The connections between the basal ganglia, i.e. the striatum, the pallidum, the substantia nigra,... more The connections between the basal ganglia, i.e. the striatum, the pallidum, the substantia nigra, and the subthalamic nucleus, as well as the connections between these structures and their input and output areas, e.g. the cortex and the thalamus, show distinct topographies. It has been proposed that this organization forms the structural basis for a number of parallel, functionally segregated basal ganglia-thalamocortical circuits (Alexander et al., 1986, 1990; Groenewegen et al., 1990). Circuits that involve the dorsolateral sector of the caudate-putamen complex, receiving predominantly inputs from the (pre)motor and somatosensory cortical areas, are associated with sensorimotor processes. Loops that involve more ventral and medial regions of the caudate nucleus and putamen, receiving inputs from the prefrontal, parietal and temporal association cortices, are involved in complex behavioral and cognitive processes. Finally, circuits involving the ventral striatum (constituted by the most ventral and medial parts of the caudate-putamen, the nucleus accumbens, and the striatal parts of the olfactory tubercle) which receives inputs from limbic cortical areas, is implicated in affective, emotional, and visceral functions. Since the ascending dopaminergic system reaches the entire striatum, this might explain, at least in part, that dopamine is implicated in a wide variety of behavioral and cognitive functions (Robbins [1992] and references therein).
Experimental studies have produced many data over the past few years which implicate nucleus accu... more Experimental studies have produced many data over the past few years which implicate nucleus accumbens in the motivational and attentional processes that are important for goal-directed behaviors (Willner and Scheel-Kruger, 1990). Moreover, disorders, such as schizophrenia, which involve the disruption of these behaviors, are thought to be specifically linked to this nucleus (Matthysse, 1981). There is experimental evidence that supports a role for dopamine (DA) in these behaviors in nucleus accumbens but the importance of other transmitter systems such as those containing gamma aminobutyric acid (GABA) and acetylcholine, has also become apparent (Costa et al., 1978; Jones et al., 1981; Matthysse, 1981; Willner and Scheel-Kruger, 1990). However, our understanding of the differential role of each of these neurochemicals is limited, especially with respect to their interactions. A major reason for this limitation is the absence of relevant data on the synaptic relationships in accumbens circuitry. We know that nucleus accumbens receives extensive projections from limbic-related parts of the cortex, the amygdala, the midline thalamic nuclei, and the ventral tegmental area (VTA) and projects to major targets in the ventral pallidum and the midbrain i.e., the substantia nigra, VTA, and retrorubral field (see Groenewegen et al., 1990, for review) but we have yet to delineate the synaptic relationships underlying this flow of information into and through the nucleus.
Cellular Imaging Techniques for Neuroscience and Beyond, 2012
The confocal laser scanning microscope (CLSM) has evolved into an indispensable image-acquisition... more The confocal laser scanning microscope (CLSM) has evolved into an indispensable image-acquisition tool for the biomedical sciences. By its physical design the instrument generates images that, compared with those obtained with a conventional fluorescence microscope, are razor sharp, offer higher radial and, in particular, higher axial resolution. High-magnification, high-resolution CLSM imaging is influenced by the statistical behavior of the emitted photons through Abbe-type diffraction that imposes limits. Through the application of several measures, these limits can be pushed back to a certain degree.
In order to determine the presence of synaptic connectivity between fibres originating from a spe... more 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.
The simultaneous use of different neuroanatomical anterograde tracers provides a potentially powe... more The simultaneous use of different neuroanatomical anterograde tracers provides a potentially powerful method to study the convergence of afferent systems in a particular brain area. However, a simple routine procedure to apply multiple anterograde tracers in conjunction with their simultaneous visualization is still missing. We report an easy and straightforward application of three sensitive anterograde tracers: Phaseolus vulgaris leucoagglutinin (PHA-L), rhodamine-conjugated dextran amine (RDA) and biotin-conjugated dextran amine (BDA). These tracers can be visualized simultaneously and permanently through a triple-staining procedure with nickel-enhanced diaminobenzidine (DAB-Ni), DAB and 1-naphthol/Azur B as chromogens. Our test model comprised the projections from the nucleus reuniens thalami and entorhinal cortex. Both projection systems show a high degree of overlap in their terminal fields in the hippocampus. Two tracers were injected in the left and right entorhinal cortex, respectively; a third tracer was injected in the nucleus reuniens. This combination of injections provided a good opportunity to compare the three tracers in one and the same animal. PHA-L, RDA and BDA, injected in either of the injection sites, turned out to be equally sensitive and revealed the morphology of the involved projection systems in great detail. The triple-staining protocol yielded an excellent, simultaneous detectability of the three tracers with a remarkably low background level. Thus, the combination of the anterograde tracers PHA-L, RDA and BDA, in conjunction with the triple-staining procedure, offers a very attractive approach for neuroanatomical research.
The axons of neurons in the CNS with their delicate ramification patterns and terminal boutons ca... more The axons of neurons in the CNS with their delicate ramification patterns and terminal boutons can be visualized with conventional neuroanatomical techniques with a high degree of accuracy. Whether identified terminal boutons form synaptic contacts with target neurons identified by a second and different marker needs resolution beyond that offered by conventional light microscopy. The morphological elements associated with synaptic connectivity consist of specialized pre- and post-synaptic junctional complexes known as the pre- and post-synaptic densities. Electron microscopy of these junctional complexes consumes much time and resources. In an attempt to increase the speed with which we can analyze networks of neurons we developed a high-resolution triple-fluorescence approach including neuroanatomical tracing, immunofluorescence, confocal laserscanning and 3D-computer reconstruction to pinpoint at the light microscopic level the three elements involved in synaptic connectivity: afferent fibers and their terminal boutons, close apposition with neurons identified by the presence of a fluorescent marker, and sandwiched in between a post-synaptic density marker. We used morphological criteria for the detection of axon terminals (swellings on fibers). Antibodies against ProSAP2/Shank3, a post-synaptic density-associated scaffolding protein, were used to pinpoint the location of the synaptic junctions. The results show the existence of sandwich-like configurations: pre-synaptic fiber, ProSAP2/Shank3, post-synaptic neuron. Thus we feel that we can minimize (and perhaps completely eliminate) the need for electron microscopy and hence dramatically increase the overall efficiency of neuroanatomical tracing and network analysis.
We studied the distribution of the calcium binding proteins calretinin and calbindin in the hippo... more 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.
The connections between the basal ganglia, i.e. the striatum, the pallidum, the substantia nigra,... more The connections between the basal ganglia, i.e. the striatum, the pallidum, the substantia nigra, and the subthalamic nucleus, as well as the connections between these structures and their input and output areas, e.g. the cortex and the thalamus, show distinct topographies. It has been proposed that this organization forms the structural basis for a number of parallel, functionally segregated basal ganglia-thalamocortical circuits (Alexander et al., 1986, 1990; Groenewegen et al., 1990). Circuits that involve the dorsolateral sector of the caudate-putamen complex, receiving predominantly inputs from the (pre)motor and somatosensory cortical areas, are associated with sensorimotor processes. Loops that involve more ventral and medial regions of the caudate nucleus and putamen, receiving inputs from the prefrontal, parietal and temporal association cortices, are involved in complex behavioral and cognitive processes. Finally, circuits involving the ventral striatum (constituted by the most ventral and medial parts of the caudate-putamen, the nucleus accumbens, and the striatal parts of the olfactory tubercle) which receives inputs from limbic cortical areas, is implicated in affective, emotional, and visceral functions. Since the ascending dopaminergic system reaches the entire striatum, this might explain, at least in part, that dopamine is implicated in a wide variety of behavioral and cognitive functions (Robbins [1992] and references therein).
Experimental studies have produced many data over the past few years which implicate nucleus accu... more Experimental studies have produced many data over the past few years which implicate nucleus accumbens in the motivational and attentional processes that are important for goal-directed behaviors (Willner and Scheel-Kruger, 1990). Moreover, disorders, such as schizophrenia, which involve the disruption of these behaviors, are thought to be specifically linked to this nucleus (Matthysse, 1981). There is experimental evidence that supports a role for dopamine (DA) in these behaviors in nucleus accumbens but the importance of other transmitter systems such as those containing gamma aminobutyric acid (GABA) and acetylcholine, has also become apparent (Costa et al., 1978; Jones et al., 1981; Matthysse, 1981; Willner and Scheel-Kruger, 1990). However, our understanding of the differential role of each of these neurochemicals is limited, especially with respect to their interactions. A major reason for this limitation is the absence of relevant data on the synaptic relationships in accumbens circuitry. We know that nucleus accumbens receives extensive projections from limbic-related parts of the cortex, the amygdala, the midline thalamic nuclei, and the ventral tegmental area (VTA) and projects to major targets in the ventral pallidum and the midbrain i.e., the substantia nigra, VTA, and retrorubral field (see Groenewegen et al., 1990, for review) but we have yet to delineate the synaptic relationships underlying this flow of information into and through the nucleus.
Cellular Imaging Techniques for Neuroscience and Beyond, 2012
The confocal laser scanning microscope (CLSM) has evolved into an indispensable image-acquisition... more The confocal laser scanning microscope (CLSM) has evolved into an indispensable image-acquisition tool for the biomedical sciences. By its physical design the instrument generates images that, compared with those obtained with a conventional fluorescence microscope, are razor sharp, offer higher radial and, in particular, higher axial resolution. High-magnification, high-resolution CLSM imaging is influenced by the statistical behavior of the emitted photons through Abbe-type diffraction that imposes limits. Through the application of several measures, these limits can be pushed back to a certain degree.
In order to determine the presence of synaptic connectivity between fibres originating from a spe... more 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.
The simultaneous use of different neuroanatomical anterograde tracers provides a potentially powe... more The simultaneous use of different neuroanatomical anterograde tracers provides a potentially powerful method to study the convergence of afferent systems in a particular brain area. However, a simple routine procedure to apply multiple anterograde tracers in conjunction with their simultaneous visualization is still missing. We report an easy and straightforward application of three sensitive anterograde tracers: Phaseolus vulgaris leucoagglutinin (PHA-L), rhodamine-conjugated dextran amine (RDA) and biotin-conjugated dextran amine (BDA). These tracers can be visualized simultaneously and permanently through a triple-staining procedure with nickel-enhanced diaminobenzidine (DAB-Ni), DAB and 1-naphthol/Azur B as chromogens. Our test model comprised the projections from the nucleus reuniens thalami and entorhinal cortex. Both projection systems show a high degree of overlap in their terminal fields in the hippocampus. Two tracers were injected in the left and right entorhinal cortex, respectively; a third tracer was injected in the nucleus reuniens. This combination of injections provided a good opportunity to compare the three tracers in one and the same animal. PHA-L, RDA and BDA, injected in either of the injection sites, turned out to be equally sensitive and revealed the morphology of the involved projection systems in great detail. The triple-staining protocol yielded an excellent, simultaneous detectability of the three tracers with a remarkably low background level. Thus, the combination of the anterograde tracers PHA-L, RDA and BDA, in conjunction with the triple-staining procedure, offers a very attractive approach for neuroanatomical research.
The axons of neurons in the CNS with their delicate ramification patterns and terminal boutons ca... more The axons of neurons in the CNS with their delicate ramification patterns and terminal boutons can be visualized with conventional neuroanatomical techniques with a high degree of accuracy. Whether identified terminal boutons form synaptic contacts with target neurons identified by a second and different marker needs resolution beyond that offered by conventional light microscopy. The morphological elements associated with synaptic connectivity consist of specialized pre- and post-synaptic junctional complexes known as the pre- and post-synaptic densities. Electron microscopy of these junctional complexes consumes much time and resources. In an attempt to increase the speed with which we can analyze networks of neurons we developed a high-resolution triple-fluorescence approach including neuroanatomical tracing, immunofluorescence, confocal laserscanning and 3D-computer reconstruction to pinpoint at the light microscopic level the three elements involved in synaptic connectivity: afferent fibers and their terminal boutons, close apposition with neurons identified by the presence of a fluorescent marker, and sandwiched in between a post-synaptic density marker. We used morphological criteria for the detection of axon terminals (swellings on fibers). Antibodies against ProSAP2/Shank3, a post-synaptic density-associated scaffolding protein, were used to pinpoint the location of the synaptic junctions. The results show the existence of sandwich-like configurations: pre-synaptic fiber, ProSAP2/Shank3, post-synaptic neuron. Thus we feel that we can minimize (and perhaps completely eliminate) the need for electron microscopy and hence dramatically increase the overall efficiency of neuroanatomical tracing and network analysis.
We studied the distribution of the calcium binding proteins calretinin and calbindin in the hippo... more 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.
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