Zinc-induced collapse of augmented inhibition by GABA in a temporal lobe epilepsy model.

In the kindling model of temporal lobe epilepsy, several physiological indicators of inhibition by gamma-aminobutyric acid (GABA) in the hippocampal dentate gyrus are consistent with an augmented, rather than a diminished, inhibition. In brain slices obtained from epileptic (kindled) rats, the excitatory drive onto inhibitory interneurons was increased and was paralleled by a reduction in the presynaptic autoinhibition of GABA release. This augmented inhibition was sensitive to zinc most likely after a molecular reorganization of GABAA receptor subunits. Consequently, during seizures, inhibition by GABA may be diminished by the zinc released from aberrantly sprouted mossy fiber terminals of granule cells, which are found in many experimental models of epilepsy and in human temporal lobe epilepsy.

Impaired electrical signaling disrupts gamma frequency oscillations in connexin 36-deficient mice.

Neural processing occurs in parallel in distant cortical areas even for simple perceptual tasks. Associated cognitive binding is believed to occur through the interareal synchronization of rhythmic activity in the gamma (30-80 Hz) range. Such oscillations arise as an emergent property of the neuronal network and require conventional chemical neurotransmission. To test the potential role of gap junction-mediated electrical signaling in this network property, we generated mice lacking connexin 36, the major neuronal connexin. Here we show that the loss of this protein disrupts gamma frequency network oscillations in vitro but leaves high frequency (150 Hz) rhythms, which may involve gap junctions between principal cells (Schmitz et al., 2001), unaffected. Thus, specific connexins differentially deployed throughout cortical networks are likely to regulate different functional aspects of neuronal information processing in the mature brain.

Axo-axonal coupling. a novel mechanism for ultrafast neuronal communication.

We provide physiological, pharmacological, and structural evidence that axons of hippocampal principal cells are electrically coupled, with prepotentials or spikelets forming the physiological substrate of electrical coupling as observed in cell somata. Antidromic activation of neighboring axons induced somatic spikelet potentials in neurons of CA3, CA1, and dentate gyrus areas of rat hippocampal slices. Somatic invasion by these spikelets was dependent on the activation of fast Na(+) channels in the postjunctional neuron. Antidromically elicited spikelets were suppressed by gap junction blockers and low intracellular pH. Paired axo-somatic and somato-dendritic recordings revealed that the coupling potentials appeared in the axon before invading the soma and the dendrite. Using confocal laser scanning microscopy we found that putative axons of principal cells were dye coupled. Our data thus suggest that hippocampal neurons are coupled by axo-axonal junctions, providing a novel mechanism for very fast electrical communication.

Proximally targeted GABAergic synapses and gap junctions synchronize cortical interneurons.

Networks of GABAergic interneurons are implicated in synchronizing cortical activity at gamma frequencies (30-70 Hz). Here we demonstrate that the combined electrical and GABAergic synaptic coupling of basket cells instantaneously entrained gamma-frequency postsynaptic firing in layers 2/3 of rat somatosensory cortex. This entrainment was mediated by rapid curtailment of gap junctional coupling potentials by GABAA receptor-mediated IPSPs. Electron microscopy revealed spatial proximity of gap junctions and GABAergic synapses on somata and dendrites. Electrical coupling alone entrained postsynaptic firing with a phase lag, whereas unitary GABAergic connections were ineffective in gamma-frequency phasing. These observations demonstrate precise spatiotemporal mechanisms underlying action potential timing in oscillating interneuronal networks.

Morphological features of the entorhinal-hippocampal connection.

The goal of this review in an overview of the structural elements of the entorhinal-hippocampal connection. The development of the dendrites of hippocampal neurons will be outlined in relation to afferent pathway specificity and the mature dendritic structure compared. Interneurons will be contrasted to pyramidal cells in terms of processing of physiological signals and convergence and divergence in control of hippocampal circuits. Mechanisms of axonal guidance and target recognition, target structures, the involvement of receptor distribution on hippocampal dendrites and the involvement of non-neuronal cellular elements in the establishment of specific connections will be presented. Mechanisms relevant for the maintenance of shape and morphological specializations of hippocampal dendrites will be reviewed. One of the significant contexts in which to view these structural elements is the degree of plasticity in which they participate, during development and origination of dendrites, mature synaptic plasticity and after lesions, when the cells must continue to maintain and reconstitute function, to remain part of the circuitry in the hippocampus. This review will be presented in four main sections: (1) interneurons-development, role in synchronizing influence and hippocampal network functioning; (2) principal cells in CA1, CA3 and dentate gyrus regions-their development, function in terms of synaptic integration, differentiating structure and alterations with lesions; (3) glia and glia/neuronal interactions-response to lesions and developmental guidance mechanisms; and (4) network and circuit aspects of hippocampal morphology and functioning. Finally, the interwoven role of these various elements participating in hippocampal network function will be discussed.

Differential expression of synaptic and nonsynaptic mechanisms underlying stimulus-induced gamma oscillations in vitro.

Gamma frequency oscillations occur in hippocampus in vitro after brief tetani delivered to afferent pathways. Previous reports have characterized these oscillations as either (1) trains of GABA(A) inhibitory synaptic events mediated by depolarization of both pyramidal cells and interneurons at least in part mediated by metabotropic glutamate and acetylcholine receptors, or (2) field potential oscillations occurring in the near absence of an inhibitory synaptic oscillation when cells are driven by depolarizing GABA responses and local synchrony is produced by field effects. The aim of this study was to investigate factors involved in the differential expression of these synaptically and nonsynaptically gated oscillations. Field effects were undetectable in control recordings but manifested when slices were perfused with hypo-osmotic solutions or a reduced level of normal perfusate. These manipulations also reduced the amplitude of the train of inhibitory synaptic events associated with an oscillation and enhanced the depolarizing GABA component underlying the post-tetanic depolarization. The resulting field oscillation was still dependent, at least in part, on inhibitory synaptic transmission, but spatiotemporal aspects of the oscillation were severely disrupted. These changes were also accompanied by an increase in estimated [K(+)](o) compared with control. We suggest that nonsynaptic oscillations occur under conditions also associated with epileptiform activity and constitute a phenomenon that is distinct from synaptically gated oscillations. The latter remain a viable model for in vivo oscillations of cognitive relevance.

Gap junctions between interneuron dendrites can enhance synchrony of gamma oscillations in distributed networks.

Gamma-frequency (30-70 Hz) oscillations in populations of interneurons may be of functional relevance in the brain by virtue of their ability to induce synchronous firing in principal neurons. Such a role would require that neurons, 1 mm or more apart, be able to synchronize their activity, despite the presence of axonal conduction delays and of the limited axonal spread of many interneurons. We showed previously that interneuron doublet firing can help to synchronize gamma oscillations, provided that sufficiently many pyramidal neurons are active; we also suggested that gap junctions, between the axons of principal neurons, could contribute to the long-range synchrony of gamma oscillations induced in the hippocampus by carbachol in vitro. Here we consider interneuron network gamma: that is, gamma oscillations in pharmacologically isolated networks of tonically excited interneurons, with frequency gated by mutual GABA(A) receptor-mediated IPSPs. We provide simulation and electrophysiological evidence that interneuronal gap junctions (presumably dendritic) can enhance the synchrony of such gamma oscillations, in spatially extended interneuron networks. There appears to be a sharp threshold conductance, below which the interneuron dendritic gap junctions do not exert a synchronizing role.

Retinal ganglion cells projecting to the accessory optic system in the rat.

The present data identify the distribution and morphological features of a homogeneous group of rat retinal ganglion cells. These cells were labelled after injection of either horseradish peroxidase or a fluorescent tracer, Fast Blue, into the medial terminal nucleus (MTN) of the accessory optic system. After retrograde fluorescent labelling, MTN-projecting retinal ganglion cells were intracellularly injected with Lucifer Yellow to reveal their complete dendritic morphology. There were on average 1,750 MTN-projecting cells fairly evenly distributed over the entire retinal ganglion cell layer. Their density ranged from 40-49 cells/mm2 in superior retina to 10-19 cells/mm2 towards the peripheral regions of both inferior and superior retina. The area of highest density formed a nasal-temporal band suggestive of a visual streak. Soma diameters ranged from 8.7 to 14.5 micron centrally and from 9.9 to 17.1 microns peripherally. Maximal dendritic field diameter ranged from 431 to 644 micron and averaged 516 micron with no obvious eccentricity dependence. The majority of MTN-projecting cells were bistratified. Dendrites stratified predominantly in the inner sublamina of the inner plexiform layer (IPL) with a varying number of branches from the remaining dendrites contained within the outer IPL, both strata presumably corresponding to the electrophysiologically determined on-off dichotomy. Cells projecting to the MTN were characterised by higher-order dendritic branching patterns that resulted in a dense dendritic tree with minor dendritic overlap. The slender dendrites had a beaded appearance and displayed spiny protrusions. The dendritic coverage of 5-6, stratification pattern, and overall morphological appearance of rat MTN-projecting cells renders them suitable candidates for on-direction--selective cells shown electrophysiologically to be linked with the MTN of the accessory optic system.

Morphological diversity of displaced retinal ganglion cells in the rat: a lucifer yellow study.

Displaced retinal ganglion cells (DRGCs) were retrogradely labelled by injections of the fluorescent dye Fast Blue into the superior colliculi of pigmented rats. Following fixation these cells were intracellularly injected with Lucifer Yellow to determine their dendritic morphology and distribution. Graphic reconstruction of Lucifer Yellow-filled prelabelled neurones revealed a heterogeneous population of DRGCs. Their stratification within the inner plexiform layer was diverse and cells were classified according to their dendritic morphology. The present sample consists largely of unistratifying neurones, the dendrites of which arborized within a narrow sublamina of the inner plexiform layer. They were characterized by a centrally located soma and densely branched dendritic network with little overlap within the branching pattern. In contrast, bistratifying DRGCs possessed a loose and sparsely branched dendritic structure, while diffusely stratifying neurones contained a high degree of dendritic crossing, culminating in a complex network, in which the soma position was biased toward the periphery. One type of DRGC bore a striking resemblance to type 1 neurones (Perry, 1979; Proc. R. Soc. Lond. [Biol.] 204:363-375) in the ganglion cell layer. They were characterized by a large soma (15.5 micron +/- 2.2 micron s.d.) and a dendritic field diameter averaging 288 micron (s.d. +/- 62 micron) and were on average larger than the rest of the displaced population but smaller than type 1 cells in the ganglion cell layer. Since the stratification patterns of the displaced and nondisplaced type 1 neurones were indistinguishable, it is reasonable to assume that the Lucifer Yellow-filled cells in the present study represent the displaced counterpart of regular type 1 ganglion cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphogenesis of the brain in the harbour porpoise.

Morphogenesis of the brain in a cetacean species has been investigated by means of reconstructions from serial sections of successive prenatal stages of the harbour porpoise (Phocoena phocoena). Four specimens ranging from 10 to 46 mm crown-rump length (CRL) were selected and three-dimensional reconstructions of the developing brains were obtained with the plate model method. External and internal characteristics, established as criteria for staging embryonic development of primates and rodents, revealed that a common ontogenetic plan regarding the chronological sequence of morphogenetic events exists in mammalian orders as different as primates and odontocetes. Comparison of the 10-mm and 11.5-mm CRL harbour porpoise brains with those in other mammalian embryos of a similar ontogenetic stage (stages 16 and 17) showed a high degree of correspondence in morphological features. This ontogenetic age group therefore might still be considered as a generalized mammalian one. However, during succeeding morphogenesis of the Phocoena brain, qualitative and quantitative divergences from other mammalian groups became manifest, such as those found in the 24-mm CRL specimen (corresponding to mammalian stages 20, 21). Early foetuses of the harbour porpoise (46 and 65 mm CRL) already exhibited a variety of typical odontocete brain features, such as absence of olfactory bulb, thick cochlear nerve, and strong progression of brainstem structures. Morphogenesis of the harbour porpoise brain is discussed from a comparative perspective, incorporating the literature on the development of mammalian brains. Part of this study has been published in abstract form (Buhl and Oelschläger: Acta Anat. (Basel) 120:15-16 (Abstract), '84).

Morphology of rabbit retinal ganglion cells projecting to the medial terminal nucleus of the accessory optic system.

Rabbit retinal ganglion cells were retrogradely labeled following injection of rhodamine-labeled microspheres into the medial terminal nucleus. The small fraction of rhodamine-labeled neurons reached their peak concentration within the visual streak and then decreased with increasing eccentricity until none were encountered in the far periphery. The same rabbits also received injections of the fluorescent tracer Fast Blue into the superior colliculus. No double-labeled neurons were observed, i.e., ganglion cells projecting to the medial terminal nucleus (MTN) had no axon collaterals to the superior colliculus. In fixed retinae rhodamine-labeled ganglion cells were intracellularly injected with the fluorescent dye Lucifer Yellow to reveal their full dendritic arborization. MTN-projecting cells had medium-sized to large somata with thin and frequently branched dendrites. The large dendritic trees had a distinct morphology and were predominantly unistratified in a narrow band that presumably corresponded to the electrophysiologically determined on-sublamina of the inner plexiform layer. Dendritic field sizes were inversely related to ganglion cell density, thus providing an eccentricity-independent, constant dendritic coverage factor. Approximately five to six dendritic fields from neighboring cells cover every point of the retina. Published reports claim that the physiological class of on-direction-selective ganglion cells provides the sole retinal input to the MTN in the rabbit. In this context morphological features of MTN-projecting cells and their presumed functional correlation with on-direction-selective ganglion cells are discussed.

Morphology of retinal ganglion cells in the flying fox (Pteropus scapulatus): a lucifer yellow investigation.

The morphology of retinal ganglion cells was determined in megachiroptera, commonly known as flying foxes. Retinal ganglion cells were intracellularly injected with the fluorescent dye Lucifer yellow in fixed retinae from adult little red flying foxes (Pteropus scapulatus) captured in their natural habitat. Ganglion cells closely resembled the three main classes of cat retinal ganglion cells, and therefore were classified into alpha-, beta-, and gamma-type cells. The size of the alpha- and beta-type somas and dendritic fields increased with increasing distance from the area centralis. However, this eccentricity dependence was not as pronounced as in the cat. The gamma-type cells were sub-divided into mono-, bi-, and diffusely stratified, in accordance with the ramification of their dendrites within the inner plexiform layer. The alpha- and beta-type cells were uni-stratified in either the sublamina of the inner plexiform layer closest to the ganglion cell layer or in that closest to the inner nuclear layer. These laminae correspond to those in the cat retina which contain the dendritic ramifications of ganglion cells whose central receptive fields respond best to onset of light (the "on-centre" cells), or to ganglion cells whose centres respond optimally to light being extinguished (the "off-centre" cells). Thus the flying fox retina contains a morphological correlate of the "on"/"off" dichotomy of alpha and beta cells in the cat retina. In general the flying fox retinal ganglion cells exhibit a degree of morphological complexity reminiscent of cat retinal cells and this may reflect similar functional properties.

Ultrastructure and aspects of functional organization of pyramidal and nonpyramidal entorhinal projection neurons contributing to the perforant path.

Identified entorhino-hippocampal projection neurons were investigated for their ultrastructure. Spinous projection neurons (pyramidal and spiny stellate cells) display common features such as symmetric axosomatic terminals on their somata, asymmetric synapses on the spines, and both types of synapses on the dendritic shafts. Their axons descend towards the white matter, branching occasionally via collaterals which establish contact with local spines and rarely on dendritic shafts and somata. The sparsely spinous projection neurons (multipolar and horizontal-bipolar) typically show deep nuclear infolds and symmetric and asymmetric synapses on their somata and dendritic shafts. Axons also collateralize in the soma vicinity and form local synapses. It is concluded that the entorhino-hippocampal projection neurons (both spiny and sparsely spinous) act locally and distally thus performing simultaneously as local-circuit and as projection neurons. In accordance with other morphological and electrophysiological reports it appears likely that the generation, modulation, and suppression of entorhinal excitation waves is mediated by these neurons through direct excitation, feed-forward and feed-back inhibition, and disinhibition.

Alpha ganglion cells in the rabbit retina.

In the rabbit retina a distinctive morphological class of large ganglion cells was demonstrated by a combination of intracellular staining with Lucifer Yellow and the quantification of reduced silver-stained preparations. The class is called alpha because of the qualitative and quantitative resemblance to the alpha cells of the cat's retina. Rabbit alpha cells change their size with location on the retina. In the high ganglion cell density region of the visual streak, their somata are about 15 micron in diameter, and their dendritic fields have diameters as small as 180-220 micron. The largest alpha cells in the inferior periphery have soma diameters of 30 micron and dendritic field diameters of 960 micron. There is a considerable scatter of sizes at any retinal location. Alpha cell density changes from about 55/mm2 in the streak to about 3/mm2 in far periphery, and the cells make up 1-1.4% of the ganglion cell population. Dendritic trees stratify in either an inner or an outer sublamina of the inner plexiform layer, suggesting an on/off dichotomy in the response to light. Each of the inner and outer branching subtypes is distributed in a regular mosaic, and the dendritic trees cover the retina completely and economically. The possibility is discussed that the alpha cells are the brisk transient/Y cells of physiology.

Disynaptic olfactory input to the hippocampus mediated by stellate cells in the entorhinal cortex.

Electrophysiological and anatomical studies indicate functional relationships between the olfactory bulb and the hippocampus, mediated by the lateral olfactory tract and perforant path. Fibres from the lateral olfactory bulb terminate in the molecular layer of the lateral entorhinal cortex, which contains stellate and pyramidal cells that project to the hippocampus. Therefore this study was performed to analyze whether a trineuronal, disynaptic chain connects the olfactory bulb and the hippocampus. In adult rats, Fast Blue was unilaterally injected into the septal hippocampus to label cells of origin of the entorhinohippocampal pathway. Lesions of the ipsilateral olfactory bulb induced anterograde terminal degeneration in the entorhinal cortex of the same animals. Fast Blue labelled, and thus hippocampally projecting entorhinal neurones in fixed vibratome slices of the operated brains were injected with Lucifer Yellow. Most of these neurones were stellate layer II and pyramidal layer III cells; in addition there were some sparsely spinous multipolar cells in layers II and III and sparsely spinous horizontal cells at the layer I/II border. Injected cells were photoconverted and processed for electron microscopy. Olfactory bulb lesions resulted in electron-dense degeneration of abundant terminal boutons in the outer zone of entorhinal layer I. The relative frequency of degenerating boutons decreased towards deeper zones of the layer. In the outer zone, degenerated terminals predominantly contacted dendritic spines. These contacts could be seen on injected stellate cells but not on pyramidal cells. This study shows that the area dentata of the rat is reached by disynaptic afferent input from the olfactory bulb and thus is likely to process olfactory information. Oligosynaptic pathways might provide the hippocampus also with visual and auditory inputs; such fast transmitted polysensory information could be essential for the proposed participation of the hippocampus in attention-related mechanisms.

Intracellular lucifer yellow injection in fixed brain slices combined with retrograde tracing, light and electron microscopy.

The present paper contains a full methodological description of iontophoretic Lucifer Yellow injections in fixed brain slices in mammals. In brief, cortical tissue was either perfused or immersion-fixed in paraformaldehyde. After Vibratome sectioning, tissue slices were transferred to epifluorescence microscopes equipped with long distance objectives. Under visual guidance, neurons were selectively impaled with Lucifer Yellow-filled electrodes and intracellularly injected until all dendrites appeared brightly fluorescent. Excellent dendritic staining was obtained in both perfusion-fixed cat visual cortex and immersion-fixed human brain biopsies. Dendritic spines, varicosities and growth cones could be readily discerned. Filling of axonal collaterals was, however, incomplete. Callosally projecting neurons in cat visual cortex were retrogradely traced with a mixture of the fluorescent tracers Fast Blue and DiI. Subsequently the morphology of labelled cells was determined by intracellular Lucifer Yellow injection. Although the Fast Blue fluorescence had become undetectable in filled neurons the granular red appearance of DiI was still discernible. Hence the neuronal composition of even relatively sparse projections can be demonstrated. To obtain permanent preparations, dye-filled neurons were immersed in a diaminobenzidine solution and irradiated with epifluorescent illumination until all visible fluorescence had faded. Photo-oxidation resulted in the intracellular formation of a homogeneously distributed brown reaction product visible with the light microscope. Brief osmication enhanced the staining contrast, thus providing a Golgi-like image. Subsequent electron microscopy of photo-converted cells showed the fine granular nature of the electron opaque reaction product, thus revealing numerous cytological features. The precipitate was homogeneously distributed throughout the entire cytoplasm and nucleus, extending into dendrites and axon. Any apparent leakage of the label into the extracellular space was not observed. Intracellular staining in fixed tissue yields a high number of neurons with extensive filling of dendritic arbors. Photo-oxidation provides stable, non-fading preparations with the option of subsequent electron microscopy. In addition, the technique can be combined with immunocytochemistry and a variety of fluorescent tracer substances. These features, combined with its high selectivity and relative methodological simplicity, render the method to be a promising alternative to classical neuroanatomical approaches.

Morphology of identified entorhinal neurons projecting to the hippocampus. A light microscopical study combining retrograde tracing and intracellular injection.

Cells of origin of the entorhinohippocampal pathway were retrogradely labeled by injection of Fast Blue into the ipsilateral hippocampus. The cells, which were located in layers I, II and III of the lateral entorhinal cortex, were then intracellularly injected with Lucifer Yellow to reveal their complete morphology. We could thus establish that among the hippocampally projecting entorhinal cells there are pyramidal and pyramid-like cells, spiny stellate cells of various shapes, sparsely spinous horizontal and multipolar cells. The involvement of horizontal and multipolar neurons in projections has not previously been recognized although all of these cell types have already been described in Golgi studies. The results indicate that the organization of the perforant path is more complex than has been assumed. Finally, they are at variance with the classical concept which subdivides cortical neurons into projection neurons (pyramidal and spiny stellate) and interneurons (non-pyramidal, local circuit neurons).

Synaptic effects of identified interneurons innervating both interneurons and pyramidal cells in the rat hippocampus.

GABAergic interneurons sculpt the activity of principal cells and are themselves governed by GABAergic inputs. To determine directly some of the sources and mechanisms of this GABAergic innervation, we have used dual intracellular recordings with biocytin-filled microelectrodes and investigated synaptic interactions between pairs of interneurons in area CA1 of the adult rat hippocampus. Of four synaptically-coupled interneuron-to-interneuron cell pairs, three presynaptic cells were identified as basket cells, preferentially innervating somata and proximal dendrites of pyramidal cells, but one differing from the other two in the laminar distribution of its dendritic and axonal fields. The fourth presynaptic interneuron was located at the border between strata lacunosum moleculare and radiatum, with axon ramifying within stratum radiatum. Action potentials evoked in all four presynaptic interneurons were found to elicit fast hyperpolarizing inhibitory postsynaptic potentials (mean amplitude 0.35 +/- 0.10 mV at a membrane potential of -59 +/- 2.8 mV) in other simultaneously recorded interneurons (n=4). In addition, three of the presynaptic interneurons were also shown to produce similar postsynaptic responses in subsequently recorded pyramidal cells (n=4). Electron microscopic evaluation revealed one of the presynaptic basket cells to form 12 synaptic junctions with the perisomatic domain (seven somatic synapses and five synapses onto proximal dendritic shafts) of the postsynaptic interneuron in addition to innervating the same compartments of randomly-selected local pyramidal cells (50% somatic and 50% proximal dendritic synapses, n=12). In addition, light microscopic analysis also indicated autaptic self-innervation in basket (12 of 12) and bistratified cells (six of six). Electron microscopic investigation of one basket cell confirmed six autaptic junctions made by five of its boutons. Together, these data demonstrate that several distinct types of interneuron have divergent output to both principal cells and local interneurons of the same (basket cells) or different type. The fast synaptic effects, probably mediated by GABA in both postsynaptic interneurons and principal cells are similar. These additional sources of GABA identified here in the input to GABAergic cells could contribute to the differential temporal patterning of distinct GABAergic synaptic networks.

Development of the nervus terminalis in mammals including toothed whales and humans.

The early ontogenesis and topography of the mammalian terminalis system was investigated in 43 microslide series of toothed whale and human embryos and fetuses. In early embryonal stages the development of the nasal pit, the olfacto-terminalis placode, and the olfactory bulb anlage is rather similar in toothed whales and humans. However, toothed whales do not show any trace of the vomeronasalis complex. In early fetal stages the olfactory bulb anlage in toothed whales is reduced and leaves the isolated future terminalis ganglion (ganglia) which contains the greatest number of cells within Mammalia. The ganglion is connected with the nasal mucosa via peripheral fiber bundles and with the telencephalon via central terminalis rootlets. The functional implications of the terminalis system in mammals and its evolution in toothed whales are discussed. Obviously, the autonomic component has been enlarged in the course of perfect adaptation to an aquatic environment.

Intracellular injection in fixed slices in combination with neuroanatomical tracing techniques and electron microscopy to determine multisynaptic pathways in the brain.

Intracellular Lucifer Yellow filling in fixed tissue has been recently introduced as a novel neuroanatomical approach to reveal the detailed morphology of individual neurons in isolated preparations of the central nervous system. Since dye injections are performed under visual control, the method is characterized by a high degree of inherent staining selectivity, thus circumventing the element of randomness often considered to be the crux of classical golgi-impregnation techniques. Moreover, the opportunity to optically monitor the injection procedure renders fixed slice preparations highly advantageous to be used in combination with retrograde fluorescent tracing. Subsequently, dye-filled neurons may be subjected to a simple photoconversion procedure leading to the intracellular formation of a stable polymer thus obtaining permanent specimens for light microscopy purposes. Due to the osmiophilic nature of the precipitate the photoconverted material is equally suitable for correlated electron microscopy, thus enabling the analysis of neuronal microcircuitry. At the ultrastructural level, sources of afferent input to identified projection neurons may be revealed by lesion-induced anterograde degeneration of synaptic terminals, therefore enabling the direct demonstration of multisynaptic links. Finally, morphologically identified neurons may be immunocytochemically characterized at the pre- and postembedding levels. It is therefore suggested that their methodological versatility and relative technical ease render intracellular fixed-slice injections a promising complement to the catalogue of anatomical techniques.