Antimutagenic and anticarcinogenic effects of Phyllanthus amarus.

This study aimed to examine the antimutagenic and anticarcinogenic potential of Phyllanthus amarus Schum. et Thonn. using the bacterial preincubation mutation assay and an in-vivo alkaline elution method for DNA single-strand breaks in hamster liver cells. The aqueous extract of the entire plant showed an antimutagenic effect against induction by 2-aminofluorene (AF2), 2-aminoanthracene (2AA) and 4-nitroquinolone-1-oxide (4-NQO) in Salmonella typhimurium strains TA98 and TA100, and in Escherichia coli WP2 uvrA/pKM101. All the results were dose-dependent; however, inhibition of N-ethyl-N-nitrosoguanidine (ENNG)-induced mutagenesis was observed only with S. typhimurium TA100. The extract also exhibited activity against 2-nitrofluorene (2NF) and sodium azide-induced mutagenesis with S. typhimurium TA98 and TA100, respectively. Based on the alkaline elution method, the plant extract prevented in vivo DNA single-strand breaks caused by dimethylnitrosamine (DMN) in hamster liver cells. When the extract was administered 30 min prior to the administration of DMN, the elution rate constant decreased more than 2.5 times, compared to that of control. These results indicate that P. amarus possesses antimutagenic and antigenotoxic properties.

Dendritic bundling in layer I of granular retrosplenial cortex: intracellular labeling and selectivity of innervation.

The extrinsic projections to and from the retrosplenial cortex have been studied in detail, but the intrinsic circuitry within this region has been characterized less completely. To further define the internal connections, small injections of the retrograde, fluorescent tracer Fluorogold were made into the retrosplenial cortex of the rat. These injections label neurons in layers II-V of the contralateral homotopic cortex. In layers III-V, the labeled neurons are present over an area much larger than the injection site, but in layer II neurons are labeled in a very precise homotopic pattern. Following these injections, only the neurons in layer II display heavily labeled apical dendrites, and these labeled dendrites form tight bundles in layer Ic and Ib of the cortex and spread out in layer Ia. An examination of Golgi-stained material demonstrates that most of the neurons in layer II are small pyramidal cells with 2-3 small basal dendrites and a single, large apical dendrite that arborizes extensively in layer Ia. To verify the structure of the layer II neurons, they were intracellularly filled with Lucifer yellow. Examination of these labeled cells confirms the observations from the Golgi-stained material and demonstrates that many apical dendrites of the layer II cells angle acutely, apparently to join a bundle and/or avoid an interbundle space. Tract tracing experiments demonstrate that the anteroventral nucleus of the thalamus appears to project selectively to the region containing the dendritic bundles, whereas intracortical projections appear to terminate in layers Ib and Ic in the 30-200 microns spaces between the bundles. Furthermore, the areas containing the bundles display dense AChE staining, but the interbundle spaces are almost free of AChE staining. These findings demonstrate a form of dendritic bundling that is input and output specific and may play an important role in the regulation of thalamic inputs to the cingulate cortex.

The laminar organization of efferent neuronal cell bodies in the retrosplenial granular cortex.

The fluorescent dye technique for retrograde axonal tracing was used to characterize the laminar organization of neurons in the rat retrosplenial granular cortex (Rg) that project to cortical or thalamic targets. The data from 40 experiments demonstrate that each of the projections studied arises from a slightly different layer of cells. The commissural projection from Rg to Rg can be divided into two categories. A point to point projection originates in the superficial layer II neurons and a somewhat more widespread projection originates from neurons in layers III, Vb and Vc. The intrinsic projection from Rg to Rg arises from cell bodies that are in the same laminar position as the cells of origin of the commissural projection, but there is an additional input from neurons in layer VIb. In contrast to the homotopic projections, the commissural projection from Rgb to retrosplenial agranular cortex (Rag) originates in layer Va, while the ipsilateral projection from Rgb to Rag originates in layer Va, Vc and VIb. All of the input to the anterior thalamic nuclei originates in layer VIa, but while the ipsilateral projection originates in both superficial and deep layer VIa neurons, contralateral projections do not arise from cells in the deepest part of layer VIa.

Thalamic projections to retrosplenial cortex in the rat.

The topographic relationships between anterior thalamic neurons and their terminal projection fields in the retrosplenial cortex of the rat were characterized by experiments with the fluorescent dye retrograde labeling technique. The results demonstrate that the anterodorsal (DAD) and anteroventral (AV) nuclei project heavily to retrosplenial granular cortex (Rg) and to a lesser extent to retrosplenial agranular cortex (Rag). In contrast, the anteromedial (AM) and lateral dorsal (LD) nuclei project heavily to Rag and more lightly to Rg. Irrespective of terminal field in Rg or Rag, the neuronal cell bodies in AD and AV are organized topographically so that the neurons in the caudal part of each nucleus project to rostral retrosplenial cortex and the neurons in the rostral portion of each nucleus project to the caudal retrosplenial cortex. Further, the ventromedial AD and AV neurons project to rostral retrosplenial cortex, whereas dorsolateral neurons in both nuclei project to caudal retrosplenial cortex. LD neurons display a different topographic organization. The neurons in the medioventral part of LD project primarily to the rostral retrosplenial cortex, and the neurons in lateral LD project to the caudal retrosplenial cortex. This latter projection to the caudal retrosplenial cortex is also contributed to by neurons residing in the mediodorsal part of caudal LD. The neurons in AM that project to the retrosplenial cortex display less segregation than the AV, AD, or LD neurons. In all experiments, a number of neurons in the dorsal ventro-anterolateral nucleus were labeled by retrosplenial injections. The largest number of cells in this nucleus were labeled after Rag injections, and these were topographically organized such that the neurons projecting to the rostral Rag were located immediately deep to the internal medullary lamina, and the neurons projecting to the caudal Rag were more ventrally located. Very few thalamic neurons have axon collaterals to different areas of the retrosplenial cortex as shown by double labeling experiments. Together, these results demonstrate a highly organized thalamic projection to the retrosplenial cortex.

Two rapid methods of counterstaining fluorescent dye tracer containing sections without reducing the fluorescence.

A method is described for counterstaining neural tissue containing cells that are retrogradely labeled by fluorescent dyes or horseradish peroxidase (HRP). Specifically, protocols are detailed for the combined use of the tracers with Methylene blue for a Nissl stain or with silver methods for the detection of acetylcholine esterase. The usefulness of these techniques is evaluated in relation to cortico-cortico and thalamocortico projections. The findings indicate that the methods do not mask the labeling of the most sensitive fluorescent dyes or by HRP. Only the yellow dyes are significantly affected by the Methylene blue counterstain. Further, Fast blue labeling in neurons is not significantly diminished by the Bodian fiber stain. The effect of coverslipping sections containing fluorescent dye labeled cells also was evaluated and found to significantly extend the life of the labeling while not reducing the sensitivity. Thus the two counterstaining techniques provide excellent structural information, do not seriously affect tracer labeling and have few of the disadvantages common to other counterstaining methods.

The cortical projection of the basolateral amygdaloid nucleus in the rat: a retrograde fluorescent dye study.

The fluorescent dye, retrograde labeling technique was used to determine the extent of the projection from the basolateral nucleus of the amygdala to the neocortex in the rat. Each rat received a single cortical injection of fast blue, and in one-half of the animals, a subsequent injection of nuclear yellow was placed in a different cortical region. An analysis of the results demonstrates that the projection to the midline cortex arises in the medial neurons within the caudal two-thirds of the basolateral nucleus. This projection is directed to the anterior cingulate cortex, but not to the posterior cingulate cortex. The primary motor cortex receives a basolateral amygdala projection which originates from neurons in two areas, (1) the medial part of the anterior one-third of the nucleus and (2) the center (in the lateral to medial axis) portion of the posterior two-thirds of the nucleus. The latter neurons are situated lateral to the neurons projecting to the cingulate cortex. Somatosensory cortex injections label many fewer basolateral nucleus neurons than do motor cortex injections, but these neurons are located in a position similar to that of those labeled by motor cortex injections. Finally the gustatory cortex, which lies just dorsal to the rhinal sulcus, receives a basolateral projection from neurons in the lateroventral one-half of the basolateral nucleus. These results demonstrate that the basolateral nucleus gives rise to a rather widespread and topographically organized projection to the anterior half of the neocortex of the rat.

The topography of the mesencephalic and pontine projections from the cingulate cortex of the rat.

A projection from the rat midline cortex to the midbrain and pons has been recognized for several years. The present study is a detailed analysis of this projection using the autoradiographic technique. Small injections of [3H]amino acids were placed within individual segments of the cingulate cortex in 68 rats. The resulting material reaffirmed the existence of the cingulo-brainstem projections and demonstrated that a precise topographical relationship exists between the cingulate cortex cells of origin and their termination fields within the brainstem. The most ventral and anterior segment of the cortex (IRaa) projects to the ventral periaqueductal gray, to the dorso-medial ventral pontine nuclei and to the lateral tegmental region. Conversely, the dorso-anterior cortex (IRca) projects to the superior colliculus, the dorso-lateral periaqueductal gray, and the medio-ventral ventral pontine nuclei. The intermediate anterior cortex projects to both dorsal and ventral periaqueductal gray, lightly to the superior colliculus, and to the medio-intermediate ventral pontine nuclei. The posterior half of the infraradiata (IR) cortex projects to the dorso-lateral periaqueductal gray, to the superior colliculus, and to the region of the ventral pontine nuclei slightly lateral to the terminal zone occupied by the anterior IR cortex. Increasingly dorsal segments of the IR beta cortex project to more increasingly ventral areas of the ventral nuclei. The posterior portion of the midline cortex (retrosplenial cortex, R) does not project to the dorsal midbrain, but it does topographically project to the ventral pontine nuclei, lateral to the terminal zone of the IR axons. Increasingly, posterior regions of the R cortex project to more lateral regions of the ventral nuclei, and increasingly, dorsal cells of the R cortex project to more dorsal regions of the ventral nuclei. These data demonstrate a very precise topography of brainstem projections which may underlie the visceral and somatic motor functions of the cingulate cortex, as well as the ability of the cingulate cortex to modulate sensory information and emotional behavior.

The indusium griseum and anterior hippocampal continuation in the rat.

The morphology and connections of the indusium griseum (IG) and anterior hippocampal continuation (AHC) suggest that this cortex contains analogues to several portions of the hippocampal formation. Whereas the outer neuronal layer of this cortex is made up of cells which are similar in structure to the neurons of the granule cell layer of the dentate gyrus, the three successively deeper layers contain morphological analogues to the neurons of the dentate hilus, Ammon's horn, and the subiculum, respectively. The neurons within each of these four layers of the AHC and IG have afferent and efferent connections which are quite similar to the connections of their hippocampal counterparts. Thus, the granule cells of the IG and AHC receive laminar inputs from the entorhinal cortex, the IG-AHC itself, and the supramammillary region. Each of these three classes of inputs ends at successively more proximal positions on the dendritic tree of these granule cells. Other inputs to this region include those from the septal nuclei and the olfactory bulb. The deeper layers of the IG and AHC receive several inputs, including those from the thalamic and septal nuclei and the entorhinal cortex. The efferent cell bodies of the IG and AHC are segregated in such a way that the granule cells appear to give rise to only short connections, while the hilar cells project to the granule cells, the intermediate pyramidal neurons project to other portions of the IG and AHC and to the olfactory bulb, and the deep pyramidal neurons project to the diencephalon. These results demonstrate that the IG-AHC is a continuation of the hippocampal formation.