Correction to : Coordinate expression of pan-neuronal and functional signature genes in sympathetic neurons.

The published online version contains mistake. We apologize for errors in the lettering of Fig. 3d and also would like to correct the legend of Fig. 2b.

Coordinate expression of pan-neuronal and functional signature genes in sympathetic neurons.

Neuron subtypes of the mature nervous system differ in the expression of characteristic marker genes while they share the expression of generic neuronal genes. The regulatory logic that maintains subtype-specific and pan-neuronal genes is not well understood. To begin to address this issue, we analyze RNA sequencing results from whole sympathetic ganglia and single sympathetic neurons in the mouse. We focus on gene products involved in the neuronal cytoskeleton, neurotransmitter synthesis and storage, transmitter release and reception and electrical information processing. We find a particular high correlation in the expression of stathmin 2 and several members of the tubulin beta family, classical pan-neuronal markers. Noradrenergic transmitter-synthesizing enzymes and transporters are also well correlated in their cellular transcript levels. In addition, noradrenergic marker transcript levels correlate well with selected pan-neuronal markers. Such a correlation in transcript levels is also seen between a number of selected ion channel, receptor and synaptic protein genes. These results provide the foundation for the analyses of the coordinated expression of downstream target genes in nerve cells.

Spinocerebellar ataxia type 1 (SCA1): new pathoanatomical and clinico-pathological insights.

AIMS: Spinocerebellar ataxia type 1 (SCA1) represents the first molecular genetically characterized autosomal dominantly inherited cerebellar ataxia and is assigned to the CAG-repeat or polyglutamine diseases. Owing to limited knowledge about SCA1 neuropathology, appropriate pathoanatomical correlates of a large variety of SCA1 disease symptoms are missing and the neuropathological basis for further morphological and experimental SCA1 studies is still fragmentary. METHODS: In the present study, we investigated for the first time serial tissue sections through the complete brains of clinically diagnosed and genetically confirmed SCA1 patients. RESULTS: Brain damage in the three SCA1 patients studied went beyond the well-known brain predilection sites of the underlying pathological process. Along with neuronal loss in the primary motor cortex, it included widespread degeneration of gray components of the basal forebrain, thalamus, brainstem and cerebellum, as well as of white matter components in the cerebellum and brainstem. It involved the motor cerebellothalamocortical and basal ganglia-thalamocortical circuits, the visual, auditory, somatosensory, oculomotor, vestibular, ingestion-related, precerebellar, basal forebrain cholinergic and midbrain dopaminergic systems. CONCLUSIONS: These findings show for the first time that the extent and severity of brain damage in SCA1 is very similar to that of clinically closely related spinocerebellar ataxias (that is, SCA2, SCA3 and SCA7). They offer suitable explanations for poorly understood SCA1 disease symptoms and will facilitate the interpretation of further morphological and experimental SCA1 studies.

Pathoanatomy of cerebellar degeneration in spinocerebellar ataxia type 2 (SCA2) and type 3 (SCA3).

The cerebellum is one of the well-known targets of the pathological processes underlying spinocerebellar ataxia type 2 (SCA2) and type 3 (SCA3). Despite its pivotal role for the clinical pictures of these polyglutamine ataxias, no pathoanatomical studies of serial tissue sections through the cerebellum have been performed in SCA2 and SCA3 so far. Detailed pathoanatomical data are an important prerequisite for the identification of the initial events of the underlying disease processes of SCA2 and SCA3 and the reconstruction of its spread through the brain. In the present study, we performed a pathoanatomical investigation of serial thick tissue sections through the cerebellum of clinically diagnosed and genetically confirmed SCA2 and SCA3 patients. This study demonstrates that the cerebellar Purkinje cell layer and all four deep cerebellar nuclei consistently undergo considerable neuronal loss in SCA2 and SCA3. These cerebellar findings contribute substantially to the pathogenesis of clinical symptoms (i.e., dysarthria, intention tremor, oculomotor dysfunctions) of SCA2 and SCA3 patients and may facilitate the identification of the initial pathological alterations of the pathological processes of SCA2 and SCA3 and reconstruction of its spread through the brain.

The human premotor oculomotor brainstem system - can it help to understand oculomotor symptoms in Huntington's disease?

Recent progress in oculomotor research has enabled new insights into the functional neuroanatomy of the human premotor oculomotor brainstem network. In the present review, we provide an overview of its functional neuroanatomy and summarize the broad range of oculomotor dysfunctions that may occur in Huntington's disease (HD) patients. Although some of these oculomotor symptoms point to an involvement of the premotor oculomotor brainstem network in HD, no systematic analysis of this functional system has yet been performed in brains of HD patients. Therefore, its exact contribution to oculomotor symptoms in HD remains unclear. A possible strategy to clarify this issue is the use of unconventional 100-microm-thick serial tissue sections stained for Nissl substance and lipofuscin pigment (Nissl-pigment stain according to Braak). This technique makes it possible to identify the known nuclei of the premotor oculomotor brainstem network and to study their possible involvement in the neurodegenerative process. Studies applying this morphological approach and using the current knowledge regarding the functional neuroanatomy of this human premotor oculomotor brainstem network will help to elucidate the anatomical basis of the large spectrum of oculomotor dysfunctions that are observed in HD patients. This knowledge may aid clinicians in the diagnosis and monitoring of the disease.

Spinocerebellar ataxia type 6 (SCA6): neurodegeneration goes beyond the known brain predilection sites.

AIMS: Spinocerebellar ataxia type 6 (SCA6) is a late onset autosomal dominantly inherited ataxic disorder, which belongs to the group of CAG repeat, or polyglutamine, diseases. Although, it has long been regarded as a 'pure' cerebellar disease, recent clinical studies have demonstrated disease signs challenging the view that neurodegeneration in SCA6 is confined to the well-known lesions in the cerebellum and inferior olive. METHODS: We performed a systematic pathoanatomical study throughout the brains of three clinically diagnosed and genetically confirmed SCA6 patients. RESULTS: This study confirmed that brain damage in SCA6 goes beyond the known brain predilection sites. In all of the SCA6 patients studied loss of cerebellar Purkinje cells and absence of morphologically intact layer V giant Betz pyramidal cells in the primary motor cortex, as well as widespread degeneration of brainstem nuclei was present. Additional damage to the deep cerebellar nuclei was observed in two of three SCA6 patients. CONCLUSIONS: In view of the known functional role of affected central nervous grey components it is likely that their degeneration at least in part is responsible for the occurrence of a variety of SCA6 disease symptoms.

Accumulation of phosphorylated I kappaB alpha and activated IKK in nodes of Ranvier.

AIMS: Nuclear factor-kappaB (NF-kappaB) is an ubiquitously expressed transcription factor that modulates inducible gene transcription crucial for the regulation of immunity, inflammatory processes, and cell survival. In the mammalian nervous system, constitutive NF-kappaB activation is considered to promote neuronal cell survival by preventing apoptosis. Increasing evidence suggests a critical role for NF-kappaB activation in acute and chronic neurodegenerative diseases. Recently, a striking enrichment of phosphorylated I kappaB alpha (pI kappaB alpha) and activated I KappaB Kinase (IKK), two key components of the NF-kappaB activation pathway, was demonstrated in the axon initial segment (AIS) of neurons. As the AIS shares fundamental features with nodes of Ranvier (NR), we examined whether pI kappaB alpha and activated IKK are also enriched in NR. METHODS: Double-immunofluorescence labelling was performed with vibratome sections of the rodent central and peripheral nervous system. Sections were analysed using confocal laser scanning microscopy and preembedding electron microscopy. RESULTS: Here we report a remarkable accumulation of pI kappaB alpha and activated IKK in NR in the central and peripheral nervous system. Immunolabelling for both proteins extended from NR into the adjacent paranode. pI kappaB alpha predominantly accumulated within the cytoplasm and was associated with fasciculated microtubules. This association was confirmed by electron microscopy. By comparison, activated IKK preferentially clustered beneath the cytoplasmic membrane. CONCLUSION: In conclusion, the coincident accumulation of pI kappaB alpha and activated IKK in AIS and NR suggests that these specific axonal compartments contribute to neuronal NF-kappaB activation.

Spinocerebellar ataxia type 7 (SCA7): widespread brain damage in an adult-onset patient with progressive visual impairments in comparison with an adult-onset patient without visual impairments.

Spinocerebellar ataxia type 7 (SCA7) represents a rare and severe autosomal dominantly inherited ataxic disorder and is among the known CAG-repeat, or polyglutamine, diseases. In contrast to other currently known autosomal dominantly inherited ataxic disorders, SCA7 may manifest itself with different clinical courses. Because the degenerative changes evolving during these different clinical courses are not well known, many neurological disease symptoms still are unexplained. We performed an initial pathoanatomical study on unconventional thick tissue sections of the brain of a clinically diagnosed and genetically confirmed adult-onset SCA7 patient with progressive visual impairments. In this patient we observed loss of myelinated fibres in distinct central nervous fibre tracts, and widespread degeneration of the cerebellum, telencephalon, diencephalon and lower brainstem. These degenerative changes offer appropriate explanations for a variety of less-understood neurological symptoms in adult-onset SCA7 patients with visual impairments: gait, stance and limb ataxia, falls, dysarthria, dysphagia, pyramidal signs, Parkinsonian features, writing problems, impairments of saccades and smooth pursuits, altered pupillary functions, somatosensory deficits, auditory deficits and mental impairments.

Involvement of the auditory brainstem system in spinocerebellar ataxia type 2 (SCA2), type 3 (SCA3) and type 7 (SCA7).

AIMS: The spinocerebellar ataxia type 2 (SCA2), type 3 (SCA3) and type 7 (SCA7) are clinically characterized by progressive and severe ataxic symptoms, dysarthria, dysphagia, oculomotor impairments, pyramidal and extrapyramidal manifestations and sensory deficits. Although recent clinical studies reported additional disease signs suggesting involvement of the brainstem auditory system, this has never been studied in detail in SCA2, SCA3 or SCA7. METHODS: We performed a detailed pathoanatomical investigation of unconventionally thick tissue sections through the auditory brainstem nuclei (that is, nucleus of the inferior colliculus, nuclei of the lateral lemniscus, superior olive, cochlear nuclei) and auditory brainstem fibre tracts (that is, lateral lemniscus, trapezoid body, dorsal acoustic stria, cochlear portion of the vestibulocochlear nerve) of clinically diagnosed and genetically confirmed SCA2, SCA3 and SCA7 patients. RESULTS: Examination of unconventionally thick serial brainstem sections stained for lipofuscin pigment and Nissl material revealed a consistent and widespread involvement of the auditory brainstem nuclei in the SCA2, SCA3 and SCA7 patients studied. Serial brainstem tissue sections stained for myelin showed loss of myelinated fibres in two of the auditory brainstem fibre tracts (that is, lateral lemniscus, trapezoid body) in a subset of patients. CONCLUSIONS: The involvement of the auditory brainstem system offers plausible explanations for the auditory impairments detected in some of our and other SCA2, SCA3 and SCA7 patients upon bedside examination or neurophysiological investigation. However, further clinical studies are required to resolve the striking discrepancy between the consistent involvement of the brainstem auditory system observed in this study and the comparatively low frequency of reported auditory impairments in SCA2, SCA3 and SCA7 patients.

Sprouting in the hippocampus is layer-specific.

Partial removal of layer-specific afferents of the hippocampus is said to induce sprouting of intact fibers from neighboring layers that invade the zone of the degenerating axons. However, recent in vivo and in vitro studies using sensitive anterograde tracers have failed to demonstrate sprouting across laminar boundaries. Sprouting does occur; but, it mainly involves unlesioned fiber systems terminating within the layer of fiber degeneration in addition to the degenerating afferents. These findings point to rigid laminar cues attracting certain fiber systems while repelling others in normal development and after partial deafferentation.

Lesion-induced plasticity of central neurons: sprouting of single fibres in the rat hippocampus after unilateral entorhinal cortex lesion.

In response to a central nervous system trauma surviving neurons reorganize their connections and form new synapses that replace those lost by the lesion. A well established in vivo system for the analysis of this lesion-induced plasticity is the reorganization of the fascia dentata following unilateral entorhinal cortex lesions in rats. After general considerations of neuronal reorganization following a central nervous system trauma, this review focuses on the sprouting of single fibres in the rat hippocampus after entorhinal lesion and the molecular factors which may regulate this process. First, the connectivity of the fascia dentata in control animals is reviewed and previously unknown commissural fibers to the outer molecular layer and entorhinal fibres to the inner molecular layer are characterized. Second, sprouting of commissural and crossed entorhinal fibres after entorhinal cortex lesion is described. Single fibres sprout by forming additional collaterals, axonal extensions, boutons, and tangle-like axon formations. It is pointed out that the sprouting after entorhinal lesion mainly involves unlesioned fibre systems terminating within the layer of fibre degeneration and is therefore layer-specific. Third, molecular changes associated with axonal growth and synapse formation are considered. In this context, the role of adhesion molecules, glial cells, and neurotrophic factors for the sprouting process are discussed. Finally, an involvement of sprouting processes in the formation of neuritic plaques in Alzheimer's disease is reviewed and discussed with regard to the axonal tangle-like formations observed after entorhinal cortex lesion.

Entorhinal cortex lesion in adult rats induces the expression of the neuronal chondroitin sulfate proteoglycan neurocan in reactive astrocytes.

The chondroitin sulfate proteoglycan neurocan is a major component of brain extracellular matrix during development. Neurocan is primarily synthesized by neurons and has the ability to interact with cell adhesion molecules involved in the regulation of cell migration and axonal growth. Within the first weeks postnatally, neurocan expression is strongly downregulated. To test whether neurocan is reexpressed in areas of axonal growth (sprouting) after brain injury, the time course of neurocan expression was analyzed in the denervated fascia dentata of the rat after entorhinal cortex lesion (12 hr; 1, 2, 4, and 10 d; 2 and 4 weeks; and 6 months after lesion). In the denervated zone, immunohistochemistry revealed neurocan-positive astrocytes by 2 d after lesion and a diffuse labeling of the extracellular matrix at all later time points. Electron microscopy confirmed the deposition of neurocan in the extracellular matrix compartment. In situ hybridization demonstrated a strong upregulation of neurocan mRNA within the denervated outer molecular layer 1 and 4 d after lesion. The combination of in situ hybridization with immunohistochemistry for glial fibrillary acidic protein demonstrated that the neurocan mRNA-expressing cells are astrocytes. These data demonstrate that neurocan is reexpressed in the injured brain. In contrast to the situation during development, astrocytes, but not neurons, express neurocan and enrich the extracellular matrix with this molecule. Similar to the situation during development, neurocan is expressed in an area of active axon growth, and it is suggested that neurocan acts to maintain the boundaries of the denervated fascia dentata after entorhinal cortex lesion.

Cerebral amyloid induces aberrant axonal sprouting and ectopic terminal formation in amyloid precursor protein transgenic mice.

A characteristic feature of Alzheimer's disease (AD) is the formation of amyloid plaques in the brain. Although this hallmark pathology has been well described, the biological effects of plaques are poorly understood. To study the effect of amyloid plaques on axons and neuronal connectivity, we have examined the axonal projections from the entorhinal cortex in aged amyloid precursor protein (APP) transgenic mice that exhibit cerebral amyloid deposition in plaques and vessels (APP23 mice). Here we report that entorhinal axons form dystrophic boutons around amyloid plaques in the entorhinal termination zone of the hippocampus. More importantly, entorhinal boutons were found associated with amyloid in ectopic locations within the hippocampus, the thalamus, white matter tracts, as well as surrounding vascular amyloid. Many of these ectopic entorhinal boutons were immunopositive for the growth-associated protein GAP-43 and showed light and electron microscopic characteristics of axonal terminals. Our findings suggest that (1) cerebral amyloid deposition has neurotropic effects and is the main cause of aberrant sprouting in AD brain; (2) the magnitude and significance of sprouting in AD have been underestimated; and (3) cerebral amyloid leads to the disruption of neuronal connectivity which, in turn, may significantly contribute to AD dementia.

Spinocerebellar ataxia type 7 (SCA7): first report of a systematic neuropathological study of the brain of a patient with a very short expanded CAG-repeat.

Spinocerebellar ataxia type 7 (SCA7) represents a very rare and severe autosomal dominantly inherited cerebellar ataxia (ADCA). It belongs to the group of CAG-repeat or polyglutamine diseases with its underlying molecular genetical defect on chromosome 3p12-p21.1. Here, we performed a systematic study of the neuropathology on unconventional thick serial sections of the first available brain tissue of a genetically confirmed late-onset SCA7 patient with a very short CAG-repeat expansion. Along with myelin pallor of a variety of central nervous fiber tracts, we observed i) neurodegeneration in select areas of the cerebral cortex, and ii) widespread nerve cell loss in the cerebellum, thalamus, nuclei of the basal ganglia, and brainstem. In addition, upon immunocytochemical analysis using the anti-polyglutamine antibody 1C2, immunopositive neuronal intranuclear inclusions bodies (NI) were observed in all cerebellar regions, in all parts of the cerebral cortex, and in telencephalic and brainstem nuclei, irrespective of whether they underwent neurodegeneration. These novel findings provide explanations for a variety of clinical symptoms and paraclinical findings of both our and other SCA7 patients. Finally, our immunocytochemical analysis confirms previous studies which described the presence of NI in obviously degenerated brain and retinal regions as well as in apparently well-preserved brain regions and retina of SCA7 patients.

3D-Reconstruction of microglia and amyloid in APP23 transgenic mice: no evidence of intracellular amyloid.

Microglia cells are closely associated with compact amyloid plaques in Alzheimer's disease (AD) brains. Although activated microglia seem to play a central role in the pathogenesis of AD, mechanisms of microglial activation by beta-amyloid as well as the nature of interaction between amyloid and microglia remain poorly understood. We previously reported a close morphological association between activated microglia and congophilic amyloid plaques in the brains of APP23 transgenic mice at both the light and electron microscopic levels [25]. In the present study, we have further examined the structural relationship between microglia and amyloid deposits by using postembedding immunogold labeling, serial ultrathin sectioning, and 3-dimensional reconstruction. Although bundles of immunogold-labeled amyloid fibrils were completely engulfed by microglial cytoplasm on single sections, serial ultrathin sectioning and three-dimensional reconstruction revealed that these amyloid fibrils are connected to extracellular amyloid deposits. These data demonstrate that extracellular amyloid fibrils form a myriad of finger-like channels with the widely branched microglial cytoplasm. We conclude that in APP23 mice a role of microglia in amyloid phagocytosis and intracellular production of amyloid is unlikely.

Synaptic connections of neuropeptide Y (NPY) immunoreactive neurons in the hilar area of the rat hippocampus.

Synaptic connections and fine structural characteristics of neuropeptide Y-immunoreactive (NPY-i) neurons in the fascia dentata were studied using an antiserum against NPY. Normal and colchicine pretreated rats were examined to study the synaptic connections of NPY-i neurons in the normal fascia dentata. The perforant pathway and fimbria fornix were transected to label afferent fibers to NPY-positive cells. Horseradish peroxidase conjugated with wheat germ agglutinin (HRP-WGA) was injected into the contralateral hippocampus to study commissural projections of hippocampal NPY-i neurons, and to search for NPY-i synaptic contacts on immunonegative commissural cells. Since earlier reports have shown that at least half of the NPY-i neurons also contain somatostatin (SS), the distribution of NPY-i neurons in the hilar area was determined and compared with that of SS-i neurons. Four types of dentate NPY-i neurons were distinguished: Type 1: large multipolar cells in the deep hilus (9%). Type 2: medium-sized multipolar and fusiform hilar neurons with dendrites occasionally reaching the outer molecular layer (64%). Type 3: pyramidal shaped cells in the granule cell layer with long apical dendrites reaching the outer molecular layer (20%). Type 4: small multipolar NPY-i cells located in the molecular layer (7%). Our results indicate two overlapping but not identical cell populations of NPY-i and SS-i neurons. Light and electron microscopic analysis of the normal fascia dentata demonstrated that the majority of NPY-i terminals are located in the outer molecular layer of the dentate gyrus, where they establish symmetric synaptic contacts on dendritic shafts and occasionally on spines of granule cells. A moderate number of NPY-i synapses were also found on dendrites in the inner molecular layer and on the cell body of granule cells. Numerous symmetric NPY-i synapses were found on dendrites and somata of neurons in the hilar area. Some NPY-i dendrites in the hilar area received mossy axon collateral input. After transection of the perforant pathway degenerated axon terminals could be found in synaptic contact with NPY-i dendrites in the outer molecular layer. Commissurotomy revealed direct commissural input to NPY-i dendrites in the inner molecular layer and in the hilus. After injection of HRP-WGA into the contralateral hippocampus 2% of hilar NPY-i neurons were retrogradely labeled and symmetric NPY-i synapses were found on the cell bodies and dendrites of unstained HRP-WGA labeled neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Associational and commissural afferents of parvalbumin-immunoreactive neurons in the rat hippocampus: a combined immunocytochemical and PHA-L study.

Nonpyramidal neurons containing the calcium-binding protein parvalbumin (PV) are one of the inhibitory elements of the hippocampal network. Previous studies have indicated that they are involved in septohippocampal disinhibitory circuits. This study analyzes the commissural and ipsilateral associational afferents of parvalbumin neurons. Injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) into the hilus of the fascia dentata labeled numerous axons in the molecular layer that established synaptic contacts with parvalbumin-immunoreactive neurons on both the injection and the contralateral side. Mossy fibers, labeled by injections into the granule cell layer, terminated on parvalbumin neurons in the hilus and in CA3. Injections of PHA-L into CA3 resulted in a dense labeling of fibers in the hilus and in CA3, CA2, and CA1 on both the injection and the contralateral side. In all these hippocampal fields, PHA-L-labeled fibers established asymmetric contacts with PV-immunoreactive, presumably GABAergic, inhibitory neurons. These observations indicate that parvalbumin-immunoreactive inhibitory neurons in the hippocampus are targets of presumably excitatory associational and commissural projections and suggest that they are involved in feed-forward and feed-back circuits.

Sprouting of crossed entorhinodentate fibers after a unilateral entorhinal lesion: anterograde tracing of fiber reorganization with Phaseolus vulgaris-leucoagglutinin (PHAL).

Fibers from the contralateral entorhinal cortex (EC) to the dentate gyrus partially replace the input lost after an ipsilateral EC lesion. To study the morphology and course of single sprouted crossed entorhinodentate fibers, the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHAL) was used. Rats that survived for 4 to 8 weeks after a unilateral entorhinal lesion received PHAL deposits into the entorhinal cortex contralateral to the lesion. Control animals received a similar PHAL deposit. Single PHAL-labeled fibers in the molecular layer of the contralateral (EC lesion) fascia dentata were drawn with a camera lucida, and an axon-branching index (branch points/100 microns axon length) was calculated for these crossed entorhinodentate fibers in controls and operated animals. In animals with EC lesions, the density of PHAL-labeled crossed entorhinodentate fibers had increased remarkably. Single crossed entorhinodentate axons showed significantly more axon branch points in experimental than in control animals. In addition, some axon segments displayed high densities of small axonal extensions. Frequently, tanglelike structures were observed in the denervated outer molecular layer. These tangles consisted of one or more PHAL-labeled axons that intertwined and formed an axon tangle filled completely with branches, extensions, and boutons. Our data indicate that crossed EC fibers sprout by forming additional collaterals, axonal extensions, and tangles. Abnormal neurite formations are a characteristic feature of plaques in Alzheimer's disease. Future studies must be done to show whether or not there is a close relationship between axonal tangles and plaques in Alzheimer's disease, which, like the present lesion paradigm, severely affects entorhinal projection neurons.

Phaseolus vulgaris-leucoagglutinin tracing of commissural fibers to the rat dentate gyrus: evidence for a previously unknown commissural projection to the outer molecular layer.

Numerous studies have shown a lamina-specific termination of commissural fibers to the dentate gyrus in the inner molecular layer. However, the exact course and arborization pattern of individual fibers remained unknown. In this study, the commissural fiber tract to the dentate gyrus of the rat has been studied using the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L), which labels individual axons and their collaterals. Following iontophoretic application of the tracer, anterogradely labeled fibers were followed through the posterior basal fornix and medial fimbria where they formed a dense fiber bundle. Labeled fibers then entered the dentate gyrus close to the medial blade of the granule cell layer where they separated and traversed the hilus. Only in those cases where the injection also involved CA3 pyramidal cells could axons arborizing in the hilus be observed. Typically, fibers that continued into the molecular layer did not arborize in the hilus. Upon their entrance into the molecular layer, these fibers changed direction, gave off several collaterals, and followed a new path parallel to the granule cell layer where they preferentially formed en passant contacts. These commissural fibers to the inner molecular layer terminated in a wide septotemporal (longitudinal) extension. However, a considerable number of fibers reached the outer molecular layer where some of them formed extensive arborizations. Moreover, these commissural fibers to the outer molecular layer appeared to be restricted to the hippocampal lamella, corresponding to the level of the contralateral injection site. These data suggest the existence of three commissural projections to the rat dentate gyrus: (1) commissural fibers to the hilus arising from CA3 neurons, (2) commissural fibers to the inner molecular layer, and, (3) commissural fibers to the outer molecular layer.

Stereological estimates of total neuron numbers in the hippocampus of adult reeler mutant mice: Evidence for an increased survival of Cajal-Retzius cells.

The cytoarchitecture of the brain is disrupted severely in reeler mice. This is caused by a deficiency in the protein, Reelin, which is essential for the normal migration and positioning of neurons during development. Although cell migration is clearly affected by the reeler mutation, it is believed that the total number of neurons is not. Thus, we were surprised to find an unusually large number of calretinin-immunopositive cells, presumably Cajal-Retzius cells, in the molecular layer of the adult reeler hippocampus (Deller et al. [1999]; Exp. Neurol. 156:239-253). This suggested that the reeler mutation affects the number of neurons in the hippocampus. In order to verify this hypothesis, unbiased stereological methods were employed. Calretinin immunostaining was used as a marker for Cajal-Retzius cells in control as well as reeler mice and Nissl staining was used to identify hippocampal principal neurons. Total numbers of calretinin-immunopositive cells, calretinin-immunoreactive Cajal-Retzius cells, and Nissl-stained neurons were estimated in different subfields of the reeler and the control hippocampus. Stereological estimates (P < 0.05) revealed that the total number of calretinin-immunopositive and Cajal-Retzius cells in reeler mice are 1.5 and 2.1 times that of controls, respectively. No significant difference in total neuron number was found in any hippocampal subfield. These data demonstrate that the reeler mutation affects the number of calretinin-immunoreactive Cajal-Retzius cells in the adult hippocampus, probably due to a reduced excitatory innervation by entorhinal terminals in the absence of reelin. However, the reeler mutation does not affect mechanisms that determine total hippocampal neuron number.