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Background: The superior hypogastric plexus (SHP) is an autonomic plexus, located ventrally to the abdominal aorta and its bifurcation, innervating pelvic viscera. It is classically described as being composed of merely sympathetic fibres. However, post-operative complications after surgery damaging the peri-aortic retroperitoneal compartment suggest the existence of parasympathetic fibres. This immunohistochemical study describes the neuroanatomical composition of the human mature SHP., Material and Methods: Eight pre-determined retroperitoneal localizations including the lumbar splanchnic nerves, the SHP and the HN were studied in four human cadavers. Control tissues (white rami, grey rami, vagus nerve, splanchnic nerves, sympathetic ganglia, sympathetic chain and spinal nerve) were collected to verify the results. All tissues were stained with haematoxylin and eosin and antibodies S100, tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP) and myelin basic protein (MBP) to identify pre- and postganglionic parasympathetic and sympathetic nerve fibres., Results: All tissues comprising the SHP and hypogastric nerves (HN) showed isolated expression of TH, VIP and MBP, revealing the presence of three types of fibres: postganglionic adrenergic sympathetic fibres marked by TH, unmyelinated VIP-positive fibres and myelinated preganglionic fibres marked by MBP. Analysis of control tissues confirmed that TH, VIP and MBP were well usable to interpret the neurochemical composition of the SHP and HN., Conclusion: The human SHP and HN contain sympathetic and most likely postganglionic parasympathetic fibres. The origin of these fibres is still to be elucidated, however surgical damage in the peri-aortic retroperitoneal compartment may cause pelvic organ dysfunction related to both parasympathetic and sympathetic denervation., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The visualization of activity in mouse brain using inversion recovery spin echo (IR-SE) manganese-enhanced MRI (MEMRI) provides unique contrast, but suffers from poor resolution in the slice-encoding direction. Super-resolution reconstruction (SRR) is a resolution-enhancing post-processing technique in which multiple low-resolution slice stacks are combined into a single volume of high isotropic resolution using computational methods. In this study, we investigated, first, whether SRR can improve the three-dimensional resolution of IR-SE MEMRI in the slice selection direction, whilst maintaining or improving the contrast-to-noise ratio of the two-dimensional slice stacks. Second, the contrast-to-noise ratio of SRR IR-SE MEMRI was compared with a conventional three-dimensional gradient echo (GE) acquisition. Quantitative experiments were performed on a phantom containing compartments of various manganese concentrations. The results showed that, with comparable scan times, the signal-to-noise ratio of three-dimensional GE acquisition is higher than that of SRR IR-SE MEMRI. However, the contrast-to-noise ratio between different compartments can be superior with SRR IR-SE MEMRI, depending on the chosen inversion time. In vivo experiments were performed in mice receiving manganese using an implanted osmotic pump. The results showed that SRR works well as a resolution-enhancing technique in IR-SE MEMRI experiments. In addition, the SRR image also shows a number of brain structures that are more clearly discernible from the surrounding tissues than in three-dimensional GE acquisition, including a number of nuclei with specific higher brain functions, such as memory, stress, anxiety and reward behavior., (Copyright © 2014 John Wiley & Sons, Ltd.)

Appropriate signaling in the brain by the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) is critical in regulation of the hypothalamic-pituitary-adrenal (HPA) axis, emotional arousal and cognitive performance. To date, few data exist on MR (and GR) expression in the brain of patients suffering from major depressive disorder (MDD). With the help of quantitative PCR we assessed MR and GR mRNA expression, including the splice variants MRα and MRβ, in tissue samples from the hippocampus, amygdala, inferior frontal gyrus, cingulate gyrus and nucleus accumbens. Expression levels were compared between tissue samples from six MDD patients and six non-depressed subjects. Relative to total GR, total MR mRNA expression was higher in hippocampus and lower in the amygdala, inferior frontal gyrus and nucleus accumbens. Both MRα and MRβ could be detected in all brain regions that were analyzed, although MRβ expression was low. Significantly lower expression levels (30-50%) were detected for MR or GR in hippocampal, inferior frontal gyrus and cingulate gyrus tissue from MDD patients (p < .05), while no differences were found in the amygdala or nucleus accumbens. The data show that both MRα and MRβ mRNA are expressed throughout the human limbic brain with highest expressions in the hippocampus. A decreased expression of corticosteroid receptors in specific brain regions of MDD patients could underlie HPA hyperactivity, mood and cognitive disturbances often observed in patients suffering from stress-related psychopathologies., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Object: Accuracy of reinnervation is an important factor that determines outcome after nerve injury and repair. Type grouping--the clustering of muscle fibers of the same type after reinnervation--can be used to investigate the accuracy of reinnervation. In this study, the degree of type grouping after crush injury in rats was compared with the clustering of muscle fibers after autografting or single-lumen nerve grafting., Methods: Twelve weeks after sciatic nerve crush injury in rats, clustering of Type I muscle fibers was analyzed in the target muscle with adenosine 5'-triphosphatase staining. In addition, the number of regenerated axons was determined in the nerve distal to the crush injury. Results were compared with that of the authors' previous study., Results: Type grouping was more abundant after crush injury than after autograft or single-lumen nerve graft repair., Conclusions: Crush injury leads to more clustered innervation of muscle fibers, probably because the Schwann cell basal lamina tubes are not interrupted as they are in autograft or artificial nerve graft repair. This finding adds to understanding the processes playing a role in nerve regeneration.

Hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis is one of the most consistent findings in major depressive disorder (MDD). Impaired HPA feedback may be due to the lower glucocorticoid receptor (GR) or mineralocorticoid receptor (MR) levels in the forebrain. GR levels are transcriptionally controlled by multiple untranslated alternative first exons, each with its own promoter providing a mechanism for tissue-specific fine-tuning of GR levels. Recently epigenetic methylation of these GR promoters was shown to modulate hippocampal GR levels. Here we investigate in post-mortem brain tissues whether in MDD HPA axis hyperactivity may be due to epigenetic modulation of GR transcript variants. Levels of GRalpha, GRbeta and GR-P transcripts were homogeneous throughout the limbic system, with GRalpha being the most abundant (83%), followed by GR-P (5-6%) while GRbeta was barely detectable (0.02%). Among the alternative first exons, 1B and 1C were the most active, while 1E and 1J showed the lowest expression and transcript 1F expressed intermediate levels of about 1%. In MDD, total GR levels were unaltered, although GRalpha was decreased in the amygdala and cingulate gyrus (p<0.05); transcripts containing exons 1B, 1C and 1F were lower, and 1D and1J were increased in some regions. NGFI-A, a transcription factor of exon 1F was down-regulated in the hippocampus of MDD patients; concomitantly exon 1F expression was reduced. Bisulphite sequencing of the alternative promoters showed low methylation levels in both MDD and control brains. Promoter 1F was uniformly unmethylated, suggesting that reduced 1F transcript levels are not linked to promoter methylation but to the observed dearth of NGFI-A. Previous studies showed high methylation levels in the 1F promoter, associated with childhood abuse. Provided our donors were not abused, our results suggest that the pathomechanism of MDD is similar but nevertheless distinct from that of abuse victims, explaining the clinical similarity of both conditions and that susceptibility to depression may be either predisposed by early trauma or developed independent of such a condition. However, this should be further confirmed in dedicated studies in larger cohorts., (Copyright 2009 Elsevier Ltd. All rights reserved.)

This monograph (Part I of two volumes) on the subthalamic nucleus (STN) accentuates the gap between experimental animal and human information concerning subthalamic development, cytology, topography and connections. The light and electron microscopical cytology focuses on the open nucleus concept and the neuronal types present in the STN. The cytochemistry encompasses enzymes, NO, glial fibrillary acidic protein (GFAP), calcium binding proteins, and receptors (dopamine, cannabinoid, opioid, glutamate, gamma-aminobutyric acid (GABA), serotonin, cholinergic, and calcium channels). The ontogeny of the subthalamic cell cord is also reviewed. The topography concerns the rat, cat, baboon and human STN. The descriptions of the connections are also given from a historical point of view. Recent tracer studies on the rat nigro-subthalamic connection revealed contralateral projections. Part II of the two volumes (volume 199) on the subthalamic nucleus (STN) starts with a systemic model of the basal ganglia to evaluate the position of the STN in the direct, indirect and hyperdirect pathways. A summary of in vitro studies is given, describing STN spontaneous activity as well as responses to depolarizing and hyperpolarizing inputs and high-frequency stimulation. STN bursting activity and the underlying ionic mechanisms are investigated. Deep brain stimulation used for symptomatic treatment of Parkinson's disease is discussed in terms of the elements that are influenced and its hypothesized mechanisms. This part of the monograph explores the pedunculopontine-subthalamic connections and summarizes attempts to mimic neurotransmitter actions of the pedunculopontine nucleus in cell cultures and high-frequency stimulation on cultured dissociated rat subthalamic neurons. STN cell models--single- and multi-compartment models and system-level models are discussed in relation to subthalamic function and dysfunction. Parts I and II are compared.

Current opinion holds that pores in synthetic nerve guides facilitate nerve regeneration. Solid factual support for this opinion, however, is absent; most of the relevant studies assessed only morphological parameters and results have been contradictory. To evaluate the effect of pores, the rat sciatic nerve was either autografted or grafted with nonporous, macroporous (10-230 mum), and microporous (1-10 microm) biodegradable epsilon-caprolactone grafts. Twelve weeks later, the grafted nerves were resected, and the electrophysiological properties were determined in vitro. Subsequently midgraft-level sections were inspected, and peroneal nerve sections were evaluated morphometrically. Finally, the gastrocnemic and tibial muscle morphometrical properties were quantified. The microporous nerve graft performed much better than the nonporous and macroporous grafts with respect to most parameters: it was bridged by a free floating bundle that contained myelinated nerve fibers, there were more nerve fibers present distal to the graft, the electrophysiological response rate was higher, and the decrease in muscle cross-sectional area was markedly smaller. Hence, the present study demonstrates the beneficial effect of synthetic nerve guide pores on nerve regeneration, although with the caveat that not pores per se, but only small (1-10 microm) pores were effective.

Type grouping signifies clustering of muscle fibres of the same metabolic type, and is a frequent finding in reinnervated muscles. To elucidate the mechanism behind it, the rat sciatic nerve was either autografted or grafted with hollow synthetic nerve grafts. Twelve weeks later the number and fibre area of the type I and type II muscle fibres in the gastrocnemic and anterior tibial muscles were determined after ATP-ase staining. The number and diameter of peroneal nerve fibres distal to the grafts were measured, and the number of Aalpha-nerve fibres was derived. Nearly all nerve and muscle morphometrical parameters changed equally in both experimental groups. However, type grouping occurred frequently only after autografting, whereas the number of nerve fibres and the number of Aalpha-nerve fibres increased in this group. Hence type grouping cannot be explained by increased intramuscular sprouting subsequent to a decrease in the number of innervating nerve fibres, as previously presumed. Regenerating axons branch along their course through the peripheral nerve. We propose that the probability of the occurrence of type grouping is related to the dispersion of sibling branches in the nerve. In the autograft, emerging branches are kept together by Schwann cell basal lamina scaffolds, in contrast to the hollow synthetic nerve grafts where the emerging branches become dispersed. Thus, in muscles reinnervated after autografting, the probability that nerve branches that arrive at a specific muscle territory are sibling branches is greater than after hollow tube grafting. Consequently, the probability that type grouping will occur is greater.

We studied electrophysiological and morphological properties of the Aalpha- and Abeta-fibers in the regenerating sciatic nerve to establish whether these fiber types regenerate in numerical proportion and whether and how the electrophysiological properties of these fiber types are adjusted during regeneration. Compound action potentials were evoked from isolated sciatic nerves 12 weeks after autografting. Nerve fibers were gradually recruited either by increasing the stimulus voltage from subthreshold to supramaximal levels or by increasing the interval between two supramaximal stimuli to obtain the cumulative distribution of the extracellular firing thresholds and refractory periods, respectively. Thus, the mean conduction velocity (MCV), the maximal charge displaced during the compound action potential (Q(max)), the mean firing threshold (V(50)), and the mean refractory period (t(50)) were determined. The number of myelinated nerve fibers and their fiber diameter frequency distributions were determined in the peroneal nerve. Mathematical modeling applied to fiber recruitment and diameter distributions allowed discrimination of the Aalpha- and Abeta-fiber populations. In regenerating nerves, the number of Aalpha-fibers increased fourfold while the number of Abeta-fibers did not change. In regenerating Aalpha- and Abeta-fibers, the fiber diameter decreased and V(50) and t(50) increased. The regenerating Aalpha-fibers' contribution to Q(max) decreased considerably while that of the Abeta-fibers remained the same. Correlation of the electrophysiological data to the morphological data provided indications that the ion channel composition of both the Aalpha- and Abeta-fibers are altered during regeneration. This demonstrates that combining morphometric and electrophysiological analysis provides better insight in the changes that occur during regeneration.

Attachment to and proliferation on the substrate are deemed important considerations when Schwann cells (SCs) are to be seeded in synthetic nerve grafts. Attachment is a prerequisite for the SCs to survive and fast proliferation will yield large numbers of SCs in a short time, which appears promising for stimulation of peripheral nerve regeneration. The aim of the present study was to compare the adhesion and proliferation of human Schwann cells (HSCs) on different substrates. The following were selected for their suitability as an internal coating of synthetic nerve grafts; the extracellular matrix proteins fibronectin, laminin and collagen type I and the poly-electrolytes poly(d-lysine) (PDL) and poly(ethylene-imine) (PEI). On all coatings, attachment of HSCs was satisfactory and comparable, indicating that this factor is not a major consideration in choosing a suitable coating. Proliferation was best on fibronectin, laminin and PDL, and worst on collagen type I and PEI. Since nerve regeneration is enhanced by laminin and/or fibronectin, these are preferred as coatings for synthetic nerve grafts seeded with SCs.

Seeding of artificial nerve grafts with Schwann cells is a promising strategy for bridging large nerve defects. The aim of the present study was to evaluate the adhesion and growth of human Schwann cells (HSCs) on 1,3-trimethylene carbonate (TMC) and epsilon-caprolactone copolymers, with the final goal of using these materials in the development of an artificial nerve graft. The adhesion, proliferation, and morphology of HSCs on copolymers containing 10 and 82 mol % of TMC and on the parent homopolymers were investigated. HSCs adhered faster and in greater numbers on the copolymer with 82 mol % of TMC and on the TMC homopolymer compared with the other (co)polymers. On all polymer films, cell adhesion was lower than on gelatin (positive control). Despite differences in cell adhesion, cells displayed exponential growth on all tested surfaces, with similar growth rates. Cell numbers doubled approximately every 3 days on all substrates. When the polymer films were coated with fibronectin, no significant differences in cell adhesion and proliferation were observed between coated polymer surfaces and gelatin. The results indicate that all tested materials support the adhesion and proliferation of HSCs and can in principle be used for the preparation of flexible and slowly degrading nerve guides., (Copyright 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 876-885, 2003)

Traumatic root avulsions of the brachial plexus constitute a devastating lesion resulting in loss of function of the upper limb and carry a large emotional and socioeconomic impact. In this literature survey, the different factors involved in root avulsion are discussed in combination with various surgical techniques for repair of experimental ventral root avulsion. Until now repair of root avulsions did not generate unequivocal proof of recovery of limb function, particularly of the hand. More experimental studies are needed to assess the efficacy of several repair techniques, the optimal timing for surgery, and the complications associated with spinal cord manipulation.

Lumbar spinal cord explants, harvested from neonatal rat pups aged between postnatal day 0 (P0) and P6, were cultured for a period of 48 hrs in the chemically defined medium R(12) on a poly-ethylene-imine (PEI) and on poly-D-lysin (PDL) coated surface. The outgrowth outside the explant was quantified. Lumbar explants from the same rat and embedded in a collagen matrix, and cortical explants from a P0 rat were used as controls. Statistical analysis demonstrated a clear relation between age-at-explantation and the number of neurites in the corona surrounding the explant. The number of outgrowing neurites decreased sharply with age-at-explantation. The average number of neurites per explant obeyed to the expression log (n) = -0.736x + 3.294 on PEI, and log (n) = -0.721x + 2.295 on PDL; x epsilon in [P0 - P6] (n, the number of neurites per explant; x, the age-at-explantation expressed in postnatal days). A similar observed age-related decrease of outgrowth has been described when culturing the lumbar explant inside a collagen matrix. The phenomenon appears to be an intrinsic property of the explant. We review growth inhibitory properties in different models and propose that the phenomenon occurs here at the interface explant-world.

Lumbar spinal cord explants, harvested from neonatal rat pups aged between postnatal day 0 (P0) and P7, were cultured for a period of 48 h in the chemically defined medium R(12) [17] (Romijn, H.J., van-Huijen, F., Wolters, P.S., Neurosci Biobehav Rev, 8 (1984) 301-334), embedded in a collagen matrix. The outgrowth into the surrounding matrix was quantified. Age-matched cortical explants were used as controls. Despite adaptations of the culture protocol, outgrowth remained variable. Statistical analysis demonstrated a clear relation between the age of the explant (at the time of explantation) and the number of neurites in the corona surrounding the explant. The number of outgrowing neurites decreased sharply with age. The average number of neurites per explant obeyed to the expression log(N)= -0.652 A+17 (N: the number of neurites per explant; A: the age expressed in gestational days; A epsilon [G23-G30]; G23 signifying gestational day 23, or P0). The observed age-related decrease of outgrowth could not be explained by progressive myelination of the spinal cord white matter, nor by the absence of trophic support from muscle, but may be related to a progressive inability of the spinal neurites to interact with collagen.

The axons that originate in the medial somatomotor cortex of the rat depart, during development, after those from the lateral somatomotor cortex, yet they arrive in the cervical spinal cord first. Either the medially originating axons elongate faster, or the laterally originating ones pause along the descent pathway. To investigate the presence of an intrinsic difference of the axonal elongation velocity between the lateral and medial somatomotor cortical areas, we cultured explants taken from these areas for 2 days, and measured the length of the outgrowth. After 2 days the explants were surrounded by a radiate corona of axons of which the longest measured 1.95 mm. A significant difference was detected between the medial and lateral somatomotor cortical areas in vitro. Axons originating from explants taken from the medial somatomotor cortical area are, after 2 days in culture, on average 0.16 mm longer than those from the lateral somatomotor cortical area. Though the observed difference is not large enough to allow for the overtaking observed in vivo, it does indicate that intrinsic differences exist within the developing rat somatomotor cortex. This in turn indicates that intrinsic cortical traits not only influence regionalization and targeting behavior of cortical projection neurons, but also their axonal elongation speed.

In order to establish a timetable for the developmental descent of supraspinal descending projections in the rat, a retrograde neuronal tracer was injected into the spinal cord of rat fetuses and neonates both at different gestational ages and at different levels of the spinal cord. From the results of these experiments a position interval could be deduced for the leading descending fibers of each spinal-projecting nucleus at each age studied. The chronological series of position intervals of each supraspinal descending projection (the descent pattern) depicts the descent of its fiber front during development and allows for easy comparison between the various projections. According to these descent patterns the descent sequences of the various spinal-projecting nuclei were established. At E17 fibers from the lateral vestibular nucleus, the raphe magnus nucleus and the gigantocellular reticular nucleus were present in the lumbosacral spinal cord; their descent along the spinal cord thus occurs before this stage. At E18 fibers from the parafascicular prerubral nucleus, the interstitial nucleus of Cajal, the mesencephalic reticular nucleus, the caudal pontine reticular nucleus, the laterodorsal tegmental nucleus, the subcoerulean nucleus, the spinal vestibular nucleus, the interpolar spinal trigeminal nucleus, the raphe obscurus nucleus and the ventral medullary reticular nucleus arrived in the lumbosacral cord. At the same stage fibers from the oral and caudal spinal trigeminal nucleus reached their caudalmost extent in the spinal cord, respectively, the lower and upper thoracic cord. At E19 fibers from the oral pontine reticular nucleus, the parvocellular reticular nucleus, the ventral gigantocellular reticular nucleus and the ambiguous nucleus first appeared in the lumbosacral cord. At E20 fibers from Darkschewitsch's nucleus, the paralemniscal and parabrachial nuclei, cell group A5, the locus coeruleus, the gigantocellular reticular nucleus-alpha, the raphe pallidus nucleus, the paramedian reticular nucleus, and from the dorsal medullary reticular nucleus arrived in the lumbosacral cord. Last to arrive in the lumbar cord during the prenatal period, at E21, were fibers from the posterior commissural nucleus, the red nucleus, the Edinger-Westphal nucleus, the paragigantocellular reticular nucleus, the medial vestibular nucleus, Roller's nucleus, and the solitary nucleus. Fibers from the paraventricular hypothalamic nucleus and from the lateral hypothalamic area only arrived in the lumbosacral cord at P1, followed by fibers from the incertal nucleus at P4. A transient spinal projection from an unknown group of neurons located immediately lateral to and partly intermingled with the mesencephalic trigeminal nucleus arrived in the lumbosacral spinal cord at E18 and had disappeared at P1. This cell group, called Gr?, closely resembled the mesencephalic trigeminal nucleus (Me5). Both were teardrop shaped; an oblong mass of neurons at the caudal end with a long and thin trailing edge. The trailing edge of Gr?, however, curved dorsad towards the dorsal midline raphe of the caudal mesencephalon, while the trailing edge of Me5 curved rostrad, parallel to the sulcus limitans of the sylvian aquaduct. The neurons of Gr? are mainly round, but in the caudal part of the nucleus some horizontally oriented fusiform neurons were observed. All neurons of Gr? were tiny. These results confirm that the generation sequence of the source nuclei is not a prime determinant of descent sequence along the spinal cord. The distance between the source nucleus and the entrance to the target seems of influence only in the most extreme cases (diencephalic source nuclei and the cerebral cortex). Descent velocity of the fiber fronts is not equal between different sources, nor is the descent velocity of specific fiber fronts constant over time. (ABSTRACT TRUNCATED)

We have explored basic rules guiding the early development of topographically organized projections, employing the rat corticopontine projection as a model system. Using anterograde in vivo tracing with 1,1',dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), we studied the distribution of labelled fibers in the pontine nuclei in relation to cortical site of origin during the first postnatal week. Labelled corticopontine fibers enter the pontine nuclei in distinct, sharply defined zones. The putative terminal fibers typically occupy lamella-like subspaces. Related to changes in cortical site of origin, we describe mediolateral, internal to external, and caudorostral distribution gradients in the pontine nuclei. Fibers originating in the anterolateral cortex occupy an internal central core, while implantations at increasing distance from the anterolateral cortex produce 1) more externally located lamellae, and 2) a caudal to rostral shift in fiber location. Previous investigations have shown that pontocerebellar neurons migrate into the ventral pons in a temporal sequence (Altman and Bayer [1987] J. Comp. Neurol. 257:529). The earliest arriving neurons occupy the central core and later arriving neurons settle in more externally and rostrally located subspaces. We hypothesize that the earliest arriving corticopontine fibers grow into the then only available zone of pontocerebellar neurons (central core), attracted by a diffusible chemotropic cue. Later arriving fibers grow into correspondingly later and more externally and rostrally located contingents of pontocerebellar neurons. Thus, we propose that the topographical organization in the early postnatal corticopontine projection is determined by simple temporal and spatial gradients operative within source (cerebral cortex) and target region (pontine nuclei).

CD15 and alkaline phosphatase are significant markers of the developing central nervous system. CD15 is known for its interaction in cell-cell contacts, while the presence of alkaline phosphatase is related to the formation of neuronal connections. This paper describes a combined immunocytochemical and enzyme histochemical technique to demonstrate both embryonic markers in the same cryostat section. The monoclonal antibody 3B9, which was used for the detection of CD15 recognizes the immunodominant carbohydrate structure alpha-1,3-Fucosyl-N-Acetyl-Lactosamine also known as X, LewisX, FAL or SSEA-1. This antigen dissolves and is easily rinsed out of the section. The procedure starts off with the detection of CD15. The results of the immunocytochemical procedure is a reaction product which is stable at a high pH, preventing loss of the immunocytochemical reaction product during the subsequent alkaline phosphatase detection. The other embryonic marker, alkaline phosphatase, can only be demonstrated enzymehistochemically if the enzyme is still active. The immunocytochemical localization procedure decided for, does not inactivate alkaline phosphatase totally. From the colorimetrical and histochemical alkaline phosphatase determinations it was concluded that the residual alkaline phosphatase activity detected with this technique, could be intensified by adding Mg(2+)-ions to both the colorimetrical and histochemical incubation media.

The circadian pacemaker in the suprachiasmatic nuclei (SCN) is entrained to the environmental light-dark cycle via a direct retinal projection to the hypothalamus. This projection is thought to use glutamate or aspartate as neurotransmitter. [3H]-D-Aspartate was microinjected in the SCN and adjacent hypothalamic nuclei of Syrian hamsters. This neuronal tracer is selectively taken up by terminals of neurons that use glutamate or aspartate as neurotransmitter and retrogradely transported to their perikarya. With autoradiography labeled cells were visualized in the retinal ganglion cell layer. Labeled cells were also found in a subset of brain nuclei known to project to the injection area. Labeled cells were detected in the bed nucleus of the stria terminalis, paraventricular nucleus of the thalamus, lateral septal nucleus, and medial amygdaloid nucleus. No labeled cells were observed in the medial septal nucleus, intergeniculate leaflet, and ventral lateral geniculate nucleus, which are also known to project to the SCN. Our results indicate that glutamatergic/aspartatergic retinal ganglion cells project to the SCN and adjacent medial hypothalamic nuclei. Moreover, the SCN may receive glutamatergic/aspartatergic input from the brain neuronal subsets that were retrogradely labeled with [3H]-D-aspartate.

This study is the first description of the descent of rubrospinal fibers through the spinal cord of the rat fetus. Either horseradish peroxidase or wheat germ agglutinin-horseradish peroxidase conjugate was injected into the spinal cord, at different levels and at different gestational ages. At embryonic day 17 (E17) fibers from all subdivisions of the nucleus ruber (NR) started their descent towards the spinal cord. At E18 fibers from the ventrolateral NR reached the lower cervical spinal cord, and those from the caudal NR reached the lower thoracic spinal cord. At E19 fibers from the dorsomedial NR and from the parvicellular NR had just reached the cervical spinal cord, while fibers from the ventrolateral and caudal NR descended to lower thoracic levels. At E21 fibers from the dorsomedial NR reached the lower cervical spinal cord. Fibers from the ventrolateral and caudal NR completed their descent through the lumbosacral spinal cord during the first three postnatal days. During their descent the rubrospinal fibers were confined to the white matter of the spinal cord. The earliest descending fibers originated in the caudal NR. Fibers from the caudal part of each magnocellular subdivision of the NR descended before their rostral counterparts. Fibers from the dorsomedial NR only reached the cervical enlargement as the fibers from the ventrolateral NR descended through the cervical enlargement. The somatotopy of the adult rubrospinal projection reflects this sequence; the dorsomedial NR (dmNR) projects to the cervical spinal cord, and the ventrolateral NR (vlNR) projects to the lumbosacral spinal cord. In general, early descending fibers originated from neurons located caudally and ventrolaterally, while later descending fibers originated from neurons located progressively more rostrally and dorsomedially in the magnocellular NR.

This study investigates the ontogeny of the spinocerebellar projection and especially focuses on the relation between temporary longitudinal patterns in the cytoarchitecture of the developing cerebellum of the chicken and the emergence of longitudinal patterning in the spinocerebellar projection, WGA-HRP was injected into the spinal cord of chicken embryos at different stages of development. It was found that spinocerebellar fibers arrive in the cerebellar "anlage" before any longitudinal cytoarchitectonic pattern can be discerned. Thereafter the organization of the spinocerebellar projection seems to develop related to the two temporary cytoarchitectonic longitudinal patterns present in the chick cerebellum. In both stages the development of the spinocerebellar projection displays a rostro-caudal gradient.

The development of the spinal projections of the nucleus paraventricularis hypothalami (PVH) was studied by injecting wheatgerm-agglutinated horseradish peroxidase into the upper thoracic spinal cord of fetal rats at developmental ages ranging from embryonic day 16 (E16) to E21, and into the thoracic spinal cord of rat pups and adult rats. At E18 the first retrogradely labeled cells are seen in the PVH. These cells are located dorsally in the PVH, adjacent to the ventricular matrix. At E20 a more ventrally located group of retrogradely labeled cells appears. This ventral group joins caudally with the dorsal group of labeled cells. The dorsal group of labeled cells is closely packed, while the ventral group of labeled cells is intermingled with unlabeled cells. After E20 this spatial configuration remains essentially constant. Therefore descending projections from all spinal projecting cell groups in the PVH have reached lower cervical spinal cord levels at E20.

The morphogenesis of the ventricular surface of the diencephalon of the rat was studied using scanning electron microscopy, cryostat serial sections and direct observations under a dissection microscope. Based on these observations a description is given of the neuromeres present within the prosencephalon and of the termination of the sulcus limitans. Two conclusions are reached. First, three neuromeres are present in the prosencephalon. Neuromere I consists of the telencephalon, the hypothalamic regions and the parencephalon anterius. Neuromere II is the parencephalon posterius, neuromere III the synencephalon. Second, the sulcus limitans terminates ventrally in the parencephalon posterius and does not continue towards the preoptic recess. No exact termination point of the sulcus limitans could be delineated.

The localization of alkaline phosphatase (E.C. 3.1.3.1.) positivity during prenatal development of the hypothalamus of the rat is described. At E12 all layers of the prosencephalon display alkaline phosphatase (AP) positivity. The AP positivity increases from dorsal to ventral. Within the hypothalamic area a second, rostro-ventral gradient exists from E14 onwards. At E18 both gradients have decreased. At E20 almost all AP positivity has disappeared from the hypothalamus, with the exception of some reaction product in the dorsal ventricular matrix of the hypothalamus. The significance of this pattern in relation to the differentiation of the hypothalamus and to the formation of hypothalamic connections is discussed. It is suggested that AP activity is related to the formation of connections.

In this paper the development of the intermediate lobe (IL) of the pituitary of the rat is described using alkaline phosphatase (AP) (EC 3.1.3.1.) enzyme histochemistry and stage-specific embryonic antigen 1 (SSEA-1) immunocytochemistry. SSEA-1 and AP are co-localized during late development and reveal the existence of two cytochemically different cell types within the IL, i.e. SSEA-1/AP-positive and SSEA-1/AP-negative cells. The SSEA-1/AP-positive cells are initially arranged along the hypophyseal lumen, in a number of longitudinally oriented zones. alpha-Melanocyte-stimulating hormone (alpha-MSH) immunoreactivity is expressed in the SSEA-1/AP-negative cells from E20 onwards. Eventually the SSEA-1/AP-positive cells develop into a layer of cells covering the luminal surface of the IL lobules. These cells represent the glio-epithelial or neuroglial cells of the IL.