Axonal Fields Research Articles

A novel single-cell staining procedure performed in vivo under electrophysiological control: morpho-functional features of juxtacellularly labeled thalamic cells and other central neurons with biocytin or Neurobiotin

We describe a novel and very effective single-cell labeling method with unique advantages for revealing the axonal and dendritic fields of any extracellularly recorded neuron. This procedure involves the use of fine glass micro-pipettes (tip diameter: ≈ 1 μm), which contain biocytin or Neurobiotin dissolved in a salt solution, for the simultaneous juxtacellular recording and tracer iontophoresis. Once a neuron is well-isolated and identified, low intensity (< 10 nA) positive-current pulses are injected by way of the micro-electrode such as to modulate its firing. Juxtacellular tracer iontophoresis may last as long as the cell electrophysiologically remains in good health, while determining some of its physiological properties. Control experiments, including the selective killing of previously injected cells, provide convincing evidence that it is the stained unit that was recorded and ‘tickled’ by the juxtamembranous iontophoretic pulses. Electrophysiological and histochemical data further show that neuronal filling could occur during an electrically induced, transient, physical micro-damage of a somatic or dendritic membrane patch. This simple, single-cell staining method has been used to label several types of rat brain neurons, including projection neurons and interneurons. Its success rate (> 86%) far exceeds that obtained by direct intracellular injections of tracers as shown by the labeling of a large sample of 100 individual cells (from 115 attempts) in the thalamic reticular (Rt) nucleus of 33 rats. We thereby demonstrate that Rt cells project to restricted regions of a single thalamic nucleus, including anterior thalamic nuclei, and that the thalamus and Rt complex have reciprocal connections. The juxtacellular procedure thus represents an ideal directed single-cell labeling tool for determination of functional properties, for subsequent identification, for delineation of overall neuronal architecture and for tracing neuronal pathways, provided care is taken to avoid the possible drawbacks and pitfalls that are illustrated and discussed in the present paper.

Functional morphology of the feedback pathway from area 17 of the cat visual cortex to the lateral geniculate nucleus

Two approaches were adopted to study the pattern of connectivity between the cat visual cortex and lateral geniculate nucleus. Fourteen individual cortico-geniculate axons were labeled and reconstructed after intracellular or extracellular injection of biocytin into regions of known receptive-field position and ocular dominance preference, and the distribution of boutons from multi-axon clusters was mapped in three dimensions and compared with the locations of strategically placed geniculate recordings made in the same tissue. The results show that the feedback has an accurate retinotopic component but that individual axons are both more extensive and more selective than described previously. Area 17 feedback axons terminate primarily in layers A and A1, but the distribution of terminal boutons is strongly biased (3:1 ratio) toward the layer that matches their eye preference. Thus, those driven by the contralateral eye preferentially target layer A, and those driven by the ipsilateral eye target layer A1. Each axon also innervates the perigeniculate nucleus (PGN), but the pattern is otherwise variable, suggesting that there are different axonal classes. The terminal fields of individual axons are much larger than described previously, with a maximum spread of 500-1500 microns. Nevertheless, the projection from a given location in area 17 has a center of maximum terminal density 400-500 microns across, which is in retinotopic correspondence with the aggregate receptive field of the cortical cells of origin. The surrounding zone of relatively sparse boutons, however, must permit corticofugal cells to influence visual processing well beyond the regions over which their own responses summate. It follows that any geniculate cell receives corticofugal input covering an equally extensive area of visual space.

Synaptic target selectivity and input of GABAergic basket and bistratified interneurons in the CA1 area of the rat hippocampus.

To assess the position of interneurons in the hippocampal network, fast spiking cells were recorded intracellularly in vitro and filled with biocytin. Sixteen non-principal cells were selected on the basis of 1) cell bodies located in the pyramidal layer and in the middle of the slice, 2) extensive labeling of their axons, and 3) a branching pattern of the axon indicating that they were not axo-axonic cells. Examination of their efferent synapses (n = 400) demonstrated that the cells made synapses on cell bodies, dendritic shafts, spines, and axon initial segments (AIS). Statistical analysis of the distribution of different postsynaptic elements, together with published data (n = 288) for 12 similar cells, showed that the interneurons were heterogeneous with regard to the frequency of synapses given to different parts of pyramidal cells. When the cells were grouped according to whether they had less or more than 40% somatic synaptic targets, each population appeared homogeneous. The population (n = 19) innervating a high proportion of somata (53 +/- 10%, SD) corresponds to basket cells. They also form synapses with proximal dendrites (44 +/- 12%) and rarely with AISs and spines. One well-filled basket cell had 8,859 boutons within the slice, covering an area of 0.331 mm2 of pyramidal layer tangentially and containing 7,150 pyramidal cells, 933 (13%) of which were calculated to be innervated, assuming that each pyramidal cell received nine to ten synapses. It was extrapolated that the intact axon probably had about 10,800 boutons innervating 1,140 pyramids. The proportion of innervated pyramidal cells decreased from 28% in the middle to 4% at the edge of the axonal field. The other group of neurons, the bistratified cells (n = 9), showed a preference for dendritic shafts (79 +/- 8%) and spines (17 +/- 8%) as synaptic targets, rarely terminating on somata (4 +/- 8%). Their axonal field was significantly larger (1,250 +/- 180 microns) in the medio-lateral direction than that of basket cells (760 +/- 130 microns). The axon terminals of bistratified cells were smaller than those of basket cells. Furthermore, in constrast to bistratified cells, basket cells had a significant proportion of dendrites in stratum lacunosum-moleculare suggesting a direct entorhinal input. The results define two distinct types of GABAergic neuron innervating pyramidal cells in a spatially segregated manner and predict different functions for the two inputs. The perisomatic termination of basket cells is suited for the synchronization of a subset of pyramidal cells that they select from the population within their axonal field, whereas the termination of bistratified cells in conjunction with Schaffer collateral/commissural terminals may govern the timing of CA3 input and/or voltage-dependent conductances in the dendrites.

1638 Movement interruption process during repetitive finger tapping. a behavioral emphasis

Antidromic microstimulation and orthodromic extracellular spike-triggered averaging were used to determine the axonal positions, divergence and terminationsof 16 axons arising from bulbospinal, inspiratory (I) neurons. Activity from these neurons was recorded in the dorsal respiratory groups (DRG) of 12 cats. Systematic tracking was done both transversely and longitudinally in the contralateral fifth and sixth cervical segments of the spinal cord. Axonal position was determined by antidromically activating axons and by recording axonal field potentials. Thirteen axons were located in the lateral funiculus, two in the ventrolateral funiculus and one in the ventral funiculus. Axonal conduction velocity (CV) was calculated from conduction distance and conduction time, the latter defined as the interval of time from the recorded action potential in the medulla to the recorded averaged axonal potential in the spinal cord. Average (±S.D.) axonal CV was52 ± 15m/s. Terminal potentials were recorded for 13 of these axons and were coincident with the location of evoked field potentials resulting from antidromic stimulation of phrenic motoneurons. In addition, terminal potentials from single I cells were recorded at multiple sites along the longitudinal axis of the phrenic motor column. These data indicate that axons of spontaneously active, DRG bulbospinal I cells descend predominantly in the lateral columns and diverge and terminate extensively within the phrenic motor column.

Hippocampal CA1 interneurons: an in vivo intracellular labeling study

Fast spiking interneurons in the CA1 area of the dorsal hippocampus were recorded from and filled with biocytin in anesthetized rats. The full extent of their dendrites and axonal arborizations as well as their calcium binding protein content were examined. Based on the spatial extent of axon collaterals, local circuit cells (basket and O-LM neurons) and long-range cells (bistratified, trilaminar, and backprojection neurons) could be distinguished. Basket cells were immunoreactive for parvalbumin and their axon collaterals were confined to the pyramidal layer. A single basket cell contacted more than 1500 pyramidal neurons and 60 other parvalbumin-positive interneurons. Commissural stimulation directly discharged basket cells, followed by an early and late IPSPs, indicating interneuronal inhibition of basket cells. The dendrites of another local circuit neuron (O-LM) were confined to stratum oriens and it had a small but high-density axonal terminal field in stratum lacunosum-moleculare. The fastest firing cell of all interneurons was a calbindin-immunoreactive bistratified neuron with axonal targets in stratum oriens and radiatum. Two neurons with their cell bodies in the alveus innervated the CA3 region (backprojection cells), in addition to rich axon collaterals in the CA1 region. The trilaminar interneuron had axon collaterals in strata radiatum, oriens and pyramidale with its dendrites confined to stratum oriens. Commissural stimulation evoked an early EPSP-IPSP-late depolarizing potential sequence in this cell. All interneurons formed symmetric synapses with their targets at the electron microscopic level. These findings indicate that interneurons with distinct axonal targets have differential functions in shaping the physiological patterns of the CA1 network.

The superior colliculus relays signals descending from the vibrissal motor cortex to the facial nerve nucleus in the rat

Electrical stimualtion of the vibrissal motor cortex (VMCx), the superior colliculus and the facial nerve nucleus elicits vibrissal movements. A possibility of the superior colliculus as one of the relay nuclei between the VMCx and the facial nerve nucleus was investigated by injecting an anterograde neural tracer, Phaseolus vulgaris leucoagglutinin (PHA-L), into the physiologically identified VMCx and a retrograde tracer, cholera toxin B subunit (M), into the facial nerve nucleus in Wistar rats. In the lateral half of the superior colliculus, the termination field of axons from the VMCx overlapped with the distribution field of projection neurons to the facial nerve nucleus. In some cases, PHA-L-labeled terminal swellings were closely apposed to the somas of CTh-labeled neurons. We conclude that signals descending from the VMCx are relayed through the lateral half of the superior colliculus to the facial nerve nucleus.

The potassium channel subunit KV3.1b is localized to somatic and axonal membranes of specific populations of CNS neurons

Potassium channels play major roles in the regulation of many aspects of neuronal excitability. These channels are particularly well suited for such multiplicity of roles since there is a large diversity of channel types. This diversity contributes to the ability of specific neurons (and possibly different regions of the same neuron) to respond uniquely to a given input. Neuronal integration depends on the local response of spatially segregated inputs to the cell and the communication of these integration centers with the axon. Therefore, the functional implications of a given set of K+ channels varies depending on their precise location on the neuronal surface. Site-specific antibodies were utilized to characterize the distribution of KV3.1b, a subunit of voltage-gated K+ channels in CNS neurons. KV3.1b subunits are expressed in specific neuronal populations of the rat brain, such as cerebellar granule cells, projecting neurons of deep cerebellar nuclei, the substantia nigra pars-reticulata, the globus pallidus, and the ventral thalamus (reticular thalamic nucleus, ventral lateral geniculate and zona incerta). The KV3.1b protein is also present in various neuronal populations involved in the processing of auditory signals, including the inferior colliculus, the nuclei of the lateral lemniscus, the superior olive, and some parts of the cochlear nuclei; as well as in several other neuronal groups in the brainstem (e.g., in the oculomotor nucleus, the pontine nuclei, the reticulotegmental nucleus of the pons, trigeminal and vestibular nuclei, and the reticular formation) and subsets of neurons in the neocortex, the hippocampus and the caudate-putamen shown by double staining to correspond to neurons containing parvalbumin. KV3.1b subunits are localized predominantly in somatic and axonal membranes (particularly in axonal terminal fields) but are much less prominent in dendritic arborizations. This distribution is different than that of other subunits of voltage gated K+ channels and is consistent with a role in the modulation of action potentials. KV3.1b proteins have a cellular and subcellular distribution different than the related KV3.2 subunits which express in Xenopus oocytes currents similar to those expressed by KV3.1b.

Corticothalamic projections from the primary visual cortex in rats: a single fiber study using biocytin as an anterograde tracer.

This study investigates the pattern of axonal projections of single corticothalamic neurons from the rat primary visual cortex. Microiontophoretic injections of biocytin were made in cortical laminae V and VI to label small pools of corticothalamic cells and their intrathalamic axonal projections. After a survival period of 48 h, the animals were perfused and the tissue was processed for biocytin histochemistry. On the basis of the intrathalamic distribution of axonal fields and the types of terminations found in the thalamus, three types of corticothalamic projections were identified. (1) Cells of the upper part of lamina VI projected to the dorsal lateral geniculate nucleus where they arborized in rostrocaudally oriented bands or "rods" parallel to the lines of projection of retinal afferents. (2) Cells of the lower part of lamina VI projected to the lateral part of the lateral posterior nucleus and they also sent collaterals to the dorsal lateral geniculate nucleus where they participated in the formation of rods. (3) The corticothalamic projection of lamina V cells originated from collaterals of corticofugal cells whose main axons reached the tectum and/or the pontine nuclei. These collaterals never terminated within the dorsal lateral geniculate nucleus; they arborized in the lateral posterior, lateral dorsal and ventral lateral geniculate nuclei. All corticothalamic cells from lamina VI displayed the same type of axonal network made of long branches decorated by terminal boutons emitted "en passant" at the tip of fine stalks. Corticothalamic fibers arising from lamina V, however, generated varicose endings in restricted regions of their target nuclei. All corticothalamic axons derived from lamina VI cells, but not those derived from lamina V cells, gave off collaterals as they traversed the thalamic reticular complex. These results demonstrate that corticothalamic fibers arising from the rat primary visual cortex display a lamina-dependent projection pattern. In the light of previous studies on the topographical organization of corticothalamic projections, it is proposed that a similar organizational plan characterizes corticothalamic relationships in other sensory systems in the rat and in other species.

Physiological subgroups of nonpyramidal cells with specific morphological characteristics in layer II/III of rat frontal cortex

Physiological and morphological properties of nonpyramidal cells in layer II/III of frontal cortex of young rats were studied in vitro by whole-cell recording and intracellular staining with biocytin. Layer II/III nonpyramidal cells could be divided into four subgroups by their firing patterns in response to depolarizing current pulses and their patterns of dendritic and axonal arborizations. (1) Fast-spiking (FS) cells (35% of the total sample) showed an abrupt episode of nonadapting repetitive discharges of shorter-duration action potentials. FS cells had local and horizontal axonal arbors which did not enter layer I. This type of FS cell was immunoreactive for parvalbumin and included some basket cells. Three chandelier cells were identified as FS cells, although one chandelier cell was not identified as an FS cell. (2) Late-spiking (LS) cells (11%) exhibited slowly developing ramp depolarizations near threshold. LS cells were neurogliaform cells. (3) Low-threshold spike (LTS) cells (5%) had prominent low-threshold spikes when stimulated from hyperpolarizations. The main axons of LTS cells ascended, and the collaterals entered into layer I. (4) The remaining cells [regular-spiking nonpyramidal (RSNP) cells] (49%) could not be categorized into the above three subgroups. In some RSNP cells depolarizing pulses from hyperpolarizations induced fast depolarizing notches with small amplitude. RSNP cells had vertically elongated axonal fields, extending from layer I to V, sometimes to layer VI. This subgroup included double bouquet cells and bipolar cells. Each subgroup with a different firing mode may differentially contribute to neocortical laminar and columnar circuitry.

Light microscopic immunocytochemical evidence of converging serotonin and dopamine terminals in ventrolateral nucleus accumbens

The mesencephalic tegmentum contains monoaminergic neurons that project to the nucleus accumbens (NAcc). These monoaminergic neurons consist of the serotonergic (5-HT) neurons of the dorsal and median raphe and the dopaminergic (DA) neurons of the ventral tegmental area (VTA). Recent neurochemical reports describe cocaine-induced alterations in dopamine and serotonin release in NAcc that has coincidental occurrence both spatially and temporally, as shown by in vivo voltammetry. There is a functional role for 5-HT-DA interactions within the NAcc in the underlying mechanism of action of cocaine as well as for 5-HT in A10 DA neurons in the basal or endogenous state whether or not cocaine-relevant reward circuits are involved. Our objective was to study the neuroanatomic localization of tyrosine hydroxylase-containing (TH) and 5-HT-containing axons in the ventrolateral region of the rat NAcc, where codetection of monoamines had been assessed. The significance of this vINAcc is its reciprocal connectivity with VTA, which contains the somatodendritic portions of the mesoaccumbens DA neurons. The results showed that, in the vINAcc, the core contained a dense terminal field of TH axons that had an extensive overlap with 5-HT axons in the periphery within the core. Because the in vivo electrochemical codetection of DA and 5-HT assessed in the ventral-most aspect of this overlap zone can be correlated with terminal release, a functional interaction of 5-HT and DA at postsynaptic sites in vINAcc is possible.

Corticothalamic Projections from the Cortical Barrel Field to the Somatosensory Thalamus in Rats: A Single‐fibre Study Using Biocytin as an Anterograde Tracer

This study investigated the pattern of axonal projections of single corticothalamic neurons from the cortical barrel field representing the vibrissae in the rat. Microiontophoretic injections of biocytin were performed in cortical layers V and VI to label small pools of corticothalamic cells and their intrathalamic axonal projections. After a survival period of 48 h, the animals were perfused and the tissue was processed for biocytin histochemistry. On the basis of the intrathalamic distribution of axonal fields and of the types of terminations found in the thalamus, four types of corticothalamic projections were identified. (i) Cells of the upper part of layer VI projected exclusively to the ventral posteromedial (VPm) nucleus, where they arborized in long rostrocaudally oriented bands or 'rods'. (ii) All cells of the lower part of layer VI projected to the medial part of the thalamic posterior group (Pom) but the vast majority of them also collateralized in VPm where they participated in the formation of rods. (iii) A minority of corticothalamic cells in the lower portion of layer VI, possibly located under the interbarrel spaces (septae), arborized exclusively in Pom. (iv) The corticothalamic projection of layer V cells originated from collaterals of corticofugal cells whose main axons ran caudally towards the brainstem. These collaterals arborized exclusively in Pom or in the central lateral nucleus. All corticothalamic cells from layer VI displayed the same type of axonal network, made of long branches decorated by terminal buttons emitted en passant at the tip of fine stalks. Corticothalamic fibres arising from layer V pyramids, however, remained smooth as they ran across the lateral thalamus and they generated in Pom one or two clusters of large boutons. All corticothalamic axons derived from layer VI cells, but not those derived from layer V cells, gave off collaterals as they traversed the thalamic reticular complex. These observations are discussed in the light of previous studies bearing on the topological organization and function of corticothalamic projections to VPm and Pom in rats. The possibility that a similar cellular specificity and a similar organizational plan may characterize corticothalamic relationships in other sensory systems is also considered.

Transcallosal connections of the distal forelimb representations of the primary and supplementary motor cortical areas in macaque monkeys.

The goal of the present neuroanatomical study in macaque monkeys was twofold: (1) to clarify whether the hand representation of the primary motor cortex (M1) has a transcallosal projection to M1 of the opposite hemisphere; (2) to compare the topography and density of transcallosal connections for the hand representations of M1 and the supplementary motor area (SMA). The hand areas of M1 and the SMA were identified by intracortical microstimulation and then injected either with retrograde tracer substances in order to label the neurons of origin in the contralateral motor cortical areas (four monkeys) or, with an anterograde tracer, to establish the regional distribution and density of terminal fields in the opposite motor cortical areas (two monkeys). The main results were: (1) The hand representation of M1 exhibited a modest homotopic callosal projection, as judged by the small number of labeled neurons within the region corresponding to the contralateral injection. A modest heterotopic callosal projection originated from the opposite supplementary, premotor, and cingulate motor areas. (2) In contrast, the SMA hand representation showed a dense callosal projection to the opposite SMA. The SMA was found to receive also dense heterotopic callosal projections from the contralateral rostral and caudal cingulate motor areas, moderate projections from the lateral premotor cortex, and sparse projections from M1. (3) After injection of an anterograde tracer (biotinylated dextran amine) in the hand representation of M1, only a few small patches of axonal label were found in the corresponding region of M1, as well as in the lateral premotor cortex; virtually no label was found in the SMA or in cingulate motor areas. Injections of the same anterograde tracer in the hand representation of the SMA, however, resulted in dense and widely distributed axonal terminal fields in the opposite SMA, premotor cortex, and cingulate motor areas, while labeled terminals were clearly less dense in M1. It is concluded that the hand representations of the SMA and M1 strongly differ with respect to the strength and distribution of callosal connectivity with the former having more powerful and widespread callosal connections with a number of motor fields of the opposite cortex than the latter. These anatomical results support the proposition of the SMA being a bilaterally organized system, possibly contributing to bimanual coordination.

Dopaminergic regulation of striatal efferent pathways

In the past year there has been a growing debate about the distribution of dopamine receptors in striatal efferent pathways. As is often the case, different approaches lead to different perspectives. Nevertheless, the available data can be reconciled with a model in which D 1 and D 2 dopamine receptors are segregated in the distal dendrites and axonal terminal fields of striatonigral and striatopallidal neurons, but intermingled in the soma and proximal dendrites.

Neurons of the medial cortex outer plexiform layer of the lizard Podarcis hispanica: Golgi and immunocytochemical studies.

The study of Golgi-impregnated lizard brains has revealed a scarce but heterogeneous neuronal population in the outer plexiform layer of the medial cortex. Some of the neuronal types detected here resemble the neurons of the dentate molecular layer of the mammalian hippocampus. According to their morphology, five intrinsic neuronal types have been clearly identified: short axon aspinous bipolar neuron (type 1, or sarmentous neuron), short axon aspinous juxtasomatic neuron (type 2, or coral neuron), short axon sparsely spinous multipolar neuron (type 3, or stellate neuron), short axon sparsely spinous juxtasomatic multipolar neuron (type 4, or deep stellate neuron), and sparsely spinous juxtasomatic horizontal neuron (type 5, or couchant neuron). Most neuronal types were identified as gamma-aminobutyric acid (GABA) and parvalbumin immunoreactive, and are thus probably involved in medial cortex inhibition. Moreover, a small fraction of them displayed beta-endorphin immunoreactivity. The distribution of these neuronal types is not uniform in the laminae of the outer plexiform layer. Type 1 (sarmentous) and type 3 (stellate) neurons overlap the axonal field projection coming from the dorsal cortex and the thalamus, whereas types 4 (deep stellate) and 5 (couchant) neurons overlap ipsi- and contralateral dorsomedial projection fields as well as raphe serotoninergic and opioid immunoreactive axonal plexi. Thus, these neuronal types may be involved in the control of specific inputs to the medial cortex by presumably feed-forward inhibition; nevertheless, feed-back inhibition may also occur regarding type 4 (deep stellate) neurons that extend deep dendrites to the zinc-rich bouton field.

Ultrastructural studies of diffuse axonal injury in humans.

Diffuse axonal injury (DAI) is observed commonly in traumatically brain injured humans. However, traditional histologic methods have proven of limited use in identifying reactive axonal change early (< 12 h) in the posttraumatic course. Recently, we have reported, in both humans and animals, that antibodies targeting neurofilament subunits are useful in the light microscopic recognition of early reactive change. In the present study, we extend our previous efforts in humans by analyzing the progression of traumatic brain injury (TBI)-induced axonal change at the ultrastructural level. This effort was initiated to follow the subcellular progression of reactive axonal change in humans and to determine whether this progression parallels that described in animals. Two commercially prepared antibodies were used to recognize reactive axonal change in patients surviving from 6 to 88 h. The NR4 antibody was used to target the light neurofilament subunit (NF-L), and the SMI32 antibody was used to target the heavy neurofilament subunit (NF-H). Plastic-embedded tissue sections were screened for evidence of reactive axonal change, and once identified, this reactive change was analyzed at the ultrastructural level. At 6 h survival, focally enlarged, immunoreactive axons with axolemmal infolding or disordered neurofilaments were seen within fields of axons exhibiting no apparent abnormality. By 12 h, some axons exhibited continued neurofilamentous misalignment, pronounced immunoreactivity, vacuolization, and, occasionally, disconnection. At later stages, specifically 30 and 60 h survival, further accumulation of neurofilaments and organelles had led to the further expansion of the axis cylinder, and clearly disconnected reactive swellings were recognized. These contained a dense core of disordered immunoreactive neurofilaments partially encompassed by a cap of less densely aggregated organelles. At 88 h, the reactive axons were larger and elongated, consistent with the continued delivery of organelles by axoplasmic transport. At the later time points, considerable heterogeneity was observed, with focally enlarged disconnected axons being observed in relation to axons showing less advanced reactive change. Our findings suggest that neurofilamentous disruption is a pivotal event in axonal injury.

Visualization and quantitation of cellular and tissue anatomy by 3D light microscopy

Many areas of biomedical research require the visualization and quantitation of 3D micro- and tissue level anatomy. Both are possible by utilizing confocal or wide-field light microscopy in combination with computational methods, including deblurring and 3D image analysis. The three-dimensionality of the specimen, required image resolution, and parameters to be quantitated dictate the methods of choice. Applications under study by our group range from automatically quantitating a few cell layers in cervical/vaginal smears, or hundreds of cell nuclei or immunologically labeled cells in thick brainslices, to tracing individual neurons for long distances in 3D space. Examples of our wide-field deconvolution methods and automated detection and counting of nuclei in thick tissue are shown.Image restoration by blind deconvolution of portions of the axonal fields of two rat hippocampal pyramidal cells injected with biocytin and contrasted with horseradish peroxidase and diaminobenzidine6 is shown in Figure 1. The original data is projected along and perpendicular to the optic axis in Fig. 1a while 1b is a similar set of restored image projections.

Functional and Structural Topography of Horizontal Inhibitory Connections in Cat Visual Cortex

The functional organization of long-horizontal inhibitory connections was studied in cat visual cortical area 17, using a combination of electrophysiological recording and anatomical tracing in the same tissue. Orientation maps were obtained by recording multiunit activity from layer III at regular intervals (100-300 microns) in a region of approximately 1.3 mm2 of cortex at a depth corresponding to the location of the basket cell axons reconstructed later. Before the physiological mapping, the neuronal tracer biocytin had been iontophoretically injected at one functionally characterized site. On the basis of light microscopic features a total of five biocytin-labelled large basket axons, BC1-BC5, were reconstructed from series of horizontal sections of two cats. The parent somata and dendritic fields of three axons (BC1, BC4 and BC5) could also be reconstructed. The axonal field of basket cell BC1 had an overall lateral spread of 1.8 mm. The axons of basket cells BC4 and BC5 spanned a distance of 3.05 and 2.85 mm, respectively. The distribution pattern of histologically reconstructed recording sites and of five labelled basket cell axons were directly compared in the same sections. The results show that a single large basket cell provides input to regions representing the whole range of orientations, i.e. iso-orientation (+/- 30 degrees), oblique orientation (+/- [30-60] degrees) and cross-orientation (+/- [60-90] degrees) to that at the basket cell's soma. Furthermore, the differential effect mediated by the same large basket cell at sites of different orientation preference was numerically estimated for two basket cells (BC4 and BC5) whose preferred orientations could be determined on the basis of recording sites adjacent to their parent somata. We counted the number of axonal terminals of these basket cells at iso-, oblique- and cross-orientation sites and found no significant difference in the average density of terminals at sites of either orientation preference. The functional topography of large basket cell axons indicates that the same basket cell can mediate iso-, oblique- and cross-orientation inhibition at different sites. Hence, we assume that large basket cells serve a complex physiological role depending on the location of target cells in the orientation map.

Physiological, morphological, and histochemical characterization of three classes of interneurons in rat neostriatum

Interneurons in lateral part of neostriatum were studied in isolated slices from juvenile rats (16-20 d postnatal) by whole-cell, current-clamp recording at 33-34 degrees C, followed by intracellular staining with biocytin and double immunocytochemical or histochemical staining for parvalbumin, ChAT, and NADPH diaphorase. Medium-sized spiny neurons (MS cells) had distal dendrites with many spines and were likely projection cells, while interneurons had dendrites with fewer spines. The neostriatal interneurons could be further divided into three classes by physiological, chemical, and morphological criteria. The first class of interneurons (fast-spiking cells, FS cells) fired very short-duration action potentials with short-duration afterhyperpolarizations at constant spike frequency during depolarizing current pulses. FS cells had more negative resting potentials and lower input resistances than the other two classes. At depolarized potentials, FS cells fired repetitive spikes in response to synaptic excitation. FS cells were immunoreactive for parvalbumin. As all parvalbumin-immunoreactive cells in the neostriatum were also immunoreactive for GABA, FS cells were considered to be GABAergic. FS cells were further divided into two morphological types: FS cells with local dendritic fields and FS cells with extended dendritic fields. The axons of both types of FS cells had their densest collateralization within or near their dendritic fields. The other two classes of interneuron, PLTS cells (persistent and low-threshold spike cells) and LA cells (long-lasting afterhyperpolarization cells), were distinguished from FS cells by longer-duration action potentials and larger input resistances, had less negative resting potentials, and had longer-lasting afterhyperpolarizations. Afterhyperpolarizations of PLTS cells had a shorter time to peak than those of LA cells. PLTS cells fired both Na(+)-dependent, persistent depolarization spikes and Ca(2+)-dependent, low-threshold spikes in addition to fast spikes. Low-threshold spikes in PLTS cells were induced only from hyperpolarized potentials. Both persistent depolarizations and low-threshold spikes could also be evoked by synaptic activation. PLTS cells were histochemically identified as NADPH diaphorase-positive cells. As all NADPH diaphorase-positive cells in the same tissue were immunoreactive for nitric oxide (NO) synthase, PLTS cells were considered to release NO. PLTS cells had the largest axonal fields. Some PLTS cells appeared to have two axonal origins from the somata and dendrites. LA cells were mostly large aspiny cells with Ca(2+)-dependent long-lasting afterhyperpolarizations and strong time-dependent hyperpolarizing rectification. As this slowly occurring anomalous rectification was blocked by 2 mM cesium, this potential was considered to be due to activation of Ih.(ABSTRACT TRUNCATED AT 400 WORDS)

β-Amyloid precursor protein (βAPP) as a marker for axonal injury after head injury

It has been demonstrated recently that β-amyloid protein (βAP), generally associated with the plaques of Alzheimer's disease, can also be found in the brains of survivors of head injury. In this study the distribution of the βAP precursor protein (βAPP) was examined immunohistochemically to determine if it is colocalized with βAP in such cases. βAPP immunoreactivity was observed in neuronal perikarya in the neocortex and in dystrophic neurites surrounding βAP immunoreactive plaques i.e. in a distribution similar to that seen in Alzheimer's disease. In addition, βAPP immunoreactivity was noted within white matter tracts where it marked damaged axons. However, no colocalisation of βAPP with βAP was observed in any white matter region. These results indicate that processing of βAPP to produce βAP occurs in the synaptic terminal field of axons and illustrate the utility of βAPP immunoreactivity as a general marker for axonal injury.

Three-dimensional transmitted brightfield imaging: pragmatic data collection and preprocessing considerations

We have been developing algorithms for 3D image reconstruction of biological specimens with absorbing stains. This is important because there are many absorbing stains which are widely used in conjunction with transmitted light brightfield (TLB) microscopy, yet most of the 3D microscopic imaging research has been directed toward fluorescence microscopy. For instance, horseradish peroxidase (HRP) is used widely in the neurosciences for its many advantages as a tracer and intracellular marker. It is readily injected into individual neurons, transported long distances, and fills both the dendritic and axonal fields, while it may double as an electron microscopy stain for correlative analysis. With such advantages, it is clear that absorbing stains will continue to be widely used. Their utility will furthermore broaden with 3D visualization and quantitation.The main principles behind our methodology are the following. Standard optical serial sectioning data collection is used. The iterative, constrained image reconstruction algorithm is designed to reconstruct the 3D optical density distribution .