Shozo Kito

Autoradiographic localization of calcitonin gene-related peptide binding sites in human and rat brains

125I-calcitonin gene-related peptide (CGRP) binding sites were mapped in the human brain and rat brains by in vitro macroautoradiography, and compared to each other. Binding experiments were made to characterize 125I-CGRP binding on the human and rat brains. Scatchard analysis of saturation experiments from slide-mounted sections of the human and rat cerebellum displayed 125I-CGRP binding sites with a dissociation constant (Kd) of 0.17 nM and 0.11 nM, respectively, and a maximal number of binding sites (Bmax) of 96.8 fmol/mg and 23.0 fmol/mg protein. 125I-CGRP binding was time-dependent, reversible and saturable with high affinity in the brains. Autoradiograms showed a discrete distribution of 125I-CGRP binding sites throughout the brains of human and rat with patterns similar to each other. In the human brain, the highest binding was seen in the cerebellum, inferior olivary nuclear complex, certain parts of the central gray matter, arcuate nuclei of the medulla oblongata and dorsal motor nucleus of the vagus, and densities of CGRP-binding sites were high in the nucleus accumbens, amygdala, tail of the nucleus caudatus, substantia nigra, ventral tegmental area, medial portion of the inferior colliculus, medial pontine nuclei, locus coeruleus, inferior vestibular nucleus, substantia gelatinosa of the spinal trigeminal nucleus, nucleus of the solitary tract and nucleus cuneatus lateralis. In the rat, high densities were found in the hippocampus pars anterior, nucleus accumbens, ventral and caudal portions of the nucleus caudatus-putamen, central and basolateral nuclei of the amygdala, caudal portion of the insular cortex, medial geniculate body, superior and inferior colliculi, certain portions of the central gray matter, locus coeruleus, inferior olivary nuclei, vagal complex, nucleus cuneatus lateralis and cerebellum. In contrast, in both species, most of the cortical areas including the hippocampus, most of the thalamus, and hypothalamus exhibited few binding sites. In addition, high quantities of the binding sites were seen on the pia mater and on walls of blood vessels in the brain and subarachnoidea. These results revealed essentially homologous locations of CGRP binding sites in the human and rat central nervous systems and well corresponding distributions of binding sites and endogenous CGRP-like immunoreactivity.

Discrete regional distributions of thyrotropin releasing hormone (TRH) receptor binding in monkey central nervous system

Thyrotropin releasing hormone (TRH) directly influences central nervous system (CNS) function, independent of its pituitary action. Although these CNS effects have been behaviorally characterized, information is not yet available on the precise regional distribution of its receptor. TRH receptor binding was examined in the monkey CNS by the radioreceptor assay for clarifying the site of TRH action. TRH was bound to brain tissue membranes via high-affinity (Kd = 5.9 x 10(-9) M) and low-affinity (Kd = 11.2 x 10(-8) M) components. TRH receptor binding varied dramatically throughout the monkey brain, with more than 40-fold variation. The limbic system contained the greatest amount of binding. The next highest areas were the cerebral cortex, hypothalamus, interpeduncular nucleus and periaqueductal gray matter of the midbrain. Receptor binding was very low or not detectable in the medial thalamus, cerebellum, brain stem, spinal cord and white matter. These data suggest that TRH has an effect on the CNS via limbic system, cerebral cortex and midbrain.

Influence of age on NMDA receptor complex in rat brain studied by in vitro autoradiography.

N-methyl-D-aspartate (NMDA) receptors are known to play an important role in learning and memory and to be involved in neuron cell death accompanying cerebral ischemia, seizures, and Alzheimers disease. The NMDA receptor complex has been considered to consist of an L-glutamate recognition site, a strychnine-insensitive glycine modulatory site, and a voltage-dependent cation channel. In the present study, effects of age on an L-glutamate recognition site and a glycine site were examined in rat brain by quantitative in vitro autoradiography with [3H]-CPP and [3H]-glycine. Both [3H]-glycine and [3H]-CPP binding sites were most abundant in the hippocampus and cerebral cortex, and they showed a similar distribution pattern throughout the brain. [3H]-glycine binding sites were severely decreased in the telencephalic regions, including the hippocampus and cerebral cortex, in aged brain. Conversely, [3H]-CPP binding sites were well preserved in these brain areas. In the mid-brain regions and cerebellum, neither [3H]-glycine nor [3H]-CPP binding sites changed in the aged brain. Our results indicate that within the NMDA receptor complex, glycine receptors are primarily affected in the aging process.

Neuropeptide Y-immunoreactive neurons receive synaptic inputs from dopaminergic axon terminals in the rat neostriatum.

Double immunocytochemistry using peroxidase-antiperoxidase and protein A-gold was performed to determine whether neuropeptide Y (NPY) immunoreactive neurons receive synaptic inputs from catecholaminergic axon terminals in the rat neostriatum. Tyrosine hydroxylase-immunoreactive axons were found to be in synaptic contact with the somas and proximal dendrites of NPY-immunoreactive neostriatal neurons. These latter neurons were medium-sized and had indented nuclei, and thus were thought to be medium aspiny interneurons. Thus nigrostriatal dopaminergic neurons may monosynaptically influence striatal NPY neurons.

Studies on familial amyloid polyneuropathy in Ogawa Village, Japan.

A newly discovered large concentration of familial amyloid polyneuropathy (FAP) in Japan is clinically outlined. The importance of the urinary secretion of IgA in these cases is stressed. Dimethyl sulfoxide administration to these patients induced clinical improvements accompanied by increased excretion of low molecular weight proteins. Amino acid composition of amyloid protein of the Ogawa village-type FAP cases was similar to that of primary amyloidosis.

Immunoreactive atrial natriuretic polypeptides in the adrenal medulla and sympathetic ganglia

Atrial natriuretic polypeptide (ANP)-like immunoreactivity was found in the rat adrenal gland by using indirect immunofluorescence and peroxidase-antiperoxidase techniques. ANP-like immunostaining was present in most of chromaffin cells with varying degrees of immunoreactivity. The majority of medullary cells displayed very intense immunostaining, and several clusters revealed weaker immunostaining. No staining was found in the adrenal cortex or in the nerve fibers in this organ. In the consecutive sections treated for dopamine-beta-hydroxylase (DBH), apparently all medullary cells had intense immunofluorescence for DBH and its distribution pattern was very similar to that for ANP-like immunoreactivity. While phenylethanolamine N-methyltransferase (PNMT) immunoreactive cells largely corresponded to the intensely stained ANP-like immunoreactive cells, suggesting that adrenaline cells contained a large amount of ANP-like substance, noradrenaline cells contained a smaller amount of this substance than adrenaline cells. Ultrastructural study showed that end-products due to the immunoreaction with the ANP antiserum were primarily associating with chromaffin granules. In addition, the presence of ANP-like immunoreactivity was investigated in several sympathetic ganglia of the rat. No principal ganglion cells were ANP-positive, whereas a few small intensely fluorescent (SIF) cells were ANP-immunoreactive. The present findings suggest that catecholamines coexist with ANP which has a natriuretic and vasodilating effect, in adrenal medullary cells and SIF cells in several rat sympathetic ganglia, but not in principal ganglion cells.

DIFFERENTIAL EXPRESSION OF c-fos mRNA IN RAT PREFRONTAL CORTEX, STRIATUM, N. ACCUMBENS AND LATERAL SEPTUM AFTER TYPICAL AND ATYPICAL ANTIPSYCHOTICS: AN IN SITU HYBRIDIZATION STUDY

The regional difference in the expression of c-fos mRNA induced by typical and atypical antipsychotics was determined in prefrontal cortex, striatum, N. accumbens and lateral septum in rats by in situ hybridization. Two typical antipsychotics, haloperidol (2 mg/kg) and fluphenazine (2 mg/kg), and three atypical antipsychotics, (-)sulpiride (100 mg/kg), clozapine (20 mg/kg) and OPC-14597 (40 mg/kg), were used. Brains were fixed with 4% paraformaldehyde 45 min after drug administration (i.p.). Brain sections of 30 microns-thickness were made in a cryostat and hybridized with 35S-labelled for c-fos oligonucleotide probe. These sections were apposed to X-ray films and the autoradiograms were semi-quantitatively analysed by computer-assisted densitometry. All antipsychotics used increased c-fos mRNA expression in N. accumbens shell, a region of the forebrain associated with limbic systems. On the other hand, two typical antipsychotics (haloperidol and fluphenazine) that cause a high incidence of acute motor side effects increased the expression of c-fos mRNA in the dorsolateral striatum, an extrapyramidal region primarily involved in motor control. Only clozapine induced c-fos mRNA in the medial prefrontal cortex and lateral septum. These results strongly suggest that the shell region of N. accumbens may be a common site of therapeutic action of antipsychotics.

Enkephalinergic projection from the ventromedial hypothalamic nucleus to the midbrain central gray matter in the rat: an immunocytochemical analysis

The projection of enkephalin-like immunoreactive (EnkI) neurons in the ventromedial hypothalamic nucleus (VMH) in the rat was investigated using immunocytochemistry. These neurons project to the dorsal portion of the midbrain central gray matter (DCG), because the destruction of the VMH resulted in a marked ipsilateral reduction of EnkI fibers there. Destruction of the dorsomedial portion of the VMH caused the appearance of fibers accumulating EnkI ventrolateral to the lesion and these fibers could be traced to the ventrolateral portion of the VMH where many EnkI neurons were concentrated. Transection of the brain at the level of the premammillary nucleus, mammillary body and superior colliculus caused the appearance of fibers accumulating EnkI in the posterior hypothalamic nucleus (PH), central gray matter medial to the fasciculus retroflexus Mynert and in the DCG, respectively. We concluded that the axons from these neurons first project dorsomedially to situate in the PH. These fibers ran caudodorsally and entered into the dorsal longitudinal fasciculus in the DCG at the midbrain.

Ontogeny of the calcitonin gene-related peptide in the nervous system of rat brain stem: An immunohistochemical analysis

The ontogeny of the calcitonin gene-related peptide in the neuron system of the rat brain stem was investigated by means of the indirect immunofluorescence technique. Calcitonin gene-related peptide-like immunoreactivity was first detected in the fibers of the nucleus of spinal tract trigeminal nerve on gestational day 18, and thereafter appeared gradually in various brain stem areas such as in the fibers of the solitary tract, gracile nucleus, cuneate nucleus, inferior colliculus, superior colliculus, medial geniculate nucleus and in the neurons of the hypoglossal nucleus, facial nucleus, superior olive, parabrachial area, superior colliculus and peripeduncular nucleus. In colchicine-untreated animals, the immunoreactive fibers increased in number and reached adult level by postnatal day 14, whereas the number of cells reached a maximum between postnatal days 2 and 6 and then decreased in number and immunoreactivity or disappeared, except in some areas such as the superior olive and peripeduncular nucleus, which showed the same immunoreactivity as for adult animals. With colchicine treatment, calcitonin gene-related peptide-like immunoreactive cells were found in more areas of the brain stem such as the abducens nucleus, parabigeminal nucleus, principal oculomotor nucleus, trochlear nucleus and central gray, along with the nuclei which had shown calcitonin gene-related peptide immunoreactivity in the untreated animals. However, the neurons in the inferior olive showed a different ontogenetical pattern of calcitonin gene-related peptide of immunoreactivity. Immunoreactivity disappeared completely by postnatal day 21 in both colchicine-untreated and -treated animals.

Peptides in the peripheral nervous system.

Publisher Summary This chapter describes the general morphological profiles of the various bioactive peptides in the peripheral nervous system, and proposes their roles in several specific organs. In addition to the peptides, the distribution of serotonin (5-HT) is described. The advances in immunocytochemical techniques have revealed that the peripheral nervous system contains an abundance of transmitter candidates including neuropeptides, monoamines, and ACH. In order to elucidate the functions of these substances, it is necessary to establish the origins of the immunoreactive fibres and projection areas of the axons arising from immunoreactive neurons. Subsequently, detailed analysis of the microcircuitry within a given target organ is required. Coexistence of neurotransmitters has been found in the peripheral nervous system as in the central nervous system. For example, ENK and ACH coexist in the preganglionic parasympathetic neurons, NA and NPY coexist in the sympathetic ganglia, ACH and CGRP coexist in the motor neurons innervating striated muscle, and SP and CGRP coexist in the primary sensory ganglionic neurons.