentate gyrus. Individual axon collaterals vary greatly in length, but most have been found to be between 100 and 300 microns long. On average, the summed lengths of the collaterals (exclusive of the parent mossy fiber) are approximately 2,300 microns. Except for an occasional collateral that is given off by a mossy fiber in the proximal part of field CA3 of the hippocampus, the collaterals of the granule cell axons are confined to the hilar region; they are rarely seen in the granule cell layer itself and have never been observed in the molecular layer. In the longitudinal dimension of the dentate gyrus, most of the collaterals are contained within a zone about 400 microns wide. The distribution of the collaterals within the hilar region is correlated with the location of the granule cell body. Those that arise from cells near the tip of the suprapyramidal blade tend to be confined to the region above field CA3; those from cells nearer the crest and from the infrapyramidal blade ramify widely throughout the hilus. Two types of varicosities are present on the collaterals. Numerous small (approximately 2 microns), round varicosities are distributed unevenly along the collaterals; in electron micrographs these varicosities can be seen to make asymmetric synaptic contacts with dendritic shafts. On average, each granule cell collateral plexus has about 160 of these varicosities. The second type of varicosity is irregular in shape and ranges from 2 to 4 microns in diameter; there is usually only one such varicosity per collateral. In all respects except size, these varicosities resemble the expansions found on the parent mossy fibers. Mossy fiber trajectories in the proximal part of field CA3 were studied after extracellular injections of HRP into localized regions of the granule cell layer. Granule cells at different locations around the blade send their mossy fibers to different depths within the pyramidal cell layer in the proximal part of field CA3. However, further distally, mossy fibers from all parts of the granule cell layer contribute to the suprapyramidal bundle that occupies the stratum lucidum.

Quantitative, three-dimensional analysis of granule cell dendrites in the rat dentate gyrus.

The three-dimensional organization of dentate granule cell dendritic trees has been quantitatively analyzed with the aid of a computerized microscope system. The dendrites were visualized by iontophoretic injection of horseradish peroxidase into individual granule cells in the in vitro hippocampal slice preparation. Selection criteria insured that the analyzed cells were completely stained and that only neurons with two or fewer cut dendrites in the distal portion of the molecular layer were analyzed. Twenty-nine of the 48 sampled granule cells had no cut dendrites. The granule cells had between one and four primary dendrites. Granule cell dendritic branches were covered with spines and most extended to the hippocampal fissure or pial surface. The mean total dendritic length was 3,221 microns with a range from 2,324 microns to 4,582 microns. The dendrites formed an elliptical plexus with the transverse spread averaging 325 microns and the spread in the septotemporal axis averaging 176 microns. On individual neurons, the maximum branch order ranged from four to eight and the number of dendritic segments ranged from 22 to 40. Approximately 63% of the dendritic branch points occurred in a zone that included the granule cell layer and the inner one-third of the molecular layer. The dendritic tree was organized so that, on average, 30% of the length was in the granule cell layer and proximal third of the molecular layer, 30% was in the middle third, and 40% was in the distal third. Comparisons were made between the dendrites of granule cells in the suprapyramidal and infrapyramidal blades of the dentate gyrus. Suprapyramidal cells had a significantly greater total dendritic length than infrapyramidal cells, their transverse spread was higher, and they had a greater number of dendritic segments. When neurons in the suprapyramidal blade were further subdivided on the basis of somal position within the depth of the cell body layer, superficial neurons were found to have a greater number of primary dendrites, more elliptical trees, and larger transverse spreads of their dendrites. There were no significant differences in dendritic segment number or total dendritic length between superficial and deep cells.

Distribution of thorny excrescences on CA3 pyramidal neurons in the rat hippocampus.

Thorny excrescences are the postsynaptic components of synapses between mossy fibers of granule cells and dendrites of CA3 pyramidal neurons in the hippocampal formation. Very little quantitative data on the number and distribution of excrescences in adult rats are available because, first, the vast majority are grouped into clusters and it is not possible to identify single excrescences within these clusters at the light microscope level. Second, clusters are of varying lengths and are distributed over hundreds of micrometers, making ultrastructural analysis prohibitively time-consuming. Here, by using three-dimensional analysis techniques at the light microscope level, we quantified the number, length, and distribution of excrescence clusters on proximal and midfield pyramidal neurons in the rat. Results indicated that proximal neurons had similar numbers of clusters on their apical and basal trees, and that cluster length was also similar. In contrast, midfield neurons had more apical than basal clusters, and apical clusters were longer. For neurons in both regions, basal clusters were located about 50% closer to somata. Overall, proximal neurons had more clusters than did midfield neurons, but the clusters were shorter; thus, proximal and midfield neurons had about the same total cluster length, and presumably the same number of single excrescences. Based on these data and on published ultrastructural measurements of single excrescences, we estimated an average of 41 excrescences/neuron, and suggest that a pyramidal neuron can be contacted by a maximum of 41 mossy fiber boutons, each from a different granule cell.

Detection of zinc in isolated nerve terminals using a modified Timm's sulfide-silver method.

An ultrastructural method for detecting the presence of zinc in isolated nerve terminals from the mammalian brain is described. This method is based on the well-known Timm's sulfide-silver technique that has been used by many investigators to detect and localize zinc-containing pathways in sections of intact brain tissue. We report here a modification of this technique that we have used to assess the homogeneity, at the electron microscopic level, of a zinc-enriched synaptosomal fraction from the rat hippocampus. This technique allows biochemical assays to be performed on samples of the same tissue if desired, and also provides the large amounts of tissue needed for synaptosomal isolation. Results indicated that all of the mossy fiber synaptosomes, identified on the basis of their large size and characteristic morphology, stained for zinc using this method, as did about one-third of the smaller synaptic profiles present in the same fraction. The method described here should be useful for determining zinc retention and localization in isolated synaptosomes from other regions of the mammalian central nervous system.

Dendritic growth and regression in rat dentate granule cells during late postnatal development.

The goal of this study was to determine whether dendritic regression occurs in granule neurons of the rat dentate gyrus during late postnatal development. In vitro hippocampal slices were prepared from rats between the ages of 14 and 60 days, and granule neurons in one portion of the suprapyramidal blade were labeled by intracellular injection of horseradish peroxidase. The dendrites of filled neurons were analyzed in both two and three dimensions directly from 400 microns thick whole-mounts. Results showed that the molecular layer expanded by approximately 50% between days 14 and 60. At every age examined, granule cell dendrites reached the top of the molecular layer, suggesting that dendrites continued to grow during this time period. In contrast, the number of dendritic segments per neuron decreased from an average of 36 to 28. Three-dimensional measurements showed that total dendritic length and surface area per granule cell did not change, suggesting that the overall dendritic tree size of granule neurons may be regulated during late postnatal development in the rodent.

Intracellular labeling of dentate granule cells in fixed tissue permits quantitative analysis of dendritic morphology.

Although cortical neurons in fixed tissue can be labeled by intracellular dye injection, it is not yet known whether the dye diffuses to the most distal tips of the dendrites, or whether all dendritic branches of a neuron are labeled. In this study we addressed these questions by injecting granule neurons from the rat dentate gyrus in fixed, 400-microns-thick hippocampal slices with the fluorescent dye Lucifer yellow and comparing the resultant dendritic morphologies with those of neurons labeled in slices maintained in vitro. Results indicated that each dendritic branch was filled to its most distal tip and that the average number of branches per neuron in the fixed tissue was the same as that seen after in vitro labeling. Thus, intracellular labeling of neurons in fixed tissue permits quantitative studies of dendritic tree structure.

Glutamate and dynorphin release from a subcellular fraction enriched in hippocampal mossy fiber synaptosomes.

A procedure is described for the isolation of intact hippocampal mossy fiber synaptosomes. Electron microscopic examination revealed numerous synaptosomal profiles which are clearly of mossy fiber origin, indicated by their large size (2-6 micron diameter) and characteristic morphology. Furthermore, this fraction is enriched in zinc and dynorphin B which appear to be concentrated in mossy fiber terminals in vivo. Synaptosomes isolated by this procedure accumulated 2-deoxyglucose and retained 88% of total lactate dehydrogenase activity after incubation at 30 degrees C for 60 minutes, indicating a high degree of membrane integrity. Oxygen consumption was stimulated 4-fold by veratridine (0.1 mM) and inhibited 90% by ouabain (1 mM), suggesting that synaptosomal metabolism remained tightly coupled to ouabain-sensitive ATPase activity. Potassium-stimulated (45 mM) release of dynorphin B was completely dependent upon the presence of extrasynaptosomal calcium, while only 30% of the evoked release of glutamate was calcium-dependent. D-aspartate, which exchanges glutamate out of the cytoplasmic pool, virtually eliminated the calcium-independent component of glutamate release. This synaptosomal preparation will be useful in identifying the factors that modulate the release of amino acid and opioid neurotransmitters from hippocampal nerve terminals and in the investigation of their presynaptic mechanisms of action.

Histamine as a neurotransmitter in the stomatogastric nervous system of the spiny lobster.

Histamine is a putative neurotransmitter in mammals and molluscs, but its role in the nervous systems of other animals is not known. This study examines the possibility that histamine is a neurotransmitter in an arthropod. Results show that first, 14 neurons in the stomatogastric ganglion of the spiny lobster respond to histamine. The response is inhibitory, is mediated by an increased conductance to chloride, and desensitizes with repeated applications of histamine. These same 14 neurons receive one type of synaptic potential from two extrinsic neurons, the "through-fibers" of the inferior ventricular nerve. This synaptic potential is also inhibitory, is mediated by an increased conductance to chloride, and is blocked when histamine receptors are desensitized. Second, assays of endogenous histamine indicate that histamine is distributed nonuniformly throughout the stomatogastric nervous system and that its distribution correlates with the axonal pathways and terminal arborizations of the inferior ventricular nerve through-fibers. Lastly, histamine is present in relatively high concentrations in the cell bodies of the through-fibers, whereas it is not detectable in other neurons in the stomatogastric system. These results suggest that histamine may be a transmitter in the lobster.

Hippocampal circuitry complicates analysis of long-term potentiation in mossy fiber synapses.

Much of the current interest in the hippocampus concerns a rapid and persistent form of synaptic plasticity, called long-term potentiation (LTP), that is a candidate substrate for some of the mnemonic functions of this structure. There are at least two kinds of LTP in the hippocampus. One form is found at the synapse between the mossy fibers of the granule cells and the pyramidal neurons of the CA3 region. Attempts to examine the mechanism underlying this form of LTP have yielded contradictory conclusions. The authors show how the complex circuitry of the dentate gyrus and adjacent hippocampus may have caused the contradictions. To overcome problems introduced by the circuitry, a specific set of procedures and criteria for evoking and identifying mossy fiber responses is proposed. Use of these or similar procedures and criteria will improve the design and interpretation of experiments on mossy fiber LTP and allow more informative comparisons among species and brain regions and across laboratories.

Comparative electrotonic analysis of three classes of rat hippocampal neurons.

We present a comparative analysis of electrotonus in the three classes of principal neurons in rat hippocampus: pyramidal cells of the CA1 and CA3c fields of the hippocampus proper, and granule cells of the dentate gyrus. This analysis used the electrotonic transform, which combines anatomic and biophysical data to map neuronal anatomy into electrotonic space, where physical distance between points is replaced by the logarithm of voltage attenuation (log A). The transforms were rendered as "neuromorphic figures" by redrawing the cell with branch lengths proportional to log A along each branch. We also used plots of log A versus anatomic distance from the soma; these reveal features that are otherwise less apparent and facilitate comparisons between dendritic fields of different cells. Transforms were always larger for voltage spreading toward the soma (V(in)) than away from it (V(out)). Most of the electrotonic length in V(out) transforms was along proximal large diameter branches where signal loss for somatofugal voltage spread is greatest. In V(in) transforms, more of the length was in thin distal branches, indicating a steep voltage gradient for signals propagating toward the soma. All transforms lengthened substantially with increasing frequency. CA1 neurons were electrotonically significantly larger than CA3c neurons. Their V(out) transforms displayed one primary apical dendrite, which bifurcated in some cases, whereas CA3c cell transforms exhibited multiple apical branches. In both cell classes, basilar dendrite V(out) transforms were small, indicating that somatic potentials reached their distal ends with little attenuation. However, for somatopetal voltage spread, attenuation along the basilar and apical dendrites was comparable, so the V(in) transforms of these dendritic fields were nearly equal in extent. Granule cells were physically and electrotonically most compact. Their V(out) transforms at 0 Hz were very small, indicating near isopotentiality at DC and low frequencies. These transforms resembled those of the basilar dendrites of CA1 and CA3c pyramidal cells. This raises the possibility of similar functional or computational roles for these dendritic fields. Interpreting the anatomic distribution of thorny excrescences on CA3 pyramidal neurons with this approach indicates that synaptic currents generated by some mossy fiber inputs may be recorded accurately by a somatic patch clamp, providing that strict criteria on their time course are satisfied. Similar accuracy may not be achievable in somatic recordings of Schaffer collateral synapses onto CA1 pyramidal cells in light of the anatomic and biophysical properties of these neurons and the spatial distribution of synapses.

Electrotonic architecture of hippocampal CA1 pyramidal neurons based on three-dimensional reconstructions.

1. The spread of electrical signals in pyramidal neurons from the CA1 field of rat hippocampus was investigated through multicompartmental modeling based on three-dimensional morphometric reconstructions of four of these cells. These models were used to dissect the electrotonic architecture of these neurons, and to evaluate the equivalent cylinder approach that this laboratory and others have previously applied to them. Robustness of results was verified by the use of wide ranges of values of specific membrane resistance (Rm) and cytoplasmic resistivity. 2. The anatomy exhibited extreme departures from a key assumption of the equivalent cylinder model, the so-called "3/2 power law." 3. The compartmental models showed that the frequency distribution of steady-state electrotonic distances between the soma and the dendritic terminations was multimodal, with a large range and a sizeable coefficient of variation. This violated another central assumption of the equivalent cylinder model, namely, that all terminations are electronically equidistant from the soma. This finding, which was observed both for "centrifugal" (away from the soma) and "centripetal" (toward the soma) spread of electrical signals, indicates that the concept of an equivalent electrotonic length for the whole dendritic tree is not appropriate for these neurons. 4. The multiple peaks in the electrotonic distance distributions, whether for centrifugal or centripetal voltage transfer, were clearly related to the laminar organization of synaptic afferents in the CA1 region. 5. The results in the three preceding paragraphs reveal how little of the electrotonic architecture of these neurons is captured by a simple equivalent cylinder model. The multicompartmental model is more appropriate for exploring synaptic signaling and transient events in CA1 pyramidal neurons. 6. There was significant attenuation of synaptic potential, current, and charge as they spread from the dendritic tree to the soma. Charge suffered the least and voltage suffered the most attenuation. Attenuation depended weakly on Rm and strongly on synaptic location. Delay of time to peak was more distorted for voltage than for current and was more affected by Rm. 7. Adequate space clamp is not possible for most of the synapses on these cells. Application of a somatic voltage clamp had no significant effect on voltage transients in the subsynaptic membrane. 8. The possible existence of steep voltage gradients within the dendritic tree is consistent with the idea that there can be some degree of local processing and that different regions of the neuron may function semiautonomously. These spatial gradients are potentially relevant to synaptic plasticity in the hippocampus, and they also suggest caution in interpreting some neurophysiological results.