The hippocampal region of rats and mice after a single i.p. dose of clioquinol: loss of synaptic zinc, cell death and c-Fos induction.

Clioquinol (CQ) is able to chelate synaptic zinc, which can modulate excitatory and inhibitory neurotransmission. In humans, CQ was associated with cases of transient global amnesia (TGA) and with the neurodegenerative syndrome subacute myelo-optico-neuropathy (SMON). We examined the CQ induced loss of synaptic zinc, cell death and c-Fos induction in rats and mice. In rats, we found a strong reduction of histochemically reactive synaptic zinc no later than 4 h after the injection of the lowest dose of CQ (50 mg/kg) and, for all doses used, a return to control levels after 48 h. There was no evidence of cell death for any dose and up to 1 week after CQ injections. Only a slight induction of c-Fos was seen in the hippocampus for the higher doses used (100-200 mg/kg). In mice injected with 100 mg/kg, CQ also resulted in a fast loss of synaptic zinc. c-Fos was induced after 4 h in cell populations of the hippocampal region and other parts of the telencephalon, and substantially increased after 24 h. One day after the injection we found a pattern of cell loss (hilus, parts of CA3, CA1 and layer III of the medial entorhinal cortex) reminiscent of that seen in models of temporal lobe epilepsy. In conjunction with published data on the behavioral effects of zinc chelation and the modulatory effects of zinc in excitatory neurotransmission, our results indicate that the loss of synaptic zinc may have been involved in TGA and the neuropathology associated with SMON.

Estimators of the precision of stereological estimates: an example based on the CA1 pyramidal cell layer of rats.

Because of the complex and dynamic structure of the brain, there is perhaps no other organ system in which the application of stereological methods can contribute so much with regard to understanding normal and pathological processes. In order to design the studies in an optimal manner, with regard to the number of individuals, sections, probes, and to be able to critically evaluate the stereological studies made by others, it is important for neuroscientists to have an understanding of the precision or reproducibility of a stereological estimation procedure. This precision or reproducibility is often referred to as the coefficient of error of the estimate, which is a statistical expression for the size of the standard error of the mean of repeated estimates, relative to the mean of the estimates. Like the 'margin of error' associated with public opinion polls, it indicates how much the estimate would vary if it were repeated numerous times. It is difficult and time consuming to empirically derive the coefficient of error of estimates made of features observed in histological preparations. To overcome this obstacle, it is common practice to try to get a feeling for the precision of an estimate by estimating the coefficient of error itself. In this paper, we will compare and discuss the coefficient of error of estimates of volume and cell number made with different numbers of sections and probes in the CA1 pyramidal cell layer of the rat hippocampus. The conclusions drawn from this analysis indicate that, using practically feasible and anatomically sensible sampling schemes, the Gundersen-Jensen coefficient of error estimator or the 'Split-Sample' coefficient of error estimator can provide useful information about the precision of stereological estimates even in highly irregular brain regions and requires little work.

Distribution of acetylcholinesterase in the hippocampal region of the mouse: II. Subiculum and hippocampus.

The distribution of acetylcholinesterase (AChE) was examined in the subiculum and hippocampus of the adult mouse (Mus musculus domesticus). A distinctly stratified AChE pattern was observed in both areas and was compared in detail with cytoarchitectural fields and layers. In the subiculum, the lateral plexiform layer was lightly stained superficially and moderately stained at depth, where it abutted the lateral, moderately stained cell layer. Medially, a moderately stained deep plexiform layer separated the darkly stained superficial plexiform layer from the equally AChE-intense cell layer. At depth, the subicular cell layer was delimited by a band of very high AChE activity. In regio superior of the hippocampus, AChE-intense bands delimited the moderately stained strata moleculare, radiatum, and oriens toward the subjacent layers. In the stratum pyramidale, precipitate insinuated between the cell bodies gave a dark appearance to the deep part of the layer. The homologous strata of regio inferior appeared darker, but the relative staining intensities corresponded largely to those in regio superior. AChE activity in the layer of mossy fibers was almost absent septally but increased gradually to very high levels temporally. The AChE staining pattern, in conjunction with cytochemical and morphological evidence, strongly suggests a division of the pyramidal cell layer of the mouse and rat into superficial and deep substrata and discourages the definition of a prosubiculum in rodents. A comparative analysis of the AChE pattern reveals that: 1) in the subiculum, differences between species are observed within a generalized pattern of medial darkly staining and lateral lightly staining portions; 2) in the hippocampus, a conservation of the AChE pattern is seen in strata associated with intrinsic hippocampal connection; while 3) numerous interspecific differences are found in the stratum moleculare.

Distribution of acetylcholinesterase in the hippocampal region of the mouse. III. The area dentata.

The distribution of acetylcholinesterase (AChE) was examined in the area dentata of the adult mouse (Mus musculus domesticus). A distinctly stratified distribution of the enzyme was observed and was compared in detail with cytoarchitectural fields and layers. In the stratum moleculare, bands of relatively high AChE activity were seen immediately beneath the pia, at the borders between the outer, middle, and deep portions of the stratum moleculare, and superficial to the granule cell layer. AChE activity was low in the intervening parts of the stratum moleculare. In contrast to the rat, three sublaminae could be discerned in the hilus of the mouse at most septotemporal levels: a limiting subzone, a hilar plexiform layer, and a deep hilar cell mass. Deep to the granule cell layer, AChE activity was high in the limiting subzone and, septally, in the hilar plexiform layer. The deep hilar cell mass stained lightly towards the septal pole of the region but darker at more temporal levels. Numerous AChE-stained cells were seen in the hilus, with the exception of the most temporal levels. A comparative analysis of the AChE pattern of the area dentata reveals that 1) AChE-intense supra- and infragranular bands are found in all mammals, whereas 2) considerable difference between various strains of mice and between species are seen in the stratum moleculare. The functional significance of the AChE pattern is discussed in relation to species differences and connectivity and also with respect to possible activities of the enzyme other than hydrolysis of ACh, which may be involved in growth-related functions and in the plastic and degenerative processes observed in Alzheimer's disease.

Comparative quantitative study of the hippocampal region of two closely related species of wild mice: interspecific and intraspecific variations in volumes of hippocampal components.

To investigate the structural changes in the hippocampal region (subiculum, Ammon's horn, and area dentata) associated with speciation, the volumes of homologous components of this region of the forebrain were compared in the two closely related murine rodent species, Apodemus flavicollis (yellow-necked wood mice) and A. sylvaticus (long-tailed field mice), and in two geographically separated groups of A. sylvaticus. With the exception of the mossy fiber zone, no significant differences were found in the relative sizes of the components of Ammon's horn. Significant interspecific differences were found in the deep subiculum, in the intermediate (medial perforant path) and deep (commissural-associational) zones of the molecular layer of the area dentata, and in the hilus. A significant intraspecific difference was found in the combined intermediate and superficial (medial and lateral perforant path) zone of the dentate molecular layer. Differences found in the relative size of the hilus and the mossy fiber zone of Ammon's horn were complementary in that the combined volumes of these zones, which are both terminal fields of dentate granule cells, did not differ in the species. This finding provides evidence that the distribution of the terminal field of a projection system can be altered while the size of the terminal field is maintained. Within the hippocampal region, components of Ammon's horn appear well suited for quantitative comparative studies that span taxonomic units beyond the species level. In agreement with previous quantitative studies, differences in the volumes of components of Ammon's horn found in species of different orders are more likely to reflect phylogenetic trends rather than changes resulting from specializations of the particular species used. This is not true for the subiculum and the components of the area dentata. Experimentally induced changes in the connectivity of the hippocampal region are discussed in terms of the structural changes which may be responsible for the quantitative differences observed between the two species studied here.

Distribution of acetylcholinesterase in the hippocampal region of the mouse: I. Entorhinal area, parasubiculum, retrosplenial area, and presubiculum.

The distribution of acetylcholinesterase (AChE) was examined in the multilayered posterior part of the hippocampal region of the adult mouse (Mus musculus domesticus), namely, the entorhinal area, the parasubiculum, the presubiculum, and those parts of the retrosplenial cortex that extend into the posterior hippocampal region (area retrosplenialis 29d and 29e). A modification of the Koelle copper thiocholine method was employed for the histochemical demonstration of AChE. The AChE staining resulted in a distinctly stratified pattern, which has been compared in detail with the fields and layers defined by cyto- and fibro-architecture. Most of the enzyme activity was located in the neuropil, but both moderately and intensely stained nerve cell bodies were observed too. In the entorhinal area two main subfields were identified, which have been designated pars medialis and pars lateralis. In pars medialis, the superficial two thirds of layer I, the interstices between the stellate cell bodies in layer II, and layers IV and VI showed moderate to high content of AChE, whereas layer V and, especially, layer III were poor in enzyme activity. A particular feature was the occurrence of cone-shaped, darkly stained areas within layer II and, occasionally, the deep part of layer I. The staining of pars laterais differed in several respects from that of pars medialis, the most prominent feature being a less conspicuous stratification. In addition, intensely stained somata occurred more frequently than in pars medialis, although they still constituted only a very small minority of the total number of nerve cell bodies. In the parasubiculum, a clear cytoarchitectural subdivision into a posterolateral parasubiculum a and an anteromedial parasubiculum b was observed. These subfields showed, however, only minor differences in AChE staining. Thus, in both subfields, layers I and IV stained intensely, whereas layers II and III showed moderate to intense staining. Layers V and VI did not differ in appearance from the corresponding layers of the entorhinal area. The retrosplenial areas 29d and 29e appeared very light in the AChE pattern, area 29e being the better stained. The presubiculum was very rich in AChE, with layers, I, III and IV being particularly intensely stained. The small nerve cell bodies of layer II were unstained, whereas the intervening neuropil was intensely stained. The distribution of AChE in the mouse was compared with that in the rat, guinea pig, and rabbit, described previously. The staining pattern is largely similar in all four species, but striking species-specific differences do exist.(ABSTRACT TRUNCATED AT 400 WORDS)

Neuron numbers in the presubiculum, parasubiculum, and entorhinal area of the rat.

Estimates of neuron numbers have been useful in studies of neurodegenerative disorders, and in their animal models, and in the computational modeling of hippocampal function. Although the retrohippocampal region (presubiculum, parasubiculum, and entorhinal area) is an integral part of the hippocampal circuitry and is affected selectively in a number of disorders, estimates of neuron numbers in the rat retrohippocampal region have yet to be published. Such data are necessary ingredients for computational models of the function of this region and will also facilitate a comparison of this region in rats and primates, which will help to determine how well we may expect rat models to predict function and dysfunction in primate brains. In the present study, we used the optical fractionator to estimate the number of neurons in the rat retrohippocampal region. The following estimates were obtained: 3.3 x 10(5) in presubicular layers II and III, 1.5 x 10(5) in parasubicular layers II and III, 2.2 x 10(5) in the combined pre- and parasubicular layers V and VI, 6.6 x 10(4) in medial entorhinal area (MEA) layer II, 1.3 x 10(5) in MEA layer III, 1.9 x 10(5) in MEA layers V and VI, 4.6 x 10(4) in lateral entorhinal area (LEA) layer II, 1.2 x 10(5) in LEA layer III, and 1.4 x 10(5) in LEA layers V and VI. A surprising finding was the large numbers of neurons in the pre- and parasubiculum, which indicate an important role of these areas in the control of the entorhino-hippocampal projection. A comparison of the numbers of neurons in the hippocampus and entorhinal areas in rats with similar estimates in humans revealed that gross input-output relations are largely conserved. Differences between rats and humans may be accounted for by more prominent entorhino-neocortical projections in primates and consequent increases in the number of neurons in the hippocampus and retrohippocampal region, which are dedicated to these projections.

Neurons of origin of zinc-containing pathways and the distribution of zinc-containing boutons in the hippocampal region of the rat.

Recent methods allow the study of neurons that contain zinc in synaptic vesicles of their boutons (Timm-stainable boutons) by the intravital precipitation (local or throughout the CNS) of the vesicular zinc with selenium compounds and its subsequent retrograde transport to the parent neurons, where the precipitate can be silver enhanced. The present study is a description of the distribution of zinc-containing neurons, their possible connections and their terminal fields within the hippocampal region of the rat. Problems inherent to the methods are addressed. Finally, based on the results and a review of literature, the possible function of zinc in the hippocampal region is considered. Neurons which contain silver-enhanced precipitates were observed in layers II, V and VI of the lateral entorhinal area and in layers V and VI of the medial entorhinal area. In the parasubiculum, labeled cells were seen in layer II/III of the parasubiculum a and in layer V. Labeled cells in the presubiculum were concentrated in layers III and V, in the hippocampal pyramidal cell layer and the dentate granule cell layer, but neurons containing precipitates were largely absent from the subiculum. Zinc-containing axonal boutons defined subpopulations within principal hippocampal neuron populations. Within layer II of the lateral entorhinal cortex and the pyramidal cell layer for regio inferior deeply situated neurons were labeled, whereas superficially placed pyramidal cells were labeled in regio superior. The neuropil staining described in the present study corresponded to that found in earlier studies. However, glial and vascular staining or unspecific background were largely absent, and the neuropil staining could unequivocally be identified light microscopically. Methodological problems are most prominently reflected in unstained mossy fibers in some animals. Based on series from animals treated with decreasing doses of sodium selenite and increased survival times, this problem can be related to small amounts of circulating reactive selenium and a competition of zinc compartments (vesicles) for the selenium. Staining will fail where the competition prevents individual compartments from reaching a threshold amount of zinc precipitate for silver amplification. A guide to evaluate histological material is provided. The distribution of zinc-containing boutons and their cells of origin indicate that zinc-containing and zinc-negative projections are not organized as parallel pathways. The mossy fibers provide an example of a pure zinc-containing pathway. Projections from regio superior to the dorsal presubiculum are likely to be zinc-negative while projections from the same area to the subiculum are zinc-containing.(ABSTRACT TRUNCATED AT 400 WORDS)

Labeling of the neurons of origin of zinc-containing pathways by intraperitoneal injections of sodium selenite.

Intraperitoneal injections of sodium selenite result in the formation of zinc-selenium complexes in zinc-containing axonal boutons ("Timm stainable boutons"), and the zinc-selenium precipitate can be rendered visible in histological sections by silver enhancement. In this work we present evidence, in the rat, that zinc-selenium precipitates formed in vivo after intraperitoneal injections of sodium selenite are translocated by colchicine-sensitive retrograde transport to neural perikarya when animals are allowed to survive 12-24 h after the selenite administration. Silver enhancement renders the perikaryal precipitates visible and thus demonstrates the perikarya of all zinc-containing neurons in the CNS simultaneously. Large populations of zinc-containing neurons identified by the method are found in layers II, III, and VI of all neocortical areas, in the superficial and deep layers of the prepyriform areas and, with a high degree of regional differentiation, in the retrosplenial, entorhinal, para- and presubicular cortices, the hippocampal formation and the amygdaloid complex. Zinc-containing cells were absent from the caudate-putamen, nucleus accumbens and septal complex. Labeled zinc-containing cells are absent in non-telencephalic parts of the brain. The findings indicate that the zinc-containing circuitry of the brain mainly serves in telencephalic information processing.

Distribution of mossy fibres in the hippocampus of two closely related species of mice.

The relative volumes of different parts of the mossy fibre projection system- the suprapyramidal mossy fibres (SP-MF) and the intra- and infrapyramidal mossy fibres (IIP-MF)- were determined in the hippocampi of Apodemus sylvaticus and A. flavicollis. The mean contribution of the IIP-MF to the mossy fibre system differed significantly between the two species. An increased relative size of the IIP-MF as compared to the SP-MF in A. sylvaticus was linked with a reduction in the relative size of the hilus found in a previous study. In addition, the two species differed significantly in the amount of intraspecific variability observed. Despite size differences, the distribution of volume along the dorsoventral axis of the hippocampus was remarkably similar for the two species. We also compared measures from five mid-septotemporal sections with results for the whole structure. Mid-septotemporal measures correctly predicted the direction of difference in relative size but overestimated its magnitude in the two species investigated.

Asymmetry in the hippocampal region specific for one of two closely related species of wild mice.

The volumes of the components of the left and right hippocampal regions were compared in male, adult specimens of the two closely related species of wild mice, Apodemus flavicollis (yellow-necked wood mice) and Apodemus sylvaticus (long-tailed wood mice). In one of the species, Apodemus flavicollis, the component containing the deep layers of the subiculum was found to be significantly larger in the hippocampal region of the left side than it was in that of the right side. No other significant asymmetries were found in the hippocampal components of the two species. These findings indicate that significant asymmetries in localized brain regions can arise during a single speciation event.

Late postnatal growth in the dentate area of the rat hippocampus compensates for volumetric changes caused by early postnatal toluene exposure.

The present report assesses the reversibility of previously described quantitative morphological alterations in the dentate area of the hippocampi of rats exposed to 500 ppm toluene for 12 h daily from postnatal day 1 (P1) to P28. Following the exposure, rats were allowed to survive until P120. The volumes of hippocampal components, as they appear in Timm-stained histological sections, were determined using the Cavalieri estimator. At P120, no differences were apparent in the volumes of the dentate components of control and experimental animals. The differences observed immediately after exposure were eliminated by significantly larger increases in the sizes of early affected components in experimental animals. Different periods of neurogenesis in rodents and primates are briefly discussed as possible confounders of rodent models of permanent toluene-induced morphological alterations in primates.

A quantitative evaluation of the neurotoxic effect of silver on the volumes of the components of the developing rat hippocampus.

The volumes of the components of the hippocampus of rats subjected to subcutaneous injections of silver during the first 4 postnatal weeks were compared to those of littermate controls. Of the 14 components measured, only the pyramidal cell layer was found to be significantly smaller in the treated animals. These findings indicate that the perikaria of the pyramidal cells are either the first elements in the developing hippocampus to show signs of silver toxicity or that they are the selective sites of silver neurotoxicity. The volumetric approach is shown to be a sensitive means by which small localized neurotoxic effects can be detected.

The effect of low-level toluene exposure on the developing hippocampal region of the rat: histological evidence and volumetric findings.

With the intention of investigating possible morphological alterations effected by toluene in the developing CNS, rat pups were exposed to 100 ppm and 500 ppm of atmospheric toluene from postnatal day 1 until sacrifice at postnatal day 28, when the hippocampal region (area dentata, Ammon's horn, subiculum) was examined light microscopically and alterations in the volumes of the layers of the subdivisions were determined. The layers of Ammon's horn and the subiculum were not affected qualitatively or quantitatively by the 500 ppm exposure. Within the area dentata, the volume of the granule cell layer was 6% smaller in animals exposed to 100 ppm and 13% smaller in animals exposed to 500 ppm than they were in controls. The volumes of the hilus, which is a terminal field of granule cell axons, and the commissural-associational zone of the dentate molecular layer, which is the terminal field of the hilar projection to the granule cells, were smaller (12% and 19%) in animals exposed to 500 ppm than they were in controls. Argyrophilic cells were found in the granule cell layer of all animals exposed to 500 ppm. Pronounced granule cell degeneration was found in one animal exposed to 500 ppm. The granule cell layer of animals exposed to 100 ppm appeared qualitatively normal. The alterations reported here support the few earlier reports of morphological alterations in the CNS of adult laboratory animals. Effects of toluene similar to those described, that is alterations in specific neuron populations and their afferent and efferent terminal fields may complement changes in neurophysiology and behavior that have been observed in prenatally and perinatally exposed rodent pups. Causal relationships, however, remain to be elucidated.

Dose-dependent bimodal effect of low-level lead exposure on the developing hippocampal region of the rat: a volumetric study.

The hippocampal region is known to accumulate experimentally applied and environmentally occurring lead and to be implicated in lead-induced behavioral deficits. With the intention of investigating morphological changes resulting from the lower range of exposures that produce behavioral alterations in laboratory animals, the volumes of hippocampal components were determined in groups of rats exposed from postnatal days 0-21 to different concentrations of lead through the maternal water supply (initially 109 ppm, subsequently replicated with 109, 218, 436 and 872 ppm). Significant effects were found in the mossy fiber zone, the granule cell layer and the commissural-associational zone of the dentate molecular layer of the group exposed to 109 ppm lead and the pyramidal cell layer of regio inferior (CA3) of Ammon's horn in the group exposed to 218 ppm lead. These results provide evidence that lead primarily affects newly formed neuronal components. In contrast to the results of previous studies, which used larger exposures to lead, the components affected in the group exposed to 109 ppm increased in volume. In addition, no significant effects were observed in the groups exposed to 436 and 872 ppm. Based on these findings and additional evidence from the literature, it is concluded that lead has a dose dependent bimodal influence on developing hippocampal components. While the volumetric changes related to lead were of the same magnitude and direction in the initial and replication studies, the absolute volumes of the affected hippocampal components were different. The differences in "baseline" values in the two studies are more likely related to differences in prenatal conditions.

Postnatal development of zinc-containing cells and neuropil in the visual cortex of the mouse.

The postnatal development of zinc-containing synaptic boutons and their cells of origin in the visual cortex of a pigmented mouse is described. Two phases can be distinguished. During the early phase zinc-containing neuropil is first apparent by postnatal day 3. By day 7 a light, but distinct neuropil staining sketches the primary and secondary visual cortices. The primary visual area contains light precipitate in layers V and VI as well as the monocular portion of layer II/III. The secondary visual areas contain slightly denser precipitate in layers II/III through VI. The transition to the second phase is marked by a large increase in precipitate density by day 11. Thereafter, the intensity of the neuropil staining increases to day 28, first in layer II/III and then in layer V, as the adult pattern of neuropil staining gradually develops. In the primary visual cortex precipitate is dense in layers II/III and V, moderate in layer VI, and sparse in layers I and IV. In the secondary visual areas the precipitate is dense in layers II/III and V and moderate in the lower portion of layer I and in layers IV and VI. Cells of origin of zinc-containing boutons are visible by the end of the second postnatal week in layer II/III of the secondary visual cortex. By 21 days of age the pattern of staining in the mature mouse is established, and cells in layers II/III and VI are labeled in both the primary and secondary visual cortices. The developmental sequence of zinc-containing cells and neuropil does not preclude an involvement of zinc in the postnatal regulation of NMDA receptor function.

Zinc-containing neurons are distinct from GABAergic neurons in the telencephalon of the rat.

We used a double-labeling protocol that combined the silver amplification of endogenous zinc with routine immunocytochemistry to determine if telencephalic neurons that exhibit GABA-, calbindin- or parvalbumin-like immunoreactivity give rise to zinc-containing boutons. We did not observe telencephalic neurons double-labeled for zinc and GABA or parvalbumin. Zinc and strong calbindin immunoreactivity were colocalized in hippocampal CA1 pyramidal cells and dentate granule cells. Other strongly calbindin-immunoreactive neurons of the telencephalon were never double-labeled. We conclude that GABAergic cells do not contain histochemically reactive zinc and, therefore, are unlikely to use this pool of zinc as a neuromodulator. This observation does not support an in vivo significance of the modulation of GABA receptors by zinc such as has been observed in vitro. In CA1 of the hippocampus, we observed the histochemical label for zinc in all visibly calbindin-immunoreactive pyramidal cells and vice versa. Thus, two markers define a subpopulation of hippocampal pyramidal cells.

Fluoro-Gold tracing of zinc-containing afferent connections in the mouse visual cortices.

To identify zinc-containing projections to the visual areas, we injected Fluoro-Gold into the occipital cortex of the mouse. Five days later, the mice underwent an intravital selenium-labeling procedure to demonstrate the somata of neurons that give rise to zinc-containing boutons. Numerous double-labeled cells were seen in the ipsi- and contralateral primary (layers II/III and VI), and secondary visual cortices (layers II/III and VI). A few double-labeled cells were apparent in other cortical areas concerned with visual processing: the orbital cortex (layers II and III), the posterior portion of the medial agranular frontal cortex (layer V/VI border), and the temporal cortex (layer VI). The cingulate, retrosplenial, perirhinal, and lateral entorhinal cortices had lamina projecting to the visual cortex and separate lamina harboring zinc-containing cells. A spatial segregation of fluorescent and zinc-containing neurons was also seen in the claustrum. This integration or segregation of projecting and zinc-containing neurons may reflect the function of the cortical areas. N-methyl-D-aspartate receptor function is antagonized by physiological concentrations of zinc in vitro. It is proposed that zinc-positive projections from areas that perform basic visual functions are less likely to be modified by N-methyl-D-aspartate receptor-mediated processes than the zinc-negative connections from associational areas.

Zinc-positive afferents to the rat septum originate from distinct subpopulations of zinc-containing neurons in the hippocampal areas and layers. A combined fluoro-gold tracing and histochemical study.

The purpose of the present study was to examine whether zinc-positive and zinc-negative hippocampal neurons in rats differed with respect to their projections to the septum. By combining retrograde axonal transport of the fluorescent tracer Fluoro-Gold with histochemical demonstration of zinc selenide complexes in zinc-containing neurons after intraperitoneal injection of sodium selenite, we were able to visualize the distribution of retrogradely Fluoro-Gold labeled neurons and zinc-containing neurons in the same sections. After unilateral injection of Fluoro-Gold into the rat septum a few retrogradely labeled cells were observed in layer IV of the ipsilateral medial entorhinal area, and numerous labeled cells were observed mainly in the superficial layers of the ipsilateral subicular areas and throughout the CA1 and CA3 pyramidal cell layers, as well as in the contralateral CA3 pyramidal cell layer. Zinc-containing neurons were observed in layers IV-VI of the medial entorhinal area, layers II and III of the parasubiculum, layers II, III and V of presubiculum, and in the superficial CA1 and deep CA3 pyramidal cell layers. Cells double-labeled with Fluoro-Gold and zinc selenide complexes were primarily located in distal (relative to the area dentata) parts of the superficial CA1 pyramidal cell layer and distal parts of the deep CA3 pyramidal cell layer and in layers II and III of presubiculum. Only a very few double-labeled cells were seen in the contralateral CA3. The result demonstrates that the hippocampo-septal projection of rats is a mixture of zinc-positive and zinc-negative fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of acetylcholinesterase and zinc in the visual cortex of the mouse.

The distributions of acetylcholinesterase (AChE) and zinc-containing boutons and their cells of origin in the visual cortex of the house mouse (Mus musculus domesticus) are described. The primary visual area is defined by both acetylcholinesterase and zinc staining. The AChE staining pattern is dark in upper layer I and layers IV and VI. It is light in layers II/III and V. The lack of a densely stained layer IV in the secondary visual cortices defines the borders between primary and secondary areas. Large, multipolar AChE-positive neurons are located throughout the cortical layers, but preferentially in layer VI. Dense zinc-positive neuropil in the primary visual cortex is apparent in layer Ib, upper layer II/III, and layers V and VI. Neurons that give rise to zinc-containing boutons are situated in layers II/III and VI. The medial and lateral borders can be distinguished by a bold contrast of staining in lower layer II/III; the secondary areas have more zinc-positive neurons, and the neuropil stains darker. A surprising observation of this study is the disparity between the mouse and rat visual cortex of the AChE staining pattern. Layer V is very light in the mouse, whereas a dark stain has been described in layer V of the rat. Layer VI stains heavily in the mouse while less AChE activity has been observed in layer VI of the rat.