Programmed cell death of developing mammalian neurons after genetic deletion of caspases.

An analysis of programmed cell death of several populations of developing postmitotic neurons after genetic deletion of two key members of the caspase family of pro-apoptotic proteases, caspase-3 and caspase-9, indicates that normal neuronal loss occurs. Although the amount of cell death is not altered, the death process may be delayed, and the cells appear to use a nonapoptotic pathway of degeneration. The neuronal populations examined include spinal interneurons and motor, sensory, and autonomic neurons. When examined at both the light and electron microscopic levels, the caspase-deficient neurons exhibit a nonapoptotic morphology in which nuclear changes such as chromatin condensation are absent or reduced; in addition, this morphology is characterized by extensive cytoplasmic vacuolization that is rarely observed in degenerating control neurons. There is also reduced terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling in dying caspase-deficient neurons. Despite the altered morphology and apparent temporal delay in cell death, the number of neurons that are ultimately lost is indistinguishable from that seen in control animals. In contrast to the striking perturbations in the morphology of the forebrain of caspase-deficient embryos, the spinal cord and brainstem appear normal. These results are consistent with the growing idea that the involvement of specific caspases and the occurrence of caspase-independent programmed cell death may be dependent on brain region, cell type, age, and species or may be the result of specific perturbations or pathology.

Transneuronal retrograde transport of attenuated pseudorabies viruses within central visual pathways.

Pseudorabies virus (PRV) has been shown to be an effective transneuronal tracer within both the peripheral and the central nervous system. The only investigations of this virus in the visual system have examined anterograde transport of PRV from injection sites in the retina. In the present study, we injected attenuated forms of PRV into the primary visual cortex of both rats and cats to determine whether transneuronal retrograde infection would occur back to the retina. In rats, we made small injections into visual cortex of a strain of PRV (Bartha Blu) that contained a beta-galactosidase promoter insert. In cats, we injected PRV-M201 into area V1 of visual cortex. After a 2- to 4-day incubation period, we examined tissue from these animals for the presence of the beta-galactosidase marker (rats) or the virus itself (cats). Cortical PRV injections resulted in transneuronal retrograde infection of the lateral geniculate nucleus (LGN), thalamic reticular nucleus (TRN), and retina. PRV was retinotopically distributed in the pathway. In addition, double-labeling experiments in cats using an antibody against gamma-aminobutyric acid (GABA) were conducted to reveal PRV-labeled interneurons within the LGN and TRN. All TRN neurons were GABA+, as was a subset of LGN neurons. Only the subset of TRN neurons adjacent to the PRV-labeled sector of LGN was labeled with PRV. In addition, a subset of GABA+ interneurons in LGN was also labeled with PRV. We processed some tissue for electron microscopy to examine the morphology of the virus at various replication stages. No mature virions were detected in terminals from efferent pathways, although forms consistent with retrograde infection were encountered. We conclude that the PRV strains we have used produce a local infection that progresses primarily in the retrograde direction in the central visual pathways. The infection is transneuronal and viral replication maintains the intensity of the label throughout the chain of connected neurons, providing a means of examining detailed circuitry within the visual pathway.

Effect of cocaine self-administration on dopamine D2 receptors in rhesus monkeys.

The present study used autoradiography to examine the effects of chronic self-administration of cocaine on the density of dopamine D2 receptors in nonhuman primates. Three rhesus monkeys intravenously self-administered an average of 1.35 mg/kg cocaine per day for 18-22 months until they were euthanized immediately after a self-administration session. Binding site density of the D2 ligand [3H]raclopride (2 nM) was assessed in these monkeys as well as three untreated controls, using quantitative in vitro receptor autoradiography. As compared to untreated controls, D2 binding site density was significantly lower in the animals that self-administered cocaine in all regions of the striatum rostral to the anterior commissure. These regions include the anterior and central regions of the caudate nucleus, putamen, olfactory tubercle, and both the shell and core of the nucleus accumbens. Within the substantia nigra and ventral tegmental area, by contrast, no differences were found in the density of D2 binding sites. These findings suggest a pervasive effect of cocaine on the regulation of D2 receptors in the striatum. The lack of change within the ventral midbrain, however, suggests a differential regulation of D2 receptors in the striatum and ventral midbrain. This study confirms and extends our knowledge of the neurobiological changes in the mesolimbic dopamine system that result from chronic exposure to cocaine.

Angiotensin-(1-7) immunoreactivity in the hypothalamus of the (mRen-2d)27 transgenic rat.

The distribution of angiotensin-(1-7) immunoreactive neurons was compared to those of vasopressin-(VP) and oxytocin-(OT) immunoreactive (IR) neurons in the hypothalamus of adult (mRen-2d)27 transgenic hypertensive and Sprague-Dawley rats. In both strains, angiotensin (Ang)-(1-7)-IR cells were found in the supraoptic nucleus (SON), and in the anterior (ap-), medial (mp-), and lateral (lp-) parvocellular, and posterior magnocellular (pm-) subdivisions of the paraventricular (PVN) nucleus. Three-dimensional reconstructions showed that cells immunoreactive to Ang-(1-7) and VP were specifically co-distributed in the SON and in the pmPVN. Double-labeling neurons for both peptides revealed that both Ang-(1-7) and VP were colocalized in a subpopulation of neurons in the pmPVN and SON. In combination with previous studies, our results suggest that Ang-(1-7) and VP are colocalized, co-released and may have a combined action at a common target. In addition, the introduction of the mouse submandibular renin (mRen-2d) transgene into Sprague-Dawley rats does not appear to have altered the fundamental organization of hypothalamic peptide systems involved in fluid homeostasis.

Effect of cocaine self-administration on striatal dopamine D1 receptors in rhesus monkeys.

An array of evidence indicates that long-term exposure to cocaine alters several components of the brain dopamine system. Because the release of dopamine in the nucleus accumbens (NAc) has been implicated in mediating the reinforcing effects of cocaine, changes in dopamine function can have profound effects on drug-seeking and drug-taking behavior. The present study examined the effects of the chronic self-administration of cocaine on the D1 family of dopamine receptors in the rhesus monkey. The brains of three rhesus monkeys that had intravenously self-administered an average of 1.35 mg/kg cocaine per day for 18-22 months were compared to the brains of three cocaine-naive controls. The in vitro quantitative autoradiographic technique was used to quantify binding densities of the D1 ligand [3H]SCH-23390 on cryostat-cut sections of fresh frozen tissue. In animals that self-administered cocaine, the density of D1 binding was significantly lower in the regions of the striatum at the level where the nucleus accumbens is most fully developed. The shell of the NAc showed the largest difference with significantly lower D1 binding also detected in adjacent regions of the caudate nucleus and the putamen. No differences were found in the rostral pole of the NAc or the dorsal striatum at that level. These findings suggest that chronic self-administration of cocaine can modulate the density of dopamine D1 receptors in specific portions of the primate striatum. Such changes might underlie some of the behavioral consequences, like drug dependence and craving, of long-term cocaine use.

An ultrastructural and morphometric study of the effect of removal of retinal input on the development of the dorsal lateral geniculate nucleus.

In normal development, cell layers in the dorsal lateral geniculate nucleus (dLGN) segregate from a relatively homogeneous cell group. If all retinal input is removed prior to this segregation, the layers fail to form. In the present study, we used ultrastructural and morphometric analyses to study dLGN development in the tree shrew following neonatal removal of retinal input. The goal of the present study was to determine whether there are differences between normal animals and enucleates in the development of dLGN cells and their interrelationships with each other and/or with the surrounding glia, which might explain the failure of cellular lamination in enucleated animals. The results indicate that although the development in enucleated animals may take place somewhat more slowly, by P90 cell size and density are not significantly different from normal. These results, coupled with the observation that the dLGN in enucleates is smaller than in normals, suggest that the removal of retinal input results in dLGN cell loss. At both the light and electron microscopic level, cells in the developing normal dLGN are arranged in bands of immediately adjacent cells. In enucleates, dLGN cells are less frequently in immediate contact and are arranged in small groups or clumps which may be separated by degenerating cells. The present data suggest that the presence of retinal input may be necessary to allow dLGN cells to maintain the intercellular relationships necessary for laminar segregation to take place.

Distribution of growth cones and synapses in developing laminar and interlaminar regions of the dorsal lateral geniculate nucleus.

In the present study, we quantified the distribution of growth cones and synapses in 2 developing layers, as well as in the intervening interlaminar space of the dorsal lateral geniculate nucleus (dLGN) in tree shrews. Our goal was to gain insight into mechanisms involved in the segregation of dLGN cells into layers during development. We sacrificed tree shrews before (P0), during (P4 and P7), and after (P15) laminar segregation as well as at maturity (P90). The dLGN from each animal was sectioned horizontally, and all tissue for analysis was blocked from the middle third of the nucleus along the dorsoventral axis. Each micrograph was coded and blindly scored for the number of growth cones and synapses in layers 4 and 5 and the intervening interlaminar space. We also measured each growth cone and classified synapse type. Statistical analyses of these data reveal that neither growth cones nor synapses are significantly more common in the interlaminar space early in the period of laminar segregation (P4). By nearly a week after the interlaminar space can first be distinguished (P7), there are more growth cones in the interlaminar space than in the layers, but this difference is no longer present at (P15). These results suggest that, although neuropil development at the laminar borders may not play a role in the onset of laminar segregation, it may contribute to the widening of the interlaminar spaces once this process has begun. In addition, growth cones continuously decrease in number and become less bulbous and more linear in shape with development. Synapses, on the other hand, continuously increase in number with age and pass through a transient period characterized by heavy spinous terminations.

Cellular interrelationships during laminar segregation in the dorsal lateral geniculate nucleus.

In order to gain insight into the mechanisms involved in the formation of groupings of functionally similar cells in the developing nervous system, we have studied the formation of cell layers in the developing dorsal lateral geniculate nucleus (dLGN). To examine the possibility that a higher affinity or adhesion between cells in individual layers may play a role in laminar segregation, we studied cellular interrelationships in the dLGN of tree shrews before (P0), during (P4 and P7), and just after (P15) laminar segregation has taken place. We compared our observations at these stages of development with similar observations in the adult. In none of the cases do we see evidence of gap junctions either between adjacent neurons or between neurons and processes in the surrounding neuropil. However, we frequently observe the presence of puncta adherentes between adjacent neurons at all stages of development. These profiles are also present between neurons and cellular processes in the neuropil. We also see subsurface cisternae in all of our cases, although these are more pronounced before and during interlaminar space formation. As with the puncta adherentes, these are found both between adjacent neurons as well as between neurons and other elements in the neuropil. We also see some evidence of what appear to be cytoplasmic bridges between adjacent neurons; these are quite rare but appear to be present only before and during laminar segregation. Finally, we frequently see cytoplasmic processes interdigitated between otherwise immediately adjacent cells. These processes also are often found oriented along other portions of the neuronal plasmalemma. Whether these processes are portions of neuronal growth cones or glial processes is impossible to determine at this time. Because of the potential role glial processes may play in the formation and maintenance of laminar cell groupings during layer formation, we have also made a preliminary survey of whether glial cells can be distinguished ultrastructurally at the stages we have studied.

Quantitative changes in hippocampal structure following long-term exposure to delta 9-tetrahydrocannabinol: possible mediation by glucocorticoid systems.

Although cannabinoids exert strong effects on brain function, there have been no extensive analyses of the long-term effects of cannabinoids on mammalian brain structure. Consequently, we conducted quantitative light and electron microscopic studies on the brains of rats treated chronically with delta 9-tetrahydrocannabinol (THC) (5 X weekly for 8 months--approximately 30% of the life-span). In these studies, we found significant THC-induced changes in hippocampal structure: specifically, THC-treated animals exhibited decreased neuronal density and increased glial cell reactivity (i.e. an increase of cytoplasmic inclusions). In addition, we confirmed prior reports of THC-induced increases in adrenal-pituitary activity, since both adrenocorticotropic hormone (ACTH) and corticosterone were elevated substantially during an acute stress. However, the animals appeared to be only minimally affected behaviorally by the doses used (highest dose: 8 mg/kg) and no effects of THC were observed on several ultrastructural variables, including synaptic density. The observed hippocampal morphometric effects of chronic THC are similar to apparent glucocorticoid-dependent changes that previously have been found to develop in rat hippocampus during normal aging. Given that cannabinoids and steroids are similar in chemical structure in several respects, therefore, the present results seem to raise the possibility that chronic THC exposure may alter hippocampal anatomical structure by interactions with, or mimicry of, adrenal steroid activity.

Ultrastructural characterization of the synapses of the crossed temporodentate pathway in rats.

The present study was undertaken to define the ultrastructure of synapses of the crossed temporodentate pathway from the entorhinal cortex to the contralateral dentate gyrus and to compare the synapses of the sparse crossed pathway with those of the massive ipsilateral temporodentate pathway. The synapses of the crossed pathway were identified by using EM degeneration and EM autoradiographic techniques. For the degeneration studies, adult male Sprague-Dawley rats were killed 1, 2, or 4 days following a unilateral entorhinal cortex lesion and prepared for electron microscopy. To identify the synapses by using autoradiographic techniques, four animals received injections of 3H-proline into the entorhinal cortex, were allowed to survive for 3 days, and were prepared for EM autoradiography. Degenerating synapses of the crossed pathway that were found in the molecular layer of the dentate gyrus contralateral to a lesion formed asymmetric synapses on spines and possessed presynaptic organelles indistinguishable from synapses of the ipsilateral temporodentate pathway. The number of degenerating synapses was very low at all survival intervals (14.80 degenerating synapses/10,000 microns2 at 1 day postlesion and 1.95 degenerating synapses/10,000 microns2 at 2 days postlesion); no degenerating synapses were found at 4 days postlesion. Ninety-eight percent of the degenerating synapses found at 1 day postlesion exhibited electron-lucent degeneration. At 2 days postlesion 83% of the degenerating synapses in the dorsal blade and 18% of those in the ventral blade showed lucent degeneration; the remainder were electron dense. EM autoradiography confirmed the degeneration studies in terms of the type of terminals that were labeled and suggested that the density of the crossed pathway was higher than the degeneration results implied. We conclude that synapses of the crossed temporodentate pathway have a similar ultrastructure to synapses of the ipsilateral temporodentate pathway but exhibit a rapid form of degeneration such that they disappear very rapidly following the lesion.

The process of reinnervation in the dentate gyrus of adult rats: an ultrastructural study of changes in presynaptic terminals as a result of sprouting.

The present study was undertaken to define the ultrastructure of synapses of the crossed temporodentate pathway after they had sprouted to reinnervate the dentate gyrus following the destruction of the normal ipsilateral temporodentate pathway. The synapses of the sprouted crossed temporodentate pathway were identified at the EM level by using autoradiographic techniques and by evaluating the degeneration of the pathway following secondary lesions. Both EM autoradiography and EM degeneration revealed that the terminals of the sprouted crossed temporodentate pathway formed asymmetric synapses on spines; individual terminals appeared to make more synaptic contacts per terminal (multiple synapses) than in the case of the normal crossed pathway. In the two lesioned animals exhibiting the best labeling, labeled terminals made an average of 3.0 +/- 2.2 and 2.0 +/- 1.3 contacts per terminal. In contrast, labeled terminals in normal animals exhibited only one contact per terminal. The terminals of the sprouted pathway were also larger than those of the normal crossed pathway. The synapses of the crossed temporodentate pathway that degenerated after a secondary lesion of the entorhinal cortex exhibited both electron-lucent and electron-dense forms of degeneration at 2 days postlesion. In two animals that were quantitatively analyzed, the density of degenerating synaptic terminals was 281 and 218/10,000 microns2 in the terminal field of the sprouted crossed pathway. These values are much higher than in normal animals, where the density of degenerating synaptic terminals was only 2.12/10,000 microns2 at 2 days postlesion.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of the cells of origin of a central pathway which sprouts following lesions in mature rats.

Following unilateral destruction of the entorhinal cortical region of the adult rat, the denervated granule cells of the dentate gyrus are reinnervated as a result of the proliferation of a pathway from the surviving contralateral entorhinal area. The present study investigates the cells of origin of this lesion-induced pathway. Following HRP injections into the reinnervated dentate gyrus, heavily labeled cells were evident in layers II and III of the contralateral entorhinal area, in marked contrast to the pattern of labeling in normal animals, where labeled cells are restricted almost entirely to layer III. The atypically labeled cells in the operated animals were found predominantly in the dorsal half of the entorhinal area, and were concentrated in the medial most portion of layer II. These atypically labeled cells in layer II of the operated animals were an average of 16% larger than their unlabeled neighbors in the same lamina. This was not related to the loading with HRP, however, since in normal animals, cells in layer II which are labeled with HRP were no different in size than unlabeled cells. The atypically labeled cells in layer II of operated animals could also be identified at the electron microscopic level, and could be distinguished from the cells in layer III which normally project to regio superior of the contralateral hippocampal formation. While labeled cells were evident in layers II and III following injections into the reinnervated dentate gyrus, no labeled cells were found in the presubiculum or parasubiculum. In combination, these results suggest (1) the pathway which reinnervates the dentate gyrus from the contralateral entorhinal area originates predominantly, if not exclusively, from the cells in layer II, (2) these cells in layer II have the same preferential distribution within the entorhinal area as the rare lightly labeled cells which can be found contralateral to an injection in normal animals and (3) cells which participate in the reinnervation are larger than their unlabeled neighbors which presumably do not give rise to fibers which reinnervate the contralateral dentate gyrus. Since the cells in layer II which sprout following lesions can be identified at both the light and elctron microscopic level, a potentially valuable model system is available in which to analyze cellular changes during sprouting.

Analysis of collateral projections with a double retrograde labeling technique.

A method is described whereby collateral innervation by a single cell type may be determined by double retrograde labeling with horseradish peroxidase (HRP) and tritiated bovine serum albumin ([(3)H]BSA). The projections from the entorhinal area to the hippocampal formation were analyzed as a model system, since layer III pyramidal cells project bilaterally to the hippocampus. Following HRP injections into the hippocampus of one side, and [(3)H]BSA injections into the opposite hippocampus, double labeled cells were found in layer III of the entorhinal cortex. These doubly labeled cells could best be viewed in plastic embedded material, whereas frozen sections were found to be inadequate for clearly distinguishing between silver grains and HRP reaction product.