Dissociation between apoptosis, neurogenesis, and synaptic potentiation in the dentate gyrus of adrenalectomized rats.

Removal of adrenal hormone corticosterone in rats aged 3-4 months results within 3 days in acceleration of apoptosis and proliferation of newborn cells in the dentate gyrus (DG). A critical question is whether such a shift in the maturity of dentate cells after adrenalectomy (ADX) affects synaptic plasticity. To address this question, male rats were adrenalectomized and synaptic potentiation was recorded in vitro in hippocampal slices, as well as in vivo, in response to high frequency stimulation of the perforant path, 3 days after ADX. At this time-point, cell loss was assessed and proliferation was examined. Based on two independent parameters, bromodeoxyuridine and Ki-67, we found that removal of the adrenal glands increases proliferation rate. This increase in proliferation was, in particular, evident in those animals that displayed substantial cell loss. The accelerated cell-turnover after ADX was accompanied by reduced synaptic potentiation, both when recorded in vitro and in vivo. Corticosterone replacement in vivo (in adrenalectomized animals), at levels that activate the mineralocorticoid receptor, prevented ADX-induced proliferation, apoptosis, and restored synaptic potentiation to control levels. Importantly, corticosterone applied to slices from adrenalectomized rats also normalized synaptic potentiation, despite increased proliferation. This suggests that changes in cell proliferation and apoptotic cell death in the DG are not necessarily key factors determining the efficacy of synaptic potentiation.

Brief treatment with the glucocorticoid receptor antagonist mifepristone normalises the corticosterone-induced reduction of adult hippocampal neurogenesis.

The glucocorticoid receptor antagonist mifepristone has been shown to rapidly and effectively ameliorate symptoms of psychotic major depression. To better understand its mechanism, we investigated mifepristone's cellular effects, and found that it rapidly reversed a chronic corticosterone-induced reduction of adult neurogenesis in rats. Unlike other antidepressants, mifepristone is particularly potent in a high corticosterone environment. These data indicate that similarly to its clinical efficacy, mifepristone's effects on adult neurogenesis are rapid and positive, and may therefore be important for its mechanism of action.

Gene expression profiles associated with survival of individual rat dentate cells after endogenous corticosteroid deprivation.

Removal of circulating corticosterone by adrenalectomy (ADX) leads to apoptosis after 3 days in a small population of rat dentate granule neurons, whereas most surrounding cells remain viable. Interestingly, a specific expression profile is triggered in surviving granule cells that may enhance their survival. Hippocampal slices prepared 1, 2 or 3 days after ADX or sham operation were stained ex vivo with Hoechst 33258, which serves to identify apoptotic neurons. After electrophysiological analysis, multiple gene expression in surviving individual granule cells was assessed by linear antisense RNA amplification and hybridization to slot blots containing various neuronal cDNAs. Hierarchical clustering and principal component analysis was performed on two physiological variables and 14 mRNA ratios from ADX cells from every time point. Our results indicate that surviving 3-day ADX granule cells display lower membrane capacitance, lower relative N-methyl-d-aspartate (NMDA) R1 mRNA expression and higher relative mineralocorticoid receptor (MR), alpha1A voltage-gated Ca-channel, Bcl-2 and NMDA R2C mRNA expression. Some 1- and 2-day ADX cells cluster with these 3-day survivors; therefore, one or more components of their mRNA expression profile may represent predictive markers for apoptosis resistance. The functional relevance of two candidate genes was tested by in vivo local over-expression in the same model system; of these, Bcl-2 conferred partial protection when induced shortly before ADX. Therefore, removal of corticosteroids triggers a specific gene expression profile in surviving dentate granule cells; key components of this profile may be associated with their survival.

Increased P27KIP1 protein expression in the dentate gyrus of chronically stressed rats indicates G1 arrest involvement.

Various chronic stress paradigms decrease new cell proliferation in the hippocampal dentate gyrus, yet the exact underlying mechanism is still unclear. In the first gap (G1) phase of the cell cycle, both stimulatory and inhibitory signals derived from the extracellular environment converge. Corticosteroids, which increase during stress and are well-known anti-mitotics, cause cells in vitro to arrest in the G1 phase. Following 3 weeks of unpredictable stress, we therefore expected a change in protein expression of various important G1 cell cycle regulators in the adult rat subgranular zone. Using quantitative immunocytochemistry, we show that particularly cyclin-dependent kinase inhibitor p27Kip1 expression is significantly increased. In addition, 3 weeks of recovery after stress normalized the numbers of p27Kip1-expressing cells, consistent with the recovered adult cell proliferation in these animals. P27Kip1-positive cells do not overlap with GFAP-staining and only to a limited extent with Ki-67-expressing cells. Numbers of cyclin E- and cyclin D1-expressing cells did not change after chronic stress. These results indicate that chronic stress causes cycling cells in the adult hippocampus to arrest in G1, thereby providing more mechanistic insight in the stress-induced decrease in cell proliferation.

Fos-like immunohistochemical identification of neurons active during the startle response of the rainbow trout.

Activity-dependent Fos-like expression was investigated immunohistochemically in rainbow trout (Oncorhynchus mykiss) that had performed vibratory-evoked startle responses. We found significantly higher numbers of Fos-like-immunoreactive neurons in the reticular formation, in the octavolateral area, and in several cranial nerve motor nuclei in the brain and in the motor column of the spinal cord of startled fish than in control fish. In one fish, in which stimulation did not evoke startle responses, substantial numbers of positive cells occurred in the brain, primarily in the magnocellular octavolateral nucleus. We observed Fos-like-immunoreactive neurons in cell groups that are known to participate in the startle response (e.g., the Mauthner cell) as well as in cell groups that have been proposed but until now not shown to be involved.

The corticosterone synthesis inhibitor metyrapone prevents hypoxia/ischemia-induced loss of synaptic function in the rat hippocampus.

BACKGROUND AND PURPOSE: Ischemia is accompanied by abundant corticosterone secretion, which could potentially exacerbate brain damage via activation of glucocorticoid receptors. We addressed whether manipulating steroid levels during ischemia affects hippocampal synaptic function along with neuronal structure. Moreover, we established whether pretreatment with the glucocorticoid receptor antagonist RU38486 is as effective in preventing deleterious effects after ischemia as is the steroid synthesis inhibitor metyrapone. METHODS: Rats underwent 20 minutes of unilateral hypoxia/ischemia (HI). Convulsions were monitored after HI, and 24 hours later, field potentials were recorded in vitro in the hippocampal CA1 area in response to stimulation of the Schaffer collateral/commissural fibers. Morphological alterations were determined in brain slices from the same animals. Data were correlated with steroid treatment before HI. RESULTS: Metyrapone suppressed plasma corticosteroid levels during HI, whereas corticosterone treatment significantly elevated plasma steroid levels. These treatments affected the incidence of visible seizures after HI: corticosterone treatment resulted in the highest incidence, whereas metyrapone attenuated the occurrence of seizures. Moreover, the HI-induced impairment in synaptic transmission in the CA1 area in vitro was exacerbated by concomitant corticosteroid treatment and alleviated by pretreatment with metyrapone. In parallel, degenerative changes in the hippocampus after HI were most pronounced after corticosterone treatment, whereas metyrapone reduced these alterations. RU38486 was effective only in reducing the incidence of seizures shortly after ischemia. CONCLUSIONS: We tentatively conclude that synaptic function along with cellular integrity is preserved after HI by preventing the ischemia-evoked rise in corticosteroid levels rather than blocking the glucocorticoid receptor.

Postischemic steroid modulation: effects on hippocampal neuronal integrity and synaptic plasticity.

Elimination of corticosteroids after ischemia, by removal of the adrenals, has been reported to preserve neuronal integrity later. To establish the therapeutic potential of this observation, the authors address two questions: first, whether clinically more relevant steroid manipulations after ischemia exert similar protective effects, and second, whether changes in synaptic functioning occur along with structural alterations. To test this, the authors treated animals immediately after hypoxia-ischemia with (1) the steroid synthesis inhibitor metyrapone, (2) the synthetic glucocorticoid receptor agonist dexamethasone, (3) the selective glucocorticoid antagonist RU 38486, or (4) corticosterone. Metyrapone, but none of the other compounds, attenuated the occurrence of seizures immediately after ischemia. Twenty-four hours after hypoxia-ischemia, CAI hippocampal field potentials in response to stimulation of Schaffer/commissural fibers were found to be reduced. The attenuation of synaptic transmission was partly prevented by metyrapone. None of the other experimental treatments influenced the impaired synaptic function. Gross morphologic analysis revealed no differences in the loss of neuronal structure between the experimental groups at this time point. Taken together, these data suggest that metyrapone preserves neuronal functioning despite loss of neuronal structure. The authors tentatively conclude that preventing the ongoing production of steroids shortly after ischemia can delay and attenuate the appearance of ischemia-related pathology.

Long term effects of spinal cord transection in zebrafish: swimming performances, and metabolic properties of the neuromuscular system.

This study concerns functional recovery of zebrafish following spinal cord transection. Spinal cords were transected at the level of the 14th vertebra, just rostral to the dorsal fin. Recovery was tested at one month after transection when descending fibers start to regrow across the transection site and at three months after transection when fish perform kick and glide swimming. To estimate the rate of regrowth across the lesion site we analysed the tyrosine hydroxylase (TH) and dorsal 5-hydroxytryptamine (5-HT) systems in distal parts of lesioned cords. Both systems have cell bodies in the brainstem and in control fish TH- and dorsal 5-HT-containing fibers descend to all spinal segments. Swimming performance was studied by subjecting lesioned fish to endurance tests in a swimming tunnel with water flowing at a constant rate of 2 or 4.5 body lengths per second (BL/s). At 2 BL/s slow myotomal muscles are active whereas at 4.5 BL/s fast myotomal muscles are recruited. Control fish endured sustained swimming at both speeds for at least 3 hours. As a measure for the condition of the neuromuscular system in trunk and tail, we analysed aerobic metabolic capacities, assessed by NADH-tetrazolium reductase (NADH-TR) histochemistry of myotomal muscle fibers and spinal lateral neuropil. We found that TH- and dorsal 5-HT-immunoreactive fibers were absent in the entire distal part of lesioned cords at one month but at two months after transection they were present at approximately 6000 microns caudally to the site of the lesion. Thus the rate of outgrowth of these fibers is at least 200 microns per day. Sustained swimming at the slow speed (2 BL/s) could be endured for about 14.4 min at one month and for 23.5 min at two months after transection; there was no further improvement in the period that followed. In contrast, in the 10 weeks following transection, fast swimming (4.5 BL/s) could be endured for about 5 to 6 minutes. A significant improvement was gained in the period of 10 to 12 weeks after transection when fish could endure the high speed for almost 15 min. The aerobic capacity of muscle fibers in distal parts of the body was not strongly affected by the lesion. The only important change in aerobic capacity was observed in the neuropil of distal parts of the cords where, at three months after transection, NADH-TR activity was increased to approximately 150% of control values. On the basis of our findings, we assume that it is not the condition of the neuromuscular system, but rather a deficient co-ordination between proximal and distal body parts of lesioned fish that accounts for the relatively poor performances in endurance tests. Furthermore, differences in timing of improvements in swimming at 2 and 4.5 BL/s indicate that the spinal circuitries serving the slow parts of the neuromuscular system recover at an earlier stage than those serving the fast parts.

Changes in the synaptology of spinal motoneurons in zebrafish following spinal cord transection.

Effects of spinal cord transection on the synaptology of zebrafish spinal motoneurons were studied. The transection was made at the level of the 14th vertebra and the synaptology of motoneuron somata and dendrites was analysed at the level of the 21st to the 23rd vertebrae at one month and three months after transection. Horseradish peroxidase, applied to the myotomal muscle, was used to label motoneuron somata and dendritic branches in central and in lateral areas of the neuropil (referred to as central and lateral dendritic profiles). Boutons impinging on motoneurons were classified according to the morphology of the vesicles. We discerned R-boutons with spherical vesicles, F-boutons with flat vesicles and DC-boutons with at least one dense core vesicle. The apposition lengths of R-, F- and DC-boutons and the circumference of labelled profiles were determined to assess the proportional covering of boutons on somata and dendrites. Ratio's of covering with R- and F-boutons (R/F ratio) for somata, central and lateral dendritic profiles were 1.1, 2.1, and 2.1 in control fish and 0.5, 0.5 and 0.9 in lesioned fish at one month after transection, respectively. The total covering of motoneurons in lesioned fish was decreased by 20% on somata and by 30% on lateral dendritic profiles, whereas central dendritic profiles did not change significantly. At three months after transection the R/F ratio's for somata, central and lateral dendritic profiles were 0.5, 0.7 and 0.6, respectively. The total covering on somata and central and lateral dendritic profiles was at control levels. The anatomical aspects of the changes in synaptology indicate that in control fish 50 to 60% of the R-boutons on the motoneuron surface originate from descending axons. In contrast, almost all F-boutons seem to be from local origin.

Organisation of the zebrafish spinal cord: distribution of motoneuron dendrites and 5-HT containing cells.

In order to find arguments for a selective innervation and modulation of the fast and slow spinal motoneurons in the zebrafish, we determined: the territories occupied by the dendritic trees of the large spinal motoneurons innervating the fast white muscle (WMNs) and those of the smaller motoneurons (RIMNs) innervating the slower red and intermediate fibers; the distribution of 5-hydroxytryptamine (5-HT) immunoreactive cells, which constitute one of the systems for the modulation of motoneuron activity. Motoneurons were either retrogradely labelled with horseradish peroxidase or iontophoretically filled with Lucifer yellow. The 5-HT cells were identified immunohistochemically. We found that the dendritic territories of the WMNs and those of the RIMNs partly overlap but they also occupy unique areas. The unique area of the WMN dendrites is dorsal to the lateral neuropil, where axon collaterals of the bulbospinal tract invade the motor column; the unique area of the RIMN dendrites lies in the ventral part of the cord, near the ventral commissural tract. 5-HT immunoreactive cells were observed in the ventromedial part of the spinal cord and among the motoneuron somata. The large overlap in dendrite territories between the WMNs and RIMNs argues for a motoneuron recruitment pattern as may be expected from the size principle; while the distinct areas occupied by dendrites of either WMNs or RIMNs hints at a selective innervation. The presence of the 5-HT cells close to the motoneuron somata and in the ventral area, where dendrites of the RIMNs ramify, suggests that the activities of the motoneuron types can be selectively modulated.

A polyclonal antibody against mammalian FOS can be used as a cytoplasmic neuronal activity marker in a teleost fish.

A polyclonal antibody raised against a conserved region of a mammalian FOS sequence was tested for its use as an activity marker in the rainbow trout. The FOS-like expression in the trout is entirely cytoplasmic and appears in a Nissl-like pattern. The reaction is specifically induced by both orthodromic and antidromic electrical stimuli and during motor responses evoked by natural stimulation, although some positive neurons are found at locations that are not obviously related to the presented stimuli. Following spinal nerve stimulation, antidromically activated motoneurons were found to be positive in the ipsilateral spinal cord. Orthodromic driving of spinal moto- and interneurons by stimulation of the medial longitudinal fasciculus (MLF) in the hindbrain evoked FOS-like immunoreactivity throughout the motor column in the spinal cord, but not in regions lying caudal to a lesion of the MLF-axons. Evoking about 25 startle responses by natural auditory stimulation gives FOS-like immunoreactivity in the Mauthner cell, which initiates the response, whereas positive Mauthner cells were never observed in control fish. The stimulation protocols that were used strongly activated the stimulated cells and so the observed FOS-like immunoreactivity might be related to an increase protein synthesis needed to restore their depleted transmitter levels.

Dopaminergic and GABAergic cerebrospinal fluid-contacting neurons along the central canal of the spinal cord of the eel and trout.

In anamniote vertebrates the central region of the spinal cord has been implicated in its regeneration. This is a complex region and so as a first step in understanding its possible regenerative role we have examined the organization of the cells that contact the lumen of the spinal cord in two teleost fishes, eel and trout, using immunohistochemical procedures and light and electron microscopy. Cell bodies immunoreacting positively with antibodies for tyrosine hydroxylase and for dopamine were located at the ventral rim of the central canal, whereas cell bodies reacting for an antibody for gamma-aminobutyric acid were more laterally located. None of the canal-contacting cells were positively immunoreactive for choline acetyltransferase. All immunopositive cells have a similar morphology: the amphora-shaped perikaryon is bipolar and has a single process that extends to the lumen of the canal, and another that branches and forms extensive lateral and ventral plexuses. Electron microscopic investigations of the ventral dopaminergic cells showed that the apical processes bear one or more cilia, which protrude into the canal lumen and which originate from within a superficial rosette of nonciliated processes. The ventral process was occasionally seen to form synapses; the cell body was also the target of synapses.

Coexistence of calcitonin gene-related peptide and choline acetyltransferase in eel efferent neurons.

We applied choline acetyltransferase, (ChAT) and calcitonin gene-related peptide (CGRP) immunocytochemistry to the efferent neurons that innervate the lateral line and the ear of the eel. Strong immunoreactivity to the ChAT antiserum was observed in neurons located within the octavolateralis efferent nucleus that could be distinguished, on the basis of their form, location and dendritic organization, from the ChAT-immunopositive motoneurons of the adjacent facial motor nucleus. Both facial motoneurons and efferent neurons were found to be immunopositive for CGRP, although the reaction was always stronger in the motoneurons. Double labelling experiments established the presence of both ChAT and CGRP in many efferent neurons. The results are evidence that cholinergic efferent neurons supplying end organs of different modalities may also produce calcitonin gene-related peptide.

Immunocytochemical analysis of the dopamine system in the brain and spinal cord of the European eel, Anguilla anguilla.

The distribution of dopamine-containing perikarya and fibres in the central nervous system of the eel, Anguilla anguilla, was determined by using a specific dopamine antiserum. Telencephalic dopamine-immunoreactive somata are located in the external cell layer of the olfactory bulb and throughout the rostrocaudal extent of the subpallium; immunoreactive fibres are located primarily in the bulb and in ventral and lateral portions of the hemispheres. Diencephalic dopamine-immunoreactive neurons are associated with the ventricles in the preoptic area and hypothalamus and in the posterior tubercle. Many of the neurons in the hypothalamus are liquor-contacting. Very few immunoreactive neurons are located in the mesencephalon, and no dopamine-containing cells are found in regions that can be homologized with the ventral tegmental area and substantia nigra of amniotes. There is a rich innervation of the medial octavolateralis nucleus and certain layers of the torus semicircularis and of the tectum. Dopamine-containing neurons are located in the vagal lobe, by the vagal motor nucleus and in the area postrema, which provides a rich dopaminergic innervation of the brainstem motor column and of the reticular formation. Immunoreactive liquor-contacting neurons line the central canal and another type of labelled neuron lies dorsally in the spinal cord.

Changes in the distribution of synapses during growth: a quantitative morphological study of the neuromuscular system of fishes.

The distribution of synaptic sites on multiply innervated muscle fibres was analysed in four teleost fish species (zebrafish, trout, goldfish and stickleback), using acetylcholinesterase histochemistry. Fishes were chosen for this study rather than other vertebrates because of their long period of growth and continuous increase of muscle fibre size. We found that length and diameter of the fibres increase linearly with fish length but that the distance between synaptic sites increases only as the square root of the fish length and of muscle fibre size. This is explained functionally in connection with the increase of the space constant of a muscle fibre that is expected to accompany the increase of its diameter. We suggest that the change in the synaptic distribution is caused by factors associated with the increasingly wider spread of postsynaptic potentials along the growing fibres, as the intersynaptic distance was found to correlate more strongly with fibre size than with other factors, such as age, speed of growth and genetical background.

Distribution of afferent fibers in the brainstem from end organs in the ear and lateral line in the European eel.

Sensory nerve fibers from the lateral line system and labyrinth of Anguilla anguilla were labeled with horseradish peroxidase and traced to various targets in the ipsilateral brainstem. The three rami of the anterior lateral line nerve and the supratemporal ramus of the posterior lateral line nerve form overlapping terminal zones in the ventral portion of nucleus medialis. The posterior lateral line nerve on the body is represented exclusively in the dorsal half of the nucleus medialis. Eighth nerve fibers from the otolithic end organs in the inner ear send fibers into dorsal portions of three octavus nuclei: anterior, magnocellular, and descending, and saccular fibers lie most medial and utricular fibers most lateral. Fibers from vestibular organs, especially the semicircular canals and utricle, end densely in ventral portions of these nuclei and in the tangential nucleus. All labyrinthine sense organs send fibers into the region of a Mauthner-like neuron, and all except the saccule terminate in the reticular formation, tangential nucleus, and eminentia granularis of the cerebellum. Primary sensory input to the octavolateralis efferent nucleus comes only from the labyrinth, and fibers from the saccule alone penetrate the region of efferent neuronal somata. Fibers from labyrinthine end organs except the saccule project to the reticular formation where they may contact the dendrites of efferent somata. Fibers from the lateral line and the eighth nerve overlap most extensively at the rostral pole of the nucleus medialis and in the eminentia granularis of the cerebellum.

Relationship between myoglobin and succinate dehydrogenase in mouse soleus and plantaris muscle fibres.

This report describes a quantitative histochemical study of myoglobin in skeletal muscle fibres. The muscle fibres were classified as fast or slow on the basis of their quantitative myofibrillar ATPase histochemistry. A large range of myoglobin absorbance values was found among fast skeletal muscle fibres. This range was relatively small among slow fibres. The concentrations of myoglobin and the activities of succinate dehydrogenase in individual muscle fibres in serial sections are weakly correlated in both the mouse soleus and plantaris muscle. The myoglobin concentration is higher in fast and slow oxidative soleus muscle fibres and the succinate dehydrogenase activity in these fibres is lower than in oxidative plantaris muscle fibres in the same range of cross-sectional area.

Quantitative histochemistry of three mouse hind-limb muscles: the relationship between calcium-stimulated myofibrillar ATPase and succinate dehydrogenase activities.

A quantitative modification of Meijer 's calcium-lead capture method, for the demonstration of calcium-stimulated myofibrillar ATPase activity at physiological pH, is described. A range of myofibrillar ATPase activities has been found among fast muscle fibres in two mouse hind-limb muscles. The myofibrillar ATPase activity of fast muscle fibres is 1.5-3 times higher than the myofibrillar ATPase activity of slow muscle fibres. Myofibrillar ATPase activities and succinate dehydrogenase activities of individual muscle fibres have been determined in serial sections. Activities of the two enzymes are correlated positively in soleus (fast and slow fibres), and negatively in plantaris (almost all fast) and extensor digitorum longus muscle (all fast). However, this correlation is not significant among the oxidative fibres in the extensor digitorum longus. The fibres of the latter muscle cannot be classified satisfactorily into two sub-types.

Acetylcholinesterase and acetylcholine receptor histochemistry on end plate regions, myotendinous junctions, and sarcolemma in the axial musculature of three teleost fish species.

In this paper we describe a rapid procedure for the identification of motor end plates in fish. We demonstrated the presence of acetylcholine receptors by means of an immune fluorescence technique with alpha-bungarotoxin. Koelle's thiocholine method was used to localize acetylcholinesterase (AChE) activity. Under carefully controlled conditions the AChE activity and the anti-alpha-bungarotoxin fluorescence showed an equal distribution. This means that in the study of motor innervation in fish the AChE reaction can be used to stain only the motor end plates, leaving the AChE rich preterminal axons unstained. Comparison of the AChE reaction pattern with the distribution of binding sites for antibodies raised against neurofilament protein revealed that in end plate regions high concentrations of AChE are only present in axons and end plates. The myotendinous junctions also possess a high receptor density and enzyme activity. A low enzymatic activity was found at the non-junctional periphery of white muscle fibres. This activity probably resides in the sarcolemma. No non-specific cholinesterase activity was found. From light microscopical analysis it appeared that a single end plate may innervate 2 adjacent muscle fibres. This was affirmed by ultrastructural observations. The dual innervation suggests that, in fish, motor units have a limited distribution through the myotome.