Contacts between medial and lateral perforant pathway fibers and parvalbumin expressing neurons in the subiculum of the rat.

The entorhinal cortex (EC) projects via the perforant pathway to all subfields in the hippocampal formation. One can distinguish medial and lateral components in the pathway, originating in corresponding medial and lateral subdivisions of EC. We analyzed the innervation by medial and lateral perforant pathway fibers of parvalbumin-expressing neurons in the subiculum. A neuroanatomical tracer (biotinylated dextran amine, BDA) was stereotaxically injected in the medial or lateral entorhinal cortex, thus selectively labeling either perforant pathway component. Transport was allowed for 1 week. Transported BDA was detected with streptavidin-Alexa Fluor 488. Parvalbumin neurons were visualized via immunofluorescence histochemistry, using the fluorochrome Alexa Fluor 594. Via a random systematic sampling scheme using a two-channel, sequential-mode confocal laser scanning procedure, we obtained image series at high magnification from the molecular layer of the subiculum. Labeled entorhinal fibers and parvalbumin-expressing structures were three dimensionally (3D) reconstructed using computer software. Further computer analysis revealed that approximately 16% of the 3D objects ('boutons') of BDA-labeled fibers was engaged in contacts with parvalbumin-immunostained dendrites in the subiculum. Both medial and lateral perforant pathway fibers and their boutons formed such appositions. Contacts are suggestive for synapses. We found no significant differences between the medial and lateral components in the relative numbers of contacts. Thus, the medial and lateral subdivisions of the entorhinal cortex similarly tune the firing of principal neurons in the subiculum by way of parvalbumin positive interneurons in their respective terminal zones.

Entorhinal projections terminate onto principal neurons and interneurons in the subiculum: a quantitative electron microscopical analysis in the rat.

The synaptic organization of projections to the subiculum from superficial layers of the lateral and medial entorhinal cortex was analyzed in the rat, using anterograde neuroanatomical tracing followed by electron microscopical quantification. Our aim was to assess the synaptic organization and whether the two projection components (lateral, medial) within the perforant pathway are qualitatively and quantitatively similar with respect to the types of synapses formed and with respect to the postsynaptic targets of these entorhinal projections. The tracer biotinylated dextran amine (BDA) was injected into the lateral and medial entorhinal cortex, respectively, and resulting anterograde labeling in the subiculum was studied. For each of the two projection components, we analyzed in four animals (2 x 2) a total of 100 synapses/animal with respect to features of the synapse type, i.e. asymmetrical or symmetrical, as well as regarding their postsynaptic target, i.e. dendritic shaft or spine. No clear differences were observed between the two pathways. The majority of the synapses were of the asymmetrical type, making contact with spines (78%) or with dendritic shafts (14%). A low percentage of symmetrical synapses targeted dendritic shafts (4.2%) or spines (1.3%). About 2.5% of the synapses remained undetermined. The findings indicate that the majority of entorhinal fibers reaching the subiculum exert an excitatory influence primarily onto principal neurons, with a much smaller feed forward inhibitory component. Only a small percentage of entorhinal fibers in the subiculum appears to be inhibitory, largely influencing interneurons.

Co-localization of calretinin and calbindin in distinct cells in the hippocampal formation of the rat.

We studied the distribution of the calcium binding proteins calretinin and calbindin in the hippocampal formation of the rat brain by means of double-label immunofluorescence - confocal laser scanning microscopy. Colocalization of calretinin and calbindin occurred mostly in large neurons located in the alveus and stratum oriens of field CA1. Some double-labeled cells were observed in the transition area between field CA1 and the subiculum. Finally, double-labeled cells were present in the deep layer of the ventral subiculum. The cells in field CA1 co-expressing both proteins resemble neurons which in neurophysiological experiments by others have been identified as O-LM cells, and we believe that these co-expressing cells should be considered a distinct subpopulation of the calretinin and calbindin populations of GABAergic hippocampal interneurons.

Neuroanatomical tracing at high resolution.

Most techniques used for the study of the fiber connectivity in the central nervous system produce results which are visualized in the conventional light microscope or fluorescence microscope. Although in some cases this may be sufficient, often proof is necessary that fibers which enter a particular brain area indeed terminate here. Alternatively, it may be necessary to determine whether the axon terminals of traced fibers form synapses with specific processes of specific neurons. With the latter neurons all cellular elements are meant which can be labeled in some way. Evidence of synaptic connectivity necessitates visualization at a higher level of resolution, that is at the electron-microscopic level. In this contribution to the Special Issue we discuss several methods currently available to visualize individual tracers, and methods developed to visualize two different markers, that is one marker attached to a fiber or an axon terminal, and the second marker attached to a presumed pre- or postsynaptic neuronal element.

Complex brain circuits studied via simultaneous and permanent detection of three transported neuroanatomical tracers in the same histological section.

Experimental neuroanatomical tracing methods lie at the basis of the study of the nervous system. When the scientific question is relatively straightforward, it may be sufficient to derive satisfactory answers from experiments in which a single neuroanatomical tracing method is applied. In various scientific paradigms however, for instance when the degree of convergence of two different projections on a particular cortical area or subcortical nucleus is the subject of study, the application of single tracing methods can be either insufficient or uneconomical to solve the questions asked. In cases where chains of projections are the subjects of study, the simultaneous application of two tracing methods or even more may be compulsory. The present contribution focuses on combinations of several neuroanatomical tract-tracing strategies, enabling in the end the simultaneous, unambiguous and permanent detection of three transported markers according to a three-color paradigm. A number of combinations of three tracers or of two tracers plus the immunocytochemical detection of a neuroactive substance can be conceived; we describe several of these combinations implemented by us using the present multitracer protocol.

Calretinin in the entorhinal cortex of the rat: distribution, morphology, ultrastructure of neurons, and co-localization with gamma-aminobutyric acid and parvalbumin.

Calretinin is a marker that differentially labels neurons in the central nervous system. We used this marker to distinguish subtypes of neurons within the general population of neurons in the entorhinal cortex of the rat. The distribution, morphology, and ultrastructure of calretinin-immunopositive neurons in this cortical area were documented. We further analyzed the co-localization of the marker with gamma-aminobutyric acid (GABA) and studied whether calretinin-positive neurons project to the hippocampal formation. Methods used included single-label immunocytochemistry at the light and electron microscopic level, retrograde tracing combined with immunocytochemistry, and double-label confocal laser scanning microscopy (CLSM). The entorhinal cortex contained calretinin-positive cells in a scattered fashion, in all layers except layer IV (lamina dissecans). Bipolar and multipolar dendritic configurations were present, displaying smooth dendrites. Bipolar cells had a uniform morphology whereas the multipolar calretinin cell population consisted of large neurons, cells with long ascending dendrites, horizontally oriented neurons, and small spherical cells. Retrograde tracing combined with immunocytochemistry showed that calretinin is not present in cells projecting to the hippocampus. Few synapic contacts between calretinin-positive axon terminals and immunopositive cell bodies and dendrites were seen. Most axon terminals of calretinin fibers formed asymmetrical synapses, and immunopositive axons were always unmyelinated. Results obtained in the CLSM indicate that calretinin co-exists in only 18-20% of the GABAergic cell population (mostly small spherical and bipolar cells). Thus, the entorhinal cortex contains two classes of calretinin interneurons: GABA positive and GABA negative. The first class is presumably a classical, GABAergic inhibitory interneuron. The finding of calretinin-immunoreactive axon terminals with asymmetrical synapses suggests that the second class of calretinin neuron is a novel type of a (presumably excitatory) interneuron.

Anatomical organization of the parahippocampal-hippocampal network.

The anatomical organization of the parahippocampal-hippocampal network indicates that it consists of different parallel circuits. Considering the topographical distribution of sensory cortical inputs, the hypothesis is that the major parallel circuits carry functionally different information. These functionally different parallel routes reach different portions of the hippocampal network along the longitudinal axis of all fields as well as along the perpendicularly oriented transverse axis of CA1 and the subiculum. In the remaining fields of the hippocampal formation, that is, the dentate gyrus and CA2/CA3, separation along the transverse axis is not present. By contrast, here the functionally different pathways converge onto the same neuronal population. The entorhinal cortex holds a pivotal position among the cortices that make up the parahippocampal region. By way of the networks of the superficial and deep layers, it mediates, respectively, the input and output streams of the hippocampal formation. Moreover, the intrinsic entorhinal network, particularly the interconnections between the deep and superficial layers, may mediate the comparison of hippocampal input and output signals. As such, the entorhinal cortex may form part of a novelty detection network. In addition, the organization of the entorhinal-hippocampal network may facilitate the holding of information. Finally, the terminal organization of the presubicular input to the medial entorhinal cortex indicates that the interactions between the deep and superficial entorhinal layers may be influenced by this input.

Sparse colocalization of somatostatin- and GABA-immunoreactivity in the entorhinal cortex of the rat.

We studied the regional and laminar distribution of neurons expressing immunoreactivity with antibodies against the neuropeptide somatostatin (SOM) in the entorhinal cortex of colchicine-treated rats. We further determined whether these neurons also express immunoreactivity with antibodies against the neurotransmitter gamma-aminobutyric acid (GABA). Frontally and horizontally cut brain sections were subjected to double immunofluorescence histochemistry and investigated in a two-laser confocal laser scanning fluorescence microscope. The exact position of each single- or double-labeled cell was obtained via the preparation of large-scale digital fluorescence images superimposed on a brightfield digital image obtained postscanning after decoverslipping and staining with cresyl violet. Three types of SOM-positive cells were found: big multipolar cells (10-15% of the SOM-positive cells), oval cells (15-20%), and small spherical cells (majority of SOM-positive cells). Most cells were seen in layer III. In addition, we found immunoreactive cells in the other layers, with the fewest cells in layers I and IV (lamina dissecans). Of the SOM-positive cells, 18% also expressed GABA immunoreactivity; of the GABA-positive cells, 8% were also immunoreactive for SOM. Double-labeled cells were mostly small spherical cells and, infrequently, multipolar. These data indicate that in the entorhinal cortex, a large proportion of the cells belonging to the SOM population do not express GABA. We speculate that there may be several subpopulations of SOM cells, of which the largest may consist of non-GABAergic, excitatory interneurons.

Multiple axonal tracing: simultaneous detection of three tracers in the same section.

Multiple neuroanatomical tract-tracing methods are important tools for elucidating the connectivity between different populations of neurons. Evaluation of the question as to whether two specific fiber inputs converge on a particular, identified population of projection neurons requires the application of a triple-staining procedure that allows the unequivocal detection of three markers in a single section. The present report deals with a combination of tracing methods using anterogradely transported Phaseolus vulgaris leucoagglutinin and biotinylated dextran amine in conjunction with retrogradely transported Fluoro-Gold. These tracers were simultaneously detected according to a three-color paradigm, which includes the use of three different peroxidase substrates (nickel-enhanced diaminobenzidine, diaminobenzidine, and Vector VIP), thus resulting in three distinct precipitates: black, brown, and purple. We illustrate this method by showing convergence of projections arising from neurons located in two separate basal ganglia-related nuclei onto identified thalamostriatal projection neurons.

Two-laser dual-immunofluorescence confocal laser scanning microscopy using Cy2- and Cy5-conjugated secondary antibodies: unequivocal detection of co-localization of neuronal markers.

The ability of the confocal laser scanning microscope (CLSM) to visualize in one focal plane the fluorescence associated with multiple markers renders this instrument extremely valuable for the study of co-localization of various markers in the somata and cellular processes of neurons. In the present protocol we deal with the question whether or not co-localization exists in neurons of two different neuronal markers. The conventionally used method towards answering this type of question is double-immunofluorescence microscopy. Fundamental to this approach, independent from whether the preparations are observed in a normal fluorescence microscope or in a CLSM, is that each of the applied fluorescent labels should not chemically interact with the other label or inadvertently be visible through the illumination/filter setup designed for the other fluorophore. In the field of double-label CLSM, three types of approach are distinguished: the single-laser, two-color approach, the two-laser, two-color approach, and the time-resolved approach (Brismar and Ulfhake, 1997). Each type of approach has its own advantages and disadvantages. In the instrument in our institute (a Zeiss LSM 410), combinations of fluorophores like fluorescein isothiocyanate (FITC) and tetramethyl rhodamine isothiocyanate (TRITC) are less useful, since TRITC produces a detectable signal in the FITC illumination/filter setup. Instead of experimenting with filter sets we have chosen to take two measures to eliminate this problem. Our first measure is to use fluorophores whose absorption/emission spectra overlap as little as possible. We have selected among the recently developed carbocyanine fluorophores one fluorescing in the visible range (Cy2) (green, in the same range as FITC and with much better resistance to fading than FITC; cf. Härtig et al., 1996), and another fluorescing in the near infrared range (Cy5, infrared; cf. Mesce et al., 1993). Our second measure to ensure excellent signal separation is the adoption of a two-laser, two-color approach. Co-localization of the calcium binding protein, calretinin, and a neurotransmitter, gamma-aminobutyric acid (GABA), in interneurons in the entorhinal cortex and the hippocampus of the rat was used as the principal test model. We compare the above two-laser, two-color approach with a single-laser, two-color CLSM approach using as markers Cy2 and the red fluorophore, Texas Red (physical characteristics resembling TRITC). In this paper considerable attention is paid to control experiments to verify the reliability of the staining procedure. The results show that our two-laser, two-color CLSM approach produces a complete and unambiguous separation of the fluorescent labels, Cy2 and Cy5. We are currently using this method to determine the degree of co-localization of neurochemical substances in CNS neurons.

Injection of dye into neurones in rat and human post-mortem brain, in combination with acetylcholinesterase histochemistry: permanent preparations.

We describe a protocol for the intracellular injection of dye into neurones in thick sections of fixed, post-mortem rat and human brain tissue. To render the sections with the intracellularly injected neurones permanent, they are sectioned again, and the resulting subsections are either immunocytochemically treated or stained histochemically for acetylcholinesterase (AChE) activity. The resultant preparations can be stored at room temperature for prolonged periods. Background staining produced by accumulation of erythrocytes in blood vessels is greatly reduced or virtually eliminated by exposure of the sections to ultraviolet radiation prior to the intracellular injection. The pattern of AChE staining is not affected by this procedure. This ability to stain sections according to a histochemical AChE procedure after the intracellular injection of dyes into striatal neurons opens the possibility to study the relationship of neuronal dendritic trees with the striosome/matrix compartmental boundaries in post-mortem (human) brain tissue of Huntington's disease patients.

Use of peroxidase substrate Vector VIP for multiple staining in light microscopy.

The study of the distribution of a fiber input to a particular brain area and the visualization of the anatomical relationships of that input with both projection- and interneurons, requires a triple-staining that allows the unequivocal distinction of each of the three components in one and the same histological section. In this regard, we investigated the properties of a recently introduced peroxidase chromogen, VIP (V-VIP; Vector Labs) in combination with two traditional substrates, standard diaminobenzidine (DAB, brown precipitate) and nickel-enhanced DAB (DAB-Ni, black). In rats, the anterograde tracer biotinylated dextran amine (BDA) and the retrograde tracer fluorogold (FG) were injected in the perirhinal cortex and hippocampus, respectively. Transported BDA was detected with an avidin-biotin-peroxidase complex, whereas the transported FG was detected via a PAP method. Tracing with BDA and FG was combined with parvalbumin- or calbindin-immunocytochemistry. We compared various combinations and staining sequences. The best results were obtained with a staining sequence comprising first the BDA stain with DAB-Ni as chromogen, second the FG protocol with the chromogen DAB and finally, parvalbumin- or calbinding-immunocytochemistry using the chromogen V-VIP. The order with which the chromogens were applied appeared to be critical. Partial or even total loss of V-VIP reaction product has been observed after standard dehydration in ethanol. As an alternative, a quick dehydration procedure in toluene yields much better staining. Colour separation is excellent and the sensitivity is high. This procedure may also be used for detection of any other combination of three different labels, taking the usual care to avoid cross-reactivity between antibodies.

GABAergic presubicular projections to the medial entorhinal cortex of the rat.

We characterized presubicular neurons giving rise to bilateral projections to the medial entorhinal cortex (MEA) of the rat. Retrograde labeling of presubiculo-entorhinal projections with horseradish peroxidase and subsequent GABA immunocytochemistry revealed that 20-30% of the ipsilaterally projecting neurons are GABAergic. No GABAergic projections to the contralateral MEA were observed. GABAergic projection neurons were observed only in the dorsal part of the presubiculum, which, when taking into account the topography of presubicular projections to MEA, indicates that only the dorsal part of MEA receives GABAergic input. The GABAergic projection neurons constitute approximately 30-40% of all GABAergic neurons present in the superficial layers of the dorsal presubiculum. Using double-label fluorescent retrograde tracing, we found that the ipsilateral and contralateral presubiculo-entorhinal projections originate from different populations of neurons. Anterograde labeling of presubiculo-entorhinal projections and electron microscopical analysis of labeled terminals substantiated the presence of a restricted GABAergic presubiculo-entorhinal projection. A small fraction of afferents to only ipsilateral dorsal MEA formed symmetrical synapses with dendritic shafts. No symmetrical synapses on spines were noted. Most afferents to the dorsal part of ipsilateral MEA, as well as all afferents to the remaining ipsilateral and contralateral MEA, formed asymmetrical synapses with both spines and dendritic shafts in an almost equal ratio. Thus, we conclude that the majority of the presubiculo-entorhinal projections exert an excitatory effect on both principal neurons and interneurons. The projections from the dorsal part of the presubiculum comprise a small inhibitory component that originates from GABAergic neurons and targets entorhinal interneurons.

Interleukin-1-induced long-lasting changes in hypothalamic corticotropin-releasing hormone (CRH)--neurons and hyperresponsiveness of the hypothalamus-pituitary-adrenal axis.

Hypothalamic CRH neurons that control ACTH secretion from the pituitary gland have secretory terminals in the external zone of the median eminence (ZEME). These neurons can coproduce vasopressin (AVP), a neuropeptide that potentiates the ACTH releasing effects of CRH. Recently, we found increased AVP production in adult rats weeks after single exposure to a stressor, which may play a role in event-induced stress disorders. Here, we describe the long-term changes in the HPA axis of adult male rats following a single exposure to a stressor, the cytokine interleukin-1 beta (IL-1 beta). The effects on storage and release of AVP and CRH were established by quantitative immunocytochemistry, the effects on ACTH and corticosterone responses by radioimmunoassay. Single administration of IL-1 beta (5 micrograms/kg i.p.) induces a delayed (at least 4 d) and a long-lasting (at least 3 weeks) increase of vasopressin (AVP) stores in CRH terminals of the ZEME without affecting the CRH stores, and a marked increase of the fraction of CRH terminals that costore AVP. Eleven days after IL-1 beta administration, a second IL-1 beta challenge causes a marked depletion of the AVP stores in the ZEME within 2 hr, which is not seen in rats treated with vehicle 11 d earlier. This is accompanied by twofold higher ACTH and corticosterone responses, as compared to those in vehicle pretreated rats. IL-1 beta-pretreated rats also showed increased ACTH and corticosterone responses to electric footshocks. We conclude that transient activation of the HPA axis by a single administration of IL-1 beta induces a delayed and long-lasting hyperproduction, hyperstorage, and hypersecretion of AVP from hypothalamic CRH neurons that results in hyperresponsiveness of the HPA axis to subsequent stimuli.

Calretinin-immunoreactivity in mitral cells of the rat olfactory bulb.

We addressed the question whether the projection neurons of the olfactory bulb, i.e. the mitral and tufted cells, are immunoreactive for the calcium-binding protein, calretinin. The following approaches were adopted: (1) light and electron microscopic calretinin-immunocytochemistry; (2) neuroanatomical tracing combined with calretinin-immunocytochemistry according to double-peroxidase and double-fluorescence protocols; (3) unilateral lesion of the olfactory bulb combined with calretinin-immunocytochemistry. The experiments were carried out in rats. Immunostaining of brain sections revealed weakly calretinin-immunopositive mitral cell bodies. Tufted cells were immunonegative. In contrast, fibers in the lateral olfactory tract were strongly immunopositive. Dense immunostaining was also present in a superficial band in layer I of the olfactory tubercle, piriform cortex, periamygdaloid cortex, and in the lateral entorhinal cortex. In electron microscopic preparations of these target areas we observed immunoreaction product in axons and axon terminals. The latter invariably formed asymmetrical synapses, mostly with dendritic spines. Injections of the neuroanatomical tracer biotinylated dextran amine (BDA) into the olfactory bulb produced labeled fibers which remained completely restricted to the superficial, calretinin-immunopositive band in layer I in the above-mentioned cortical forebrain areas. We noted colocalization of transported BDA and calretinin-immunoreactivity in mitral cells, in fibers in the lateral olfactory tract and in fibers in the piriform cortex. Olfactory bulb lesions produced depletion of calretinin-immunoreactivity in the lateral olfactory tract and the superficial band in the olfactory cortex-related areas. Together these data firmly indicate that mitral cells and their axons are calretinin-immunoreactive.(ABSTRACT TRUNCATED AT 250 WORDS)

Parvalbumin-immunoreactive neurons in the entorhinal cortex of the rat: localization, morphology, connectivity and ultrastructure.

We studied the distribution, morphology, ultrastructure and connectivity of parvalbumin-immunoreactive neurons in the entorhinal cortex of the rat. Immunoreactive cell bodies were found in all layers of the entorhinal cortex except layer I. The highest numbers were observed in layers II and III of the dorsal division of the lateral entorhinal area whereas the lowest numbers occurred in the ventral division of the lateral entorhinal area. Most such neurons displayed multipolar configurations with smooth dendrites. We distinguished a type with long dendrites and a type with short dendrites. We also observed pyramidal immunoreactive neurons. A dense plexus of immunoreactive dendrites and axons was prominent in layers II and III of the dorsal division of the lateral entorhinal area and the medial entorhinal area. None of the parvalbumin-immunoreactive cells became retrogradely labelled after injection of horseradish peroxidase into the hippocampal formation. By electron microscopy, immunoreactivity was observed in cell bodies, dendrites, myelinated and unmyelinated axons and axon terminals. Immunoreactive dendrites and axons occurred in all cortical layers. We noted many myelinated immunoreactive axons. Immunoreactive axon terminals were medium sized, contained pleomorphic synaptic vesicles, and established symmetrical synapses. Both horseradish peroxidase labelled and unlabelled immunonegative cell bodies often received synapses from immunopositive axon terminals arranged in baskets. Synapses between immunoreactive axon terminals and unlabelled dendritic shafts and spines were abundant. Synapses with initial axon segments occurred less frequently. In addition, synaptic contacts were present between immunopositive axon terminals and cell bodies and dendrites. Thus, the several types of parvalbumin-containing neuron in the entorhinal cortex are interneurons, connected to one another and to immunonegative neurons through a network of synaptic contacts. Immunonegative cells projecting to the hippocampal formation receive axo-somatic basket synapses from immunopositive terminals. This connectivity may form the morphological substrate underlying the reported strong inhibition of cells in layers II and III of the entorhinal cortex projecting to the hippocampal formation.

Phagocytic activity of macrophages and microglial cells during the course of acute and chronic relapsing experimental autoimmune encephalomyelitis.

The ED1 monoclonal antibody recognizes an antigen in lysosomal membranes of phagocytes. The expression of this antigen in cells increases during phagocytic activity. Here we describe the expression of ED1-immunoreactivity during the various stages of both acute (monophasic) and chronic relapsing experimental autoimmune encephalomyelitis (EAE) in the Lewis rat. During the first attack of acute and chronic relapsing EAE, ED1-immunoreactivity was present in macrophages and in cells which displayed morphologic features of activated microglial cells (i.e., cells with thick short processes). At the ultrastructural level these cells were seen to contain phagocytosed myelin structures in lysosomes. ED1-immunoreactivity in these cells was present in the cytoplasm near lysosomes. During the remission phase of acute EAE and the relapse phase of chronic relapsing EAE, ED1-positive cells with dendritic morphology not only were present in or nearby lesions, but were also found at sites distant from lesions throughout large parts of the brain. These cells had a morphology comparable to microglial cells in normal brain. A major difference between animals which were in remission and animals which on day 25 were suffering from a relapse, was that the latter showed the presence of lesions with darkly stained round ED1-positive macrophages and activated microglial cells. These results indicate that during a relapse, newly recruited blood-borne macrophages infiltrate the brain and, together with activated lymphocytes and microglial cells, recommence a new demyelination process.

Light and electron microscopic characterization of cholinergic and dopaminergic structures in the striatal complex and the dorsal ventricular ridge of the lizard Gekko gecko.

The purpose of the present study was to visualize the morphological substrate underlying acetylcholine-dopamine interactions in the striatal complex of the lizard Gekko gecko and to compare the results with data obtained by others in mammals. The results are also discussed in the light of data obtained previously by us on neurochemical aspects of acetylcholine-dopamine interactions in Gekko and in rats. The study is part of a large research program in which the cholinergic and dopaminergic elements of the striatum of rats and reptiles are studied at morphological and neurochemical levels. We employed light microscopic immunocytochemistry, using single-label staining with antibodies against choline acetyltransferase (ChAT) and dopamine (DA) and double-staining with antibodies against ChAT and tyrosine hydroxylase (TH). A detailed analysis of ultrastructural characteristics of ChAT- and DA-immunolabeled striatal tissue was undertaken. The morphology and synaptic relations of the ChAT-immunopositive neurons in the basal forebrain of the lizard Gekko gecko are very similar to those of the cholinergic cells in the striatum of mammals. Probably, the cholinergic cells are in both mammals and reptiles interneurons that receive inputs of intrinsic or extrinsic origin and project upon output neurons. The location of ChAT-immunopositive somata outside the patches of high TH- or DA-immunoreactivity is at odds with the situation in the striatum of mammals and suggests the possibility of axoaxonal or axodendritic contacts at the level of these patches. We found no essential differences between the synaptic relations of the dopaminergic fibers in the striatal complex of Gekko and the conditions described for rats. In conclusion, we found little evidence for the presence of synaptic interaction between the cholinergic and dopaminergic systems in the striatum of this reptile. The possibility of nonsynaptic interaction, however, remains open.

Combined anterograde tracing with biotinylated dextran-amine, retrograde tracing with fast blue and intracellular filling of neurons with lucifer yellow: an electron microscopic method.

In order to determine the presence of synaptic connectivity between fibres originating from a specific source and neurones with a known morphology and known fibre projection, we have introduced a method for electron microscopy that combines three techniques: retrograde fluorescent tracing, anterograde tracing using biotinylated dextran-amine and intracellular injection of Lucifer Yellow (LY) in lightly fixed brain slices. Neurones in the rat entorhinal cortex that project to the infralimbic cortex and that might be in synaptic contact with fibres originating in the dorsal subiculum served as a model. After surgical application of the tracers and a survival period enabling transport, the brain was fixed and vibratome slices 300 microns thick were prepared in which retrogradely labelled cells were intracellularly injected with LY. This substance and the transported biotinylated dextran-amine were converted into different electron-dense labels. First, LY immunocytochemistry was conducted, then followed by silver-gold enhancement of the immunoprecipitate. Subsequently, the tissue sections were treated with an avidin-biotin-horseradish peroxidase complex and subjected to a diaminobenzidine-peroxide reaction. This protocol resulted in labelling of biotinylated dextran-amine-positive fibres and terminals that could easily be differentiated from the LY-positive neuronal elements and also showed well preserved ultrastructural detail.