Parvalbumin- and calbindin-containing neurons express c-fos protein in primary and secondary (mirror) epileptic foci of the rat neocortex.

The present experiments aimed at the description and further immunocytochemical characterization of activated neocortical neurons expressing the c-fos gene. Focal seizures were induced by the topical application of isotonic, isohydric 4-aminopyridine solution to the frontal neocortex of adult anesthetized Wistar rats. The EEG of both hemispheres was recorded from the surface of the skull. The animals were perfused with fixative, coronal plane vibratome sections were cut and stained with cocktails containing polyclonal c-fos and monoclonal calbindin or parvalbumin antibodies. The polyclonal c-fos antibody was tested with Western blotting and the diffusion of 4-aminopyridine investigated with autoradiography of [3H]4-aminopyridine. The c-fos protein was detected in every layer of the neocortex (primary focus) and in some allocortical areas of the treated hemisphere. Scattered immunostained nuclei were observed in layers II, III, IV and VI of the contralateral neocortex (mirror focus). Several parvalbumin- and calbindin-positive neurons contained the c-fos protein in both foci. The medium-sized non-pyramidal parvalbumin neurons were found in layers II-IV and VI of the neocortex and in stratum multiforme of the prepiriform cortex. The c-fos protein was colocalized with calbindin mainly in layers II and III in small and medium-sized non-pyramidal neurons. The results prove that focal epileptiform activity of the neocortex activates diverse inhibitory neuronal populations. As concluded, the inhibitory control is probably more effective in the contralateral hemisphere (mirror focus) than on the side of 4-APY treatment (primary focus).

In vitro detection of calretinin immunoreactivity in chicken embryo dorsal root ganglion neurons: a possible developmental marker.

Calretinin, a cytosolic calcium-binding protein, is widely expressed in mammalian and chicken neurons, including subpopulations of dorsal root ganglion neurons. In chicken embryo spinal ganglion cells, calretinin first appears on the 9th day of incubation. To determine whether the expression of this protein is maintained in vitro and depends on the developmental stage, dissociated dorsal root ganglion cultures from chick embryos at E6 and E10 (before or after establishing connections) were processed for calretinin immunohistochemistry. Cultured neurons from E6 embryos never showed calretinin immunoreactivity at any culture duration, whereas neurons from E10 embryos displayed strong immunostaining immediately after attaching to the culture dish. Quantitative evaluation revealed that the percentage of calretinin-positive neurons increased until day 3 in culture and afterwards declined in parallel the total cell number. These results indicate that primary sensory neurons express calretinin in vitro similarly as in vivo and the gene expression depends from the establishment of connections with peripheral targets.

Parvalbumin and calretinin in the avian thymus.

The avian thymic hormone, known to support the maturation of T-lymphocytes, is biochemically similar to parvalbumin. However, its exact cellular distribution in the thymus is unknown. We have therefore studied the occurrence of parvalbumin and other related calcium-binding proteins in this organ by immunohistochemistry during development and after hatching. Parvalbumin immunoreactivity appears in the epithelial cells on embryonic day 9, and is expressed in the cortical reticular cells in the adult. Calretinin is found in the clusters of medullary epithelial cells from embryonic day 11, whereas calbindin D-28k is absent from this organ. Thus, calcium binding-proteins are excellent markers for different compartments of the avian thymus in embryonic life and after hatching, and their expression seems to coincide with their functional maturation.

Ontogeny of calretinin in chick dorsal root ganglion neurons.

Calretinin immunostaining was performed on chick lumbosacral dorsal root ganglia during embryonic development. Calretinin-immunopositive neurons were first observed at around the 9th day of incubation. Quantitative evaluation revealed a close correlation between the number of immunopositive cells and the duration of incubation. Morphometric measurements disclosed that calretinin-immunoreactive cells belong in the large or intermediate categories of dorsal root ganglion neurons. It was concluded that the appearance of calretinin immunopositivity in spinal ganglion cells during development may be associated with both the morphological and functional maturation of this particular population of primary sensory neurons.

Possible morphological correlates of capsaicin desensitization.

The changes in the fine structure of axonal profiles of the guinea-pig ureter were examined after exposure to capsaicin in vitro. In the ureters exposed to capsaicin (1-10 microM), about 60%, of all axonal profiles exhibited conspicuous ultrastructural impairment. It is suggested that these alterations might interfere with the release of peptides from these particular nerve endings and therefore contribute significantly to the mechanisms of capsaicin desensitization.

Neuronal carbonic anhydrase activity in the central nervous system of the rat: light- and electron histochemical investigations of the islands of Calleja.

Results obtained with a new semipermeable method provided evidence for the neuronal localization of carbonic anhydrase in the olfactory tubercle of the rat brain. Examination of serial coronal sections revealed the carbonic anhydrase content of the granule cells in the islands of Calleja. The enzyme was localized in the nucleus and the cytoplasm of these neurons and did not show binding to any of the intracellular organelles. The large neurons of the olfactory tubercle did not contain the enzyme. The enzyme activity of the granule cells was totally abolished by 10(-4) mol/l acetazolamide. Since, up to now, carbonic anhydrase activity has not been demonstrated with histochemical techniques in any central neurons, our results are the first indication of the presence of this enzyme in mammalian central neurons.

A modified semipermeable membrane technique for carbonic anhydrase histochemistry of the nervous system.

We present a modification of Hansson's method for the demonstration of carbonic anhydrase activity. Using a semipermeable membrane together with a fluid incubation medium, frozen sections of aldehyde-fixed tissue were incubated without floating or dipping. Thin sections (thickness, 20-40 microns) were mounted on the outer surface of a tubular-shaped, semipermeable cellophane dialysis membrane containing the incubation fluid. After incubation for 25-30 min at room temperature, the sections were rinsed in buffer and treated with 0.5% (NH4)2S solution. The histochemical reaction was fully inhibited by 10(-4) M acetazolamide.

The effects of cyproterone acetate on statural growth in children with precocious puberty.

Forty-four patients (42 f, 2 m) with precocious puberty (31 idiopathic, 1 familial, 7 cerebral, 5 McCune-Albright) were treated with cyproterone acetate for periods of 1-8.75 years in different (P less than 0.05) daily dosages of 117 +/- 6.1 mg/m2 per day (mean +/- SEM, group A, N = 20) and 60.8 +/- 2.42 mg/m2 per day (group B, N = 24). Thirty-three girls had experienced menarche before therapy at a mean age of 4.89 +/- 0.42 years. Treatment was started at a chronologic age of 5.45 +/- 0.33 years in the girls and 5.74 +/- 1.34 years in the boys. At the time of evaluation, 31 of our patients had reached final height. With respect to the effects of treatment on statural growth, the Standard Deviation Scores were retrospectively determined for height, weight, and growth velocity. The initial Bayley-Pinneau height predictions were compared with final height and target height, and the skeletal maturation was studied. There were no significant differences between those parameters in the patients of group A and B or between treated and untreated subjects as far as final height and target height were concerned. It is concluded that cyproterone acetate administered orally at daily doses from 50-150 mg/m2 does not improve statural growth of patients with precocious puberty.

Neurotoxic effect of capsaicin in mammals.

Capsaicin is now widely used to explore and/or prove the role of peptide-containing primary afferent neurones in different somato- and viscerosensory functions. The present paper deals with the morphological effects of capsaicin administered according to currently used experimental paradigms. As it has been repeatedly confirmed in the recent literature, administration of capsaicin to newborn mammals results in a highly selective degeneration of a particular population of small sized, B-type primary afferent neurones located in spinal and cranial sensory ganglia. Chemosensitive i.e. capsaicin sensitive primary sensory neurones (CPSNs) correspond to primary sensory ganglion cells which contain neuropeptides. The permanent functional impairments and the decrease in the peptide contents of the sensory neurones observed after neonatal capsaicin treatment may be accounted for an irreversible loss of CPSNs. Direct application of capsaicin to peripheral nerves results in an apparently irreversible functional impairment of unmyelinated afferent fibres implicated in nociceptive, viscerosensory and neurogenic inflammatory mechanisms. Morphological observations indicate that perineural treatment with capsaicin initiates a selective but delayed degeneration process of unmyelinated afferent nerve fibres presumably due to an inhibition of intraneuronal transport mechanisms. In contrast with perineural capsaicin treatment affecting the chemistry and function of the whole sensory neurone, injection of capsaicin into the subarachnoid space results in an irreversible abolition of the "afferent" but not the "efferent" function of CPSNs. Accordingly, noxious thermal or chemical stimuli applied to the peripheral innervation areas of the trigeminal nucleus caudalis or the affected segments of the spinal cord fail to induce nociceptive reflexes because of the degeneration of the central terminals of CPSNs. However, in these same skin areas, application of chemical irritants invariably evoked the neurogenic inflammatory response, indicating that CPSNs deprived of their central terminals maintain their capacity to synthesize and release the peptide(s) responsible for the initiation of that response. In contrast with previous findings, our recent studies furnished evidence for a selective neurodegenerative action of systemically injected capsaicin in adult mammals, as well. Therefore, some of the irreversible functional impairments produced by capsaicin in adult animals may result from the degeneration of a particular subpopulation of CPSNs.(ABSTRACT TRUNCATED AT 400 WORDS)

A glutamate-sensitive neuronal system originating from the area postrema terminates in and transports acetylcholinesterase to the nucleus of the solitary tract.

The possible cellular mechanism of action of systemically administered monosodium-L-glutamate and the projections of glutamate-sensitive area postrema neurons have been studied in rats. Parenteral administration of monosodium-L-glutamate induced a selective degeneration of a particular population of AChE-containing area postrema neurons. Electron microscopic cytochemistry and X-ray microanalysis revealed the presence of calcium-containing electron-dense deposits in the mitochondria of degenerating area postrema neurons indicating the possible pathogenetic role of an enhanced intracellular calcium level in the mechanism of monosodium-L-glutamate-induced nerve cell degeneration. Degeneration of area postrema neurons was followed by the appearance of degenerating axon terminals in a well-defined region of the nucleus of the solitary tract, the area subpostrema. Degenerating area postrema neurons and axon terminals were rapidly engulfed by phagocytes predominantly of microglial character. AChE activity, localized to the basal lamina of the capillaries of the area subpostrema under normal conditions, could no longer be detected in rats treated with monosodium-L-glutamate 3-4 weeks previously. These findings provide evidence for the existence of a particular population of glutamate-sensitive, AChE-containing area postrema neurons which project and transport AChE to the nucleus of the solitary tract. This specific neuronal pathway connecting the area postrema with the nucleus of the solitary tract may play an important role in some of the functions attributed to the area postrema. The results also strengthen the hypothesis that brain capillary AChE activity may be of neuronal origin.

Selective degeneration by capsaicin of a subpopulation of primary sensory neurons in the adult rat.

The morphological effects of systemic capsaicin treatment have been studied in adult rats. Light and electron microscopy revealed that a subpopulation of small-to-medium sized B-type primary sensory neurons, representing about 17% of the total neuronal population in the 4th lumbar spinal ganglion, underwent rapid degeneration after the administration of capsaicin. Quantitative electron microscopy demonstrated a decrease of about 45% in the number of unmyelinated axons in the saphenous nerve. Light microscopy showed extensive axon terminal degeneration in the brainstem and spinal cord confined to the central projection areas of capsaicin-sensitive afferent fibers, as has already been revealed in the newborn rat. The present results furnish evidence for a hitherto unrecognized selective neurodegenerative action of capsaicin in the adult rat.

Neurotoxin induced nerve cell degeneration: possible involvement of calcium.

Neurotoxin induced nerve cell degeneration has been studied in sensory ganglia of newborn and in the area postrema of adult rats following the administration of the selective sensory neurotoxin, capsaicin and the amino acid excitotoxin, glutamic acid, respectively. Light microscopic histochemical, autoradiographic, electroncytochemical and X-ray microanalytical studies revealed that degeneration of certain small-sized, type B primary sensory neurons, induced by capsaicin, was associated with a marked accumulation of calcium predominantly in mitochondria of the damaged ganglion cells. Similarly, monosodium glutamate treatment resulted in the appearance of calcium-containing electron-dense granules in mitochondria of degenerating area postrema neurons. In addition, after a combined administration of 45Ca2+ and capsaicin or monosodium glutamate, significantly higher levels of radioactivity have been detected by liquid scintillation spectroscopy in the Gasserian ganglia and the area postrema, respectively. It is concluded that an enhancement in intracellular calcium level may be intimately involved in the process of neuronal cell death and may represent a common basic mechanism responsible for the development of cellular events leading ultimately to the degeneration of nerve cells.

Cutaneous nerve regeneration in the rat: reinnervation of the denervated skin by regenerative but not by collateral sprouting.

The functional regeneration of cutaneous chemosensitive nerves responsible for the initiation of the neurogenic inflammatory response has been studied in rat hind paw skin. The qualitative and quantitative estimation of plasma extravasation induced by mustard oil, a skin irritant which induces an inflammatory response by the neurogenic route, proved to be a reliable and objective method for studying the regeneration of these cutaneous nerves. The results indicate a complete functional reinnervation by chemosensitive sensory nerves of the skin area previously denervated by crushing the corresponding peripheral nerve. It is concluded that these particular unmyelinated sensory nerves regenerate by means of regenerative sprouting but apparently lack the ability for collateral sprouting. The significance of the present findings in relation to the mechanisms of cutaneous nerve regeneration is discussed.

Immunohistochemical studies on the effect of capsaicin on spinal and medullary peptide and monoamine neurons using antisera to substance P, gastrin/CCK, somatostatin, VIP, enkephalin, neurotensin and 5-hydroxytryptamine.

After neonatal treatment of rats with capsaicin, the spinal cord, the spinal trigeminal nucleus and spinal and trigeminal ganglia were analysed with immunohistochemistry using antisera to several peptides and 5-hydroxytryptamine. A marked decrease was observed in substance P-, cholecystokinin-, somatostatin- and VIP-like immunoreactivity present in the central branches of primary sensory neurons in the spinal cord and in substance P- and somatostatin-like immunoreactivity in sensory ganglion cells. No definite depleting effect of capsaicin could be established on 5-hydroxytryptamine and peptides, such as enkephalin and neurotensin, present in centrally originating fibres in the dorsal horn of the spinal cord. The results demonstrate that the effects of capsaicin are not confined to substance P immunoreactive primary sensory neurons. The possibility is discussed that capsaicin effects specifically functioning rather than chemically specific primary sensory neurons.

Sensory neurotoxins: chemically induced selective destruction of primary sensory neurons.

Neonatal capsaicin treatment has been shown to cause selective degeneration of chemosensitive primary sensory neurons involved in the mediation of chemogenic pain and in neurogenic inflammatory responses. In the present study the neurotoxic effect of capsaicin congeners was investigated in the newborn rat. Some quantitative data on the selective neurotoxic action of capsaicin are also reported. Electron microscopy indicates that some pungent congeners of capsaicin also induce the selective degeneration of type 'B' sensory ganglion cells. At high doses the distribution pattern of axon terminal degeneration within the spinal cord and brain stem was equivalent to that observed after neonatal capsaicin treatment. The neurotoxic potency of capsaicin congeners, unlike desensitizing activity, is closely related to the sensory irritant property of these compounds. It is concluded that primary sensory neurons degenerating after the administration of these capsaicin congeners may correspond to substance P-containing chemosensitive primary sensory neurons involved in the transmission of nociceptive impulses.

Evidence for the neuronal origin of brain capillary acetylcholinesterase activity.

Acute administration of monosodium-L-glutamate (MSG) to rats induces the degeneration of acetylcholinesterase (AChE)-positive neurons of the area postrema (AP). Chronic treatment with MSG results in the disappearance of AChE activity of area subpostrema (ASP) capillaries. It is concluded that processes of AChE-positive AP neurons terminate within the ASP and may contribute to the AChE activity of ASP capillaries.

Preferential vulnerability of dendrites to lithium ion in rat brain and in nerve cell culture.

Preferential swelling and vacuolation of dendrites were observed electron microscopically in different brain regions of rats treated with LiCl in a dose of 6-18 mmol/kg for 1-6 days. The most severe fine structural changes were revealed in the hippocampus. Low-dose (0.33 mmol/kg) lithium treatment lasting for a year did not cause any morphologically detectable alterations in the rat brain. In vitro studies showed a reduction of dendro-axonal process network of neurons measured by morphometric means after lithium exposure. Using different cultures of neuronal and glial cell populations, higher lithium uptake was observed for neuron-enriched cultures.

Direct evidence for an axonal site of action of capsaicin.

Local application of capsaicin to the sciatic nerve of rats induced a long-lasting increase in the nociceptive threshold as tested by the hot-plate method, and prevented neurogenic inflammation in the lateral part of the dorsal skin of the rat's paw. Application of capsaicin to the saphenous nerve prevented the neurogenic inflammatory response, induced either by antidromic electrical stimulation of the saphenous nerve or by painting the skin with mustard oil, in the medial part of the dorsal skin of the paw. The functional impairment induced by local capsaicin treatment of saphenous or sciatic nerves was strictly confined to the skin area supplied by the corresponding nerve. It is suggested that local capsaicin treatment of peripheral nerves selectively damages the chemosensitive nerve fibres presumably by depleting their substance P content.