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Age-related changes in astrocytes, tanycytes and the microvasculature were investigated in the median eminence (ME) of the anterior hypothalamo-hypophyseal system of 2- and 24-month old rats by means of immunocytochemistry and scanning electron microscopy. A computer-assisted image processing system showed a significant increase of the glial fibrillary acidic protein (GFAP) immunoreactivity in the internal layer (IL) and the perivascular region of the external layer (EL) of the ME with age. However, no remarkable changes in either layer could be detected employing vimentin immunoreactivity. An immunoelectron microscopic study revealed that the number of the GFAP-immunoreactive processes markedly increased with age in regions surrounding the capillaries of the EL. Therefore, it may be considered that, in both layers, the astrocytic cell bodies and associated glial processes exhibit a significant increase with age. Investigation of the microvasculature under a scanning electron microscope demonstrated the development and the sinusoidal progress of the external capillary plexus with age, but these changes were not observed in the internal capillary network of the ME. In conclusion, the results of the present study showed a close correlation between the increase of the GFAP-immunoreactive astrocytes and the development of the external capillary plexus. In addition, there was a close correlation between the increase in the GFAP-immunoreactive astrocytes and the sinusoidal progress.

Astrocytes are abundant in the hypothalamic suprachiasmatic nucleus (SCN), particularly in the retinorecipient region. Using glial fibrillary acidic protein (GFAP) immunocytochemistry, we investigated the effect of light on the development of astrocytes in the SCN housing under light-dark (LD) or constant dark (DD) conditions after birth. GFAP immunoreactivity in the DD group showed lower levels than those in the LD group at P50. However, there was no difference in density of retinohypothalamic tract (RHT) terminals in the SCN between the DD and LD groups. After the adult pattern of GFAP immunoreactivity was established at P30, transferring rats to different LD conditions produced changes in GFAP immunoreactivity evident when rats were sacrificed at P50. We next examined, using a primary culture of hypothalamic astrocytes, whether neurotransmitters of RHT such as glutamate and pituitary adenylate cyclase activating polypeptide (PACAP) can stimulate GFAP expression directly. PACAP-38 increased the length and number of astrocytic processes but glutamate did not. These findings indicate that the functional aspects of RHT such as the light stimulated release of neurotransmitters is important for the development of astrocytes in rat SCN. Dynamic plasticity of astroglial elements in the SCN occurs even after GFAP shows an adult pattern.

VIP-like immunoreactive (VIP-LIR) and vasopressin-like immunoreactive (VP-LIR) neuronal elements located in the monkey suprachiasmatic nucleus (SCN) were investigated by light and electron microscopic immunocytochemistry. Both populations of neurons in the monkey SCN show a similar distribution to those of the rat except for some subtle differences. In addition to the ordinary distribution of VIP-LIR and VP-LIR neurons, VIP-LIR neurons of similar or some smaller size to those in the ventrolateral portion of the nucleus were also observed in the periventricular nucleus adjacent to the third ventricle. VP-LIR neurons with similar size and either strong or weak immunoreactivity compared with those located in the dorsomedial area were detected in the periventricular nucleus in which magnocellular VP-LIR neurons with strong immunoreactivity were distributed. Both VIP-LIR and VP-LIR neuronal perikarya were found to contain well developed cell organelles such as rER and mitochondria as well as immunoreactive dense granules. VIP-LIR neuronal perikarya were generally surrounded by astroglial processes. However, non-immunoreactive axons were frequently found to make synaptic contact with VIP-LIR dendrites or dendritic spines. Thus the VIP-LIR neurons are thought to receive neuronal inputs from other parts of CNS mainly from the dendrites or dendritic spines. VIP-LIR and VP-LIR axons were seen to form synapses on non-immunoreactive dendrites. VIP-LIR axons were also found to form axo-axonic synapses on non-immunoreactive axons. Synaptic contacts between VIP-LIR neuronal elements were also occasionally observed. The present study has demonstrated that both the VIP-LIR and VP-LIR neurons are at least partly intrinsic neurons in the SCN, and also projecting elements to the other neurons of the CNS.

We found differential expression of two neuronal markers in nerve fibers that came from the same neuronal somata. Prolactin releasing peptide (PrRP) and tyrosine hydroxylase (TH), a catecholamine synthesis rate-limiting enzyme, were differentially distributed in distinct fibers derived from the same neuronal cell body located in the rat medulla oblongata. All PrRP-immunoreactive neurons were catecholaminergic at the cell body level in the nucleus tractus solitarius (NTS) and in the ventral and lateral reticular nuclei (VLRN). However, in the terminal areas such as the bed nucleus of the stria terminalis (BNST) and paraventricular nucleus (PVN), where fibers are projected from both PrRP and TH positive neurons in the NTS and VLRN, dense PrRP immunoreactive fibers were observed that did not contain TH. In the medulla oblongata, proximal fibers near a neuronal cell body that contained both TH and PrRP were diverged into mainly PrRP-positive or mainly TH-positive fibers. These results suggest that TH and PrRP are differentially expressed between fibers from the same cell body. The fibers containing predominantly PrRP have been detected at birth, so the machinery to transport PrRP and TH separately may be established at an embryonic stage.

The present study examined a novel function of PRL-releasing peptide (PrRP) on the neuroendocrine. PrRP-immunoreactive nerve fibers and nerve terminals were located in the vicinity of the somatostatin (SOM)-neurons in the hypothalamic periventricular nucleus (PerVN). Immuno-electron microscopy revealed that PrRP-immunoreactive nerve terminals made synaptic contacts with nonimmunoreactive neuronal elements in the PerVN. Intracerebroventricular (icv) administration of PrRP induced immediate early gene, NGFI-A, in SOM-neurons in the PerVN. Double-labeling in situ hybridization showed that some parts of SOM-neurons in the PerVN expressed PrRP receptor messenger RNA. Therefore, some parts of SOM-neurons in the PerVN are considered to be directly innervated by PrRP via PrRP receptor. In addition to the above morphological characteristics, icv administration of PrRP decreased plasma GH levels. Such inhibitory effects of PrRP on the secretion of GH from the anterior pituitary were diminished by depletion or neutr...

The relationship between neuronal nitric oxide synthase (n-NOS), and heme oxygenase-2 (HO-2), the enzyme synthesizing CO, in the myenteric plexuses of the canine esophagus were examined by means of double staining technique of NADPH-diaphorase (NADPH-d) histochemistry and HO-2 immunohistochemistry. The coexistent ratio was examined regarding three portions of the esophagus; the cervical, intrathoracic and subdiaphragmatic portions. In the myenteric neurons of all portions, n-NOS frequently coexisted with HO-2 but HO-2 only partially coexisted with n-NOS. Thus, NO cooperates with CO but CO has some functions independent of NO. Many NADPH-d reactive nerve fibers were detected but there were very few HO-2 immunoreactive nerve fibers around the blood vessels and exocrine glands in the esophagus. As a neurotransmitter, NO may be more important than CO because many NADPH-d reactive nerve fibers could be detected in the esophagus. Another possibility is that CO may act as a neuromodulator, rather than as a neurotransmitter.

Using behavioral, immunohistochemical and pharmacological studies, we report here that large thoracic burn injuries remotely induce hindpaw hyperalgesia during the healing stage. During 2-3 weeks after thoracic burn injury when the skin was regenerating from the wound, we observed by formalin test that the number of flinching behaviors significantly increased and simultaneously we observed by von Frey test that rats developed mechano-hyperalgesia in the foot. In the dorsal horn of the lumbar spinal cord in burn injured rats, c-Fos expression was significantly augmented after plantar formalin injection. The expression of μ-opioid receptor in burn injured rats was significantly decreased compared with that in sham operated rats. The expression of substance P and CGRP in the lumbar dorsal horn was not different between burn and sham operated animals. We also observed that intrathecal administration of glutamate receptor antagonists (MK801 and CNQX) but not cyclooxigenase-2 antagonist (NS-398) reversed the threshold of von Frey test on the foot up to the control level at 2 weeks after injury. Collectively, we analyzed a new pain model showing foot hyperalgesia after thoracic burn injury and demonstrated that neurotransmission of glutamate was enhanced at the lumbar spinal cord level by immunocytochemistry and intrathecal administration of NMDA and non NMDA antagonists. Although the precise mechanism of how remote hyperalgesia at the healing stage developed in this model remains to be confirmed, substances such as trophic factors released from the regenerating skin may cause systemic hyperalgesia including in the foot.

The cell body sizes and succinate dehydrogenase (SDH) activities of motoneurons in the retrodorsolateral region of the ventral horn in the spinal cord innervating the soleus muscle in mice, rats, and cats were compared using quantitative enzyme histochemistry. There was an inverse relationship between cell body size and SDH activity of motoneurons in the three species. The mean cell body sizes of both gamma and alpha motoneuron pools were in the rank order of mice rats > cats. It is concluded that smaller motoneurons innervating the soleus muscle have higher SDH activities than larger motoneurons, irrespective of the species, and that motoneuron pools innervating the soleus muscle in smaller animals have smaller mean cell body sizes and higher mean SDH activities than those in larger animals.

To investigate the possibility of the neurosecretion of Bax-like protein in the adult rat, we examined its distribution in the hypothalamo-posterior pituitary system using light and electron microscopic immunohistochemistry. We also measured the serum Bax-like protein using an enzyme immunoassay. Neurons in the paraventricular nucleus (PVN) and supraoptic nucleus of the hypothalamus exhibited intense Bax-like immunoreactivity (Bax-IR). Bax-like immunoreactive axons projected to the posterior pituitary through the internal layer of the median eminence. By immunoelectron microscopy, Bax-IR was observed in the granulated vesicles and in the membrane of the roughsurfaced endoplasmic reticulum in the neuronal perikarya in the medial magnocellular division of PVN. It was also detected in the secretory vesicles in the nerve terminals of the posterior lobe of the pituitary gland. Double-labeling histochemistry revealed that almost all Bax-like immunoreactive substances were present in the oxytocin neurons, and many Bax-like immunoreactive neurons were nitro-oxidergic neurons. The enzyme immunoassay revealed that the average Bax-like protein concentration in the serum was 39.0±9.6 pmol/ml in the adult male rat. Colchicine treatment significantly increased the densities of Bax-IR in the PVN and decreased the serum Bax-like protein levels. From these results, Bax-like protein is considered to be produced in the hypothalamic oxytocin neurons, transported through axoplasmic flow and secreted into systemic circulation at the posterior pituitary as a "neurohormone"

The development of astroglial cells and the effect of the retinohypothalamic tract on it were studied by vimentin and glial fibrillary acidic protein (GFAP) immunocytochemistry in the suprachiasmatic nucleus (SCN) of the rat. At the embryonic stage, vimentin-immunoreactive (VIM-IR) radial glia, precursors of astrocytes, were dominant. However, their filaments vanished in the first few postnatal days. Instead of VIM-IR glial filaments, GFAP-immunoreactive (GFAP-IR) astrocytes appeared at E20 and grew rapidly from the P3 stage. GFAP immunoreactivity in the ventrolateral portion of the SCN (VLSCN) was measured using a computer-assisted image analyzing system. In normal rats, GFAP immunoreactivity showed a stepwise pattern with two slopes at P3-P4 and P20-P25. Bilaterally eye-enucleated rats operated on the day of birth showed lower GFAP immunoreactivity than normal rats and the GFAP immunoreactivity did not increase between P20 and P25 when GFAP-IR glial processes rapidly expand. Electron microscopic investigation at P50 (adult stage) revealed that neurons in the VLSCN had often direct apposition without astroglial processes and the frequency of this finding was significantly higher in eye-enucleated rats than in the control rats. These findings strongly suggest that the postnatal development of astroglial elements, particularly the expansion of GFAP-IR processes in the SCN, is regulated by retinohypothalamic projection.

Only a few RFamide peptides have been identified in mammals, although they have been abundantly found in invertebrates. Here we report the identification of a human gene that encodes at least three RFamide-related peptides, hRFRP-1-3. Cells transfected with a seven-transmembrane-domain receptor, OT7T022, specifically respond to synthetic hRFRP-1 and hRFRP-3 but not to hRFRP-2. RFRP and OT7T022 mRNAs are expressed in particular regions of the rat hypothalamus, and intracerebroventricular administration of hRFRP-1 increases prolactin secretion in rats. Our results indicate that a variety of RFamide-related peptides may exist and function in mammals.

Cross−sectional areas(CSAs) and succinate dehydrogenase(SDH) activities of different types of fibers in deep, middle, and superficial regions of the tibialis anterior muscle in mice and rats were determined using quantitative histochemistry. In both mice and rats, mean CSAs of fibers were in the rank order of type IIa type IIx >type IIb, irrespective of the muscle region. In mice, mean CSAs of the corresponding type of fibers were in the rank order of deep middle=superficial. Muscle fibers in mice had smaller CSAs and higher SDH activities than the corresponding type of fibers in rats. These results indicate species−specific patterns and regional differences in the CSA and the SDH activity of fibers in the muscle even within the same fiber type defined by myosin heavy chain isoform expressions

We studied NGFI-A gene expression in response to photic stimulation in the rat suprachiasmatic nucleus (SCN) using in situ hybridization histochemistry. This gene expression spread within the SCN and extended dorsally into the anterior hypothalamus after 30 min-1 h of light exposure at circadian time (CT) CT18. It appeared first in the ventrolateral SCN where the retinohypothalamic tract (RHT) innervates, then it expanded dorsomedially in the SCN and beyond the SCN to the anterior hypothalamus. However, stimulation for 2 h light exposure decreased its expression in the SCN. NGFI-A expression in the somatostatin neurons in the periventricular nucleus increased from 8.7% to 41% with increasing exposure time from 5 to 30 min. NGFI-A mRNA expression in the SCN was suppressed by pretreatment with baclofen, the GABAB receptor agonist. The spread of photic information from the retina to the SCN was visualized at immediate early gene level not only in the SCN but also in the area beyond the SCN. Somatostatin neurons in the periventricular nucleus which project to the external layer of the median eminence and are involved in regulation of growth hormone release showed NGFI-A gene expression corresponding to the duration of photic stimulation. Photic-induced NGFI-A gene expression in the SCN was also shown to be regulated by GABAergic transmission via GABAB receptors. These NGFI-A gene-expressing cells in the SCN may be involved in the circadian entrainment by light and some of those outside the SCN may participate in the regulation of neuroendocrine function.

This study was performed to examine the differences in expression of heme oxygenase protein with age using immunocytochemistry. We compared the contents of HO-1 and HO-2 between young and aged rats using immunocytochemical methods. Stronger HO-1 expression was detected in the internal layer of the median eminence (ME) of aged than of young rats. Moreover, the cells expressing HO-1 were larger in the aged than the young animals. Electron microscopy indicated these cells with HO-1-like immunoreactivity (HO-1-LI) to be astrocytes. These findings suggested that the expression of HO-1 increased in the ME with age. The significance of this increased expression of HO-1 with age will be discussed briefly.

Serine proteases are considered to play several important roles in the brain. In an attempt to find novel brain-specific serine proteases (BSSPs), motopsin (PRSS-12) was cloned from a mouse brain cDNA library by polymerase chain reaction (PCR). Northern blot analysis demonstrated that the postnatal 10-day mouse brain contained the most amount of motopsin mRNA. At this developmental stage, in situ hybridization histochemistry showed that motopsin mRNA was specifically expressed in the following regions: cerebral cortical layers II/III, V and VIb, endopiriform cortex and the limbic system, particularly in the CA1 region of the hippocampal formation. In addition, in the brainstem, the oculomotor nucleus, trochlear nucleus, mecencephalic and motor nuclei of trigeminal nerve (N), abducens nucleus, facial nucleus, nucleus of the raphe pontis, dorsoral motor nucleus of vagal N, hypoglossal nucleus and ambiguus nucleus showed motopsin mRNA expression. Expression was also found in the anterior horn of the spinal cord. The above findings strongly suggest that neurons in almost all motor nuclei, particularly in the brainstem and spinal cord, express motopsin mRNA, and that motopsin seems to have a close relation to the functional role of efferent neurons.

Arginine vasopressin peptide and messenger RNA expression were examined at the cellular level in the magnocellular and parvocellular neurons in the rat paraventricular nucleus after dehydration and rehydration, employing immunocytochemistry andin situ hybridization histochemistry on the same tissue sections. Most magnocellular vasopressinergic neurons of control animals expressed both vasopressin-like immunoreactivity and messenger RNA. However, neurons negative for vasopressin-like immunoreactivity but expressing messenger RNA were also detected, and their number increased during dehydration. In contrast, almost all of the parvocellular vasopressinergic neurons of dehydrated animals expressed vasopressin messenger RNA alone, with continued increase in their number after rehydration, despite return of the number of magnocellular vasopressinergic neurons to the control level. Vasopressin messenger RNA and corticotropin releasing factor-like immunoreactivity were co-localized in the same parvocellular neurons, and vasopressin-immunoreactive nerve terminals were detected in the external zone of the median eminence. These findings suggest that magno- and parvocellular vasopressinergic neurons are differentially activated during dehydration/rehydration. Osmotic stimuli activate all magnocellular vasopressinergic neurons, but the effect is not simultaneous in all of these neurons. Parvocellular vasopressinergic neurons are also activated by the stress of dehydration which effect appears to last longer than in the magnocellular system.

The induction and functional involvement of macrophages and postganglionic sympathetic fibers have been reported in rat neuropathic pain models. In the present study, we examined the morphology and structural relationship among macrophages, postganglionic sympathetic fibers, the dorsal root ganglion (DRG) neurons, and the satellite cells by light and electron microscopic examination. We used the rat spinal nerve ligation model reported by Kim & Chung. On postoperative day (POD) 1, when sympathetically maintained pain was confirmed, macrophages invaded from the nerve ligation site and reached the DRG neuronal area. Macrophages might have induced the demyelination of the spinal nerve and the degeneration of the satellite cells, and then the DRG neurons, from which the satellite cells were detached, lost the insulation from other neurons or nerve fibers. After the invasion of macrophages, postganglionic sympathetic fibers with TH-like immunoreactivity migrated into the same area, and occupied sites around the neuronal soma which were surrounded by satellite cells under normal conditions. At POD 180, the sympathetically maintained pain had disappeared, and the DRG neurons and spinal nerve showed normal structure without any invasive macrophages or postganglionic sympathetic fibers. The above changes in the morphological arrangements of macrophages and postganglionic sympathetic fibers in the DRG were thus elucidated and we suggest that this process may be part of the mechanisms underlying the development of the sympathetically maintained pain.

We investigated the effects of dizocilpine maleate (MK-801), an NMDA receptor antagonist, on arginine vasopressin heterogeneous nuclear RNA expression in the supraoptic nucleus in the rat hypothalamus. MK-801 treatment completely blocked the osmotic stimulus-induced increase in AVP hnRNA expression, but had no effect on basal AVP hnRNA expression in the SON. These observations indicate that the NMDA receptor is essential for regulation of AVP gene transcription in response to osmotic stimulation, but has no effect on steady-state AVP gene transcription.

There have been no systematic comparisons of skeletal muscle adaptations in response to voluntary wheel running under controlled loading conditions. To accomplish this, a voluntary running wheel for rats and mice was developed in which a known load can be controlled and monitored electronically. Five-week-old male Sprague-Dawley rats (10 rats/group) were assigned randomly to either a 1) sedentary control group (Control); 2) voluntary exercised with no load (Run-No-Load) group; or 3) voluntary exercised with additional load (Run-Load) group for 8 wk. The load for the Run-Load group was progressively increased to reach ∼60% of body weight during the last week of training. The proportions of fast glycolytic (FG), fast oxidative glycolytic (FOG), or slow oxidative (SO) fibers in the plantaris were similar in all groups. The absolute and relative plantaris weights were greater in the Run-Load group compared with the Control and Run-No-Load groups. The mean fiber cross-sectional areas of FG, FOG, and SO fibers were 20, 25, and 15% greater in the Run-Load than in Control rats. In addition, these fiber types were 16, 21, and 12% larger in Run-Load than in Run-No-Load rats. The muscle weights and mean cross-sectional areas of each fiber type were highly correlated with the average running distances and total work performed in the Run-Load, but not the Run-No-Load, group. The slope of the relationship between fiber size and running distance and total work performed was significant for each fiber type but was higher for FG and FOG fibers compared with SO fibers. These data show that the load on a rat running voluntarily can determine the magnitude of a hypertrophic response and the population of motor units that are recruited to perform at a given loading condition.

The morphological interactions between astroglial and neuronal elements were elucidated in the rat suprachiasmatic nucleus (SCN) by light and electron microscopic immunocytochemistry using antibodies against glial fibrillary acidic protein (GFAP), vasoactive intestinal peptide (VIP) and arginine-vasopressin (AVP). Throughout the SCN, particularly in its ventral portion, GFAP-like-immunoreactive (GFAP-LI) astroglial elements were found. These astrocytes displaying GFAP-like immunoreactivity occasionally contained fairly well-developed organelles. Some of these astrocytes were found as satellite cells in close contact with non-immunoreactive neuronal perikarya and processes. Around the neurons, GFAP-LI astroglial processes were also observed to cover some portions of presynaptic and postsynaptic elements. In addition, these astroglial elements were seen between two neuronal somata and pericytes of blood capillaries as glial endfeet. By double labeling immunoelectron microscopy using antibodies against GFAP/VIP and GFAP/AVP, some portions of VIP-like-immunoreactive or AVP-like-immunoreactive neuronal somata and processes were found to be engulfed by GFAP-LI astroglial processes. The possible functional roles of the morphological interactions between astroglial and neuronal elements are discussed.

Mammalian skeletal muscle fibers have several types of myosin heavy chain (MHC) isoforms. Immunohistochemical techniques enable us to identify the MHC isoform type in a single muscle fiber, i.e., MHCI, MHCIIa, MHCIIx, or MHCIIb. In addition, hybrid single fibers having more than one MHC isoform type can be classified using immunohistochemistry. The presence of different MHC isoform(s) in a single fiber correlates with the maximal contractile velocity and the enzyme histochemical reaction of the myofibrillar adenosine triphosphatase. The adaptive response of the skeletal muscle is the basis of the fiber-type transformation depending on the phenotypic expression pattern in several MHC isoforms. In this review article, we address the availability of the monoclonal antibodies which recognize specific MHC isoform(s) in a single fiber.

We investigated the characteristics of GABAergic neurons in the rat suprachiasmatic nucleus (SCN) in normal untreated rats by examination of co-expressed peptides. We adopted double labeling in situ hybridization using a digoxigenin-labeled glutamic acid decarboxylase (GAD) riboprobe and 35S-labeled peptide riboprobes. GAD mRNA-positive neurons were distributed throughout the SCN from the rostral to the caudal pole. In the dorsomedial part of the SCN, most GAD mRNA-positive neurons co-expressed arginine vasopressin mRNA. In the ventrolateral part of the SCN, about two thirds of GAD mRNA-positive neurons co-expressed vasoactive intestinal peptide (VIP) mRNA. Co-expression of GAD and somatostatin mRNA was observed in virtually all neurons of the intermediate part of the SCN. In contrast, these peptidergic traits were poorly expressed in hypothalamic GABAergic neurons outside the SCN. Vasopressin mRNA-positive cells in the supraoptic nucleus did not express GAD mRNA, and co-expression of somatostatin mRNA and GAD mRNA was rare in the periventricular hypothalamic nucleus. Similarly, the VIP mRNA co-expression ratio of GABAergic neurons in the cerebral cortex was far lower than that in the SCN.

The suprachiasmatic nucleus (SCN) is considered to be a circadian oscillator that regulates a set of phys iological aspects of behavior, including sleep-wakefulness and hormone release in mammalian species. In this review, we describe recent research that has begun to reveal the functional organization of the SCN. The SCN, which consists of a bilateral pair of tiny nuclei located just above the optic chiasm, contains several kinds of peptidergic neurons, but vasoactive intestinal peptide (VIP), arginine vasopressin (AVP), and somatostatin (SOM) neurons are the main components. VIP neurons and AVP neurons show distinctly different locations in the SCN; the former are found in the ventrolateral portion, whereas the latter are localized in the dorsomedial portion. VIP neurons receive all neuronal inputs from other regions of the CNS, such as those evoked by photic stimulation via the retinal ganglion cells and those relayed by 5HT inner vation from the raphe nuclei. VIP neurons relay their information to other kinds of neurons in the SCN, such as AVP and SOM neurons. VIP neurons, thus, may play a significant role in entrainment of circadian rhythm. VIP, AVP, SOM, and their mRNAs show rhythmic fluctuations that are predicted by this model; VIP and its mRNA show diurnal variation under the influence of photic stimulation, whereas AVP, SOM, and their mRNAs show endogenous rhythms. Immediate early genes (lEGs), such as c-fos mRNA, are also expressed in VIP neurons in the SCN, and IEG expression in the cells appears to be modified by photic stimuli. Together with transplantation studies showing that exogenous SCN tissue tends to restore circadian rhythm in arrhythmic animals, these results are beginning to clarify the function of the SCN in setting, maintaining, and resetting the biological clock. NEUROSCIENTIST 3:215-225, 1997

Photic induction of NGFI-A gene expression was investigated in the rat suprachiasmatic nucleus (SCN) using in situ hybridization histochemistry. Following light exposure for 30 min, NGFI-A mRNA appeared in the ventral portion of the rostral SCN, in the ventrolateral and in part of the dorsomedial portion at the middle level, and in the lateral portion of the caudal SCN. The distribution of NGFI-A mRNA was wider than that of c-fos mRNA which was confined to the ventrolateral portion at the middle level of the SCN. By double labeling in situ hybridization, approximately half of NGFI-A mRNA-positive cells in the SCN were shown to coexpress vasoactive intestinal peptide (VIP) mRNA, while 16% of cells positive for c-fos mRNA coexpressed VIP mRNA. These findings indicate that the broadness of NGFI-A mRNA and c-fos mRNA expression after photic stimulation are different. NGFI-A gene induced in these cells of the SCN including VIP neurons may be involved in circadian entrainment by light.

Lesion-induced induction of neuronal nitric oxide synthase (nNOS) was examined in the rat cerebellum. The stab-lesioned cerebellar cortex was examined with NADPH-diaphorase (NADPH-d) histochemistry and in situ hybridization using nNOS cRNA probe at 1, 3, 7, 14, 35 days post-lesion. NADPH-d- and nNOS mRNA-positive Purkinje cells appeared adjacent to the lesion by 3 days after the lesion. The area of distribution expanded and the number of positive cells increased at 7 days after the lesion, and at 14 days post-lesion, shrunken NADPH-d-positive Purkinje cells with irregular surface appeared. NADPH-d activity and nNOS mRNA signal could not be detected in Purkinje cells after 35 days post-lesion. Combined NADPH-d histochemistory and in situ hybridization using glutamic acid decarboxylase (GAD) cRNA probe revealed that nNOS-expressing Purkinje cells showed fewer GAD mRNA signals than those in normal Purkinje cells. The atrophic contour and the lower expression of GAD mRNA signals in NADPH-d positive Purkinje cells suggest that nNOS is expressed under a degenerating process.

We identified a novel gene, kf-1, highly expressed in the normal cerebellum but not in the cerebral cortex, the expression of which could have been augmented in the cerebral cortex of a sporadic Alzheimer's disease patient. We cloned human and mouse entire kf-1 cDNAs encoding conserved 79 kDa proteins containing a zinc-binding RING-H2 finger motif at the carboxy-terminus as found in acetylcholine receptor-associated protein (RAPsyn). The 3′-untranslated regions are highly conserved between human and mouse as to constitute a common mRNA secondary structure. In situ hybridization analysis of mouse brain sections revealed strong kf-1 expression in the cerebellum and hippocampus. We propose that KF-1 is involved in membranous protein-sorting apparatus similarly to RAPsyn. We mapped the human kf-1 gene to 2p11.2.

Mitochondrial density in neurons of the dorsolateral region of the ventral horn at the L5 spinal cord segment in rats was examined using electron microscopy. The gamma motoneurons had a higher density of mitochondria (25.1 ± 4.2%, n = 19) in the cytoplasm compared to the alpha motoneurons which had a mitochondrial density of 19.4 ± 4.5% (n = 38). An inverse relationship between cell body size and mitochondrial density was found for alpha (n = 38) and alpha plus gamma (n = 57), but not for gamma (n = 19), motoneuron populations. The higher densities of mitochondria in the smaller neurons correspond well with their metabolic properties since the smaller neurons have the highest oxidative enzyme activities.

In the present study, interactions between lutenizing hormone releasing hormone (LHRH) -like immunoreactive (LHRH-LI) nerve endings and processes of tanycytes and/or astrocytes in the median eminence (ME) particularly its external layer were investigated by double labeling immunoelectron microscopy employing antibodies against glial fibrillary acidic protein (GFAP) and LHRH. GFAP-like immunoreactivity was observed in some tanycytes throughout the ME and/or astrocytes in the subependymal layer of the ME. In the palisade layer of the ME, GFAP-like immunoreactive (GFAP-LI) elements were frequently seen to make intimate contact with LHRH-LI nerve endings. LHRH-LI nerve endings and processes of the tanycytes and/or astrocytes were also found to direct contact with the basement membrane of the pericapillary space. And they were partially engulfed each other in the vicinity of the perivascular region. The above findings strongly suggest that the processes of tanycytes and/or astrocytes with intimate communication of cerebrospinal fluid in the third ventricle may play a role in regulation of secretion of LHRH into the portal vessels.

Albumin immunoreactivity in the liver was examined on days 2, 5 and 10 after two-thirds partial hepatectomy by light and ultrastructural immunoperoxidase methods and the ultrastructural area of the rough endoplasmic reticulum (ER) in hepatocytes was measured. Albumin immunoreactivity was seen in the rough ER and Golgi apparatus of all hepatocytes in the hepatectomized liver and ultrastructural analysis showed a significantly greater area of rough ER on day 5 than on days 2 or 10. Albumin mRNA was studied by the in situ hybridization technique using radioisotopes and their numbers were determined visually. Albumin mRNA was present as grains in all hepatocytes and the grains varied in number during regeneration of the liver, being more abundant on day 5 than on days 2 or 10. The activity of [3H]-leucine incorporated into albumin synthesis, an indicator of translational activity, was higher on days 5 and 10 than on day 2 and was highest on day 5. In conclusion, albumin synthesis varied during liver regeneration after partial hepatectomy, being reduced at the peak of cell proliferation on day 2 and being most active on day 5.

The participation of vasoactive intestinal polypeptide (VIP) or calcitonin gene-related peptide (CGRP) in the nitrergic innervation of the canine laryngeal glands was investigated using a double-staining technique of NADPH-diaphorase (NADPHd) histochemistry and VIP or CGRP immunohistochemistry. NADPHd-positive nerve fibers with varicosities appeared to terminate in some acinar cells. Double staining revealed that NADPHd reactivity and VIP- or CGRP-like immunoreactivity were colocalized in some nerve fibers distributed around the acini. A cluster of NADPHd-positive cells were occasionally found in the larynx. Many NADPHd-positive cells had VIP-like immunoreactivity and no NADPHd-positive cells were CGRP-like immunoreactive. These findings suggest that nitric oxide participates in the neural control of the laryngeal exocrine secretion in cooperation with intrinsic VIP and/or extrinsic CGRP.

Accumulating evidence indicates that somatostatin (SS) is a key substance for the circadian rhythm of rodents. In the present study, we investigated whether SS mRNA coexists with arginine-vasopressin (AVP) mRNA, vasoactive intestinal peptide/peptide histidine isoleucine amide (VIP/PHI) mRNA and glutamate decarboxylase (GAD) mRNA in neurons of the rat suprachiasmatic nucleus (SCN) by double labeling in situ hybridization technique. SS mRNA-positive neurons were scattered in the whole region of rostral SCN, in the intermediate region between dorsomedial and ventrolateral region at the middle level, and in the mid to lateral region at the caudal level. These neurons were located in the close vicinities of the dorsomedial AVP and ventrolateral VIP/PHI mRNA-positive cell clusters. They rarely coexpressed AVP mRNA or VIP/PHI mRNA, but mostly coexpressed GAD mRNA. Thus, SS-synthesizing neurons are GABAergic and form a distinct cell group different from AVP or VIP/PHI cell groups.

Several substances have been reported as candidates for the neurotransmitter in the laryngeal afferent system. In the present study we demonstrated that catecholamine is also a candidate neurotransmitter in the canine laryngeal afferent system using tyrosine hydroxylase (TH) immunochemistry in combination with retrograde labelling with cholera toxin B in subunit-conjugated gold (CTBG). A few cells in the nodose ganglion labelled by application of CTBG to the internal branch of the superior laryngeal nerve were also TH-immunoreactive. These cells were also labelled following application of CTBG to the nucleus of the solitary tract. These results indicate that some of the TH-IR cells in the nodose ganglion could be primary afferent neurones for the canine larynx.

Nitrergic ganglionic cells located in the canine intrinsic laryngeal muscle were studied by NADPH-diaphorase (NADPH-d) histochemistry and neuronal nitric oxide synthase (nNOS) immunohistochemistry. Cells intensely stained by NADPH-d histochemistry were found between the striated muscle fibers of the intrinsic laryngeal muscle. Most of these cells were bipolar or pseudounipolar in form. Some NADPH-d negative cells were observed to be enveloped in a mesh by varicose NADPH-d positive nerve fibers. The findings obtained by nNOS immunohistochemistry corresponded well with those obtained by NADPH-d histochemistry, indicating that NADPH-d activity in the ganglion in the intrinsic laryngeal muscle is nNOS. The present findings clearly indicate that some of the ganglion cells located in the canine intrinsic laryngeal muscle are nitrergic, and that the ganglionic cells synapse together with the participation of nitric oxide in integrating ganglionic cells.

The topography-dependent vulnerability of midbrain dopaminergic neurons to neonatal intracranial exposure to 6-hydroxydopamine (6-OHDA) was investigated at adult age by the quantitative analysis of cell counts of tyrosine hydroxylase-immunopositive neurons. In all cases of intracisternal 6-OHDA treatment, A9 dopaminergic neurons in the substantia nigra (SN) were much more vulnerable to death than more medially located A10 dopaminergic neurons. Moreover, within each cell group, there were also lateromedial topographic gradients. In the A9 neuronal group, cells located in the pars lateralis of the SN and the lateral part of the pars compacta of the SN were more susceptible to 6-OHDA toxicity than those located more medially. In the A10 neuronal group, cells located in the medial part of the ventral tegmental area were more resistant to toxicity than those located more laterally, and dopaminergic cells in the midline midbrain areas (interfascicular nucleus and rostral linear nucleus of raphe) were completely spared from 6-OHDA toxicity. These findings revealed that 6-OHDA is not equally toxic to all midbrain dopaminergic neurons in neonates and that the lateromedial vulnerability pattern shows similarities to those reported in Parkinson's disease.

To investigate the possible role of the brainstem in cysteamine-induced peptic ulceration, we examined the expression of VGF mRNA, which is induced in PC12 cells following application of nerve growth factor [23], in the nucleus tractus solitarius (NTS)/dorsal motor nucleus of vagus (DMV) complex of the medulla oblongata by in situ hybridization histochemistry. In control saline-treated rats, weak VGF mRNA signals were only rarely detected in neurons of the NTS and none were observed in those in the DMV. After 12 h of cysteamine administration (450 mg/kg, s.c.), the time at which duodenal ulcer was detected in all cases, heavily labeled VGF mRNA-expressing neurons appeared in the NTS and DMV. By quantitative analysis on macroautoradiogram, the VGF mRNA signals of the NTS/DMV complex in cysteamine-treated rats were twice as much as those in saline-treated rats. In situ hybridization histochemistry combined with the use of the retrograde neuronal tracer cholera toxin-B subunit revealed that the induced VGF mRNA-expressing neurons of the DMV projected directly to the stomach. The present results suggest that ulceration accompanies the induction of VGF mRNA in neurons of vagal afferent and efferent areas of the brainstem.

Neuronal activity of the suprachiasmatic nucleus (SCN) is known to be regulated by two major extrinsic factors conveyed by three anatomically distinct pathways to the SCN: photic stimulus by the direct retinohypothalamic tract (RHT) and the indirect geniculohypothalamic tract (GHT), and information from the brainstem by ascending forebrain serotonergic (5-hydroxytryptamine: 5-HT) tract. It has been shown that VIP mRNA level in neurons of the SCN is altered by external light, but remains stable in constant darkness. In the present study, by using the in situ hybridization technique combined with computer-assisted image analysis, we examined VIP mRNA expression in the SCN of rats in which the two major factors were eliminated, i.e. photic stimulus by exposing animals in total darkness and 5-HT transmission by three-day successive administration of p-chlorophenyl-alanine methylester (an inhibitor of tryptophan hydroxylase, 200 mg/kg, daily). In saline-treated controls, VIP mRNA levels remained almost constant throughout the day. In contrast, in PCPA-treated rats, a significant rhythm of VIP mRNA was observed with a peak at CT 4 and a trough at CT 20. These observations suggest that the removal of photic and 5-HT influence induces VIP mRNA rhythm in the SCN, indicating that VIP mRNA is controlled not only by photic information but also by the circadian clock.

Biochemical and immunocytochemical changes after unilateral 6-hydroxydopamine (6-OHDA) injection into the striatum were investigated in the rat nigro-striatal dopamine (DA) neuron system. Four weeks after 6-OHDA injection into the striatum, concentrations of DA and its metabolites were specifically decreased in the substantia nigra (SN), as well as in the striatum, ipsilateral to the injection. Immunocytochemistry of tyrosine hydroxylase (TH) revealed a marked decrease in the number of TH-immunoreactive neuronal cell bodies in the SN ipsilateral to the injection; this effect appeared 2 weeks after the injection and remained even 10 months after the injection. Electron microscopic study of these periods demonstrated degenerative neurons in the SN pars compacta, suggesting that the degenerative changes persisted for a long time after a single injection of 6-OHDA into the striatum. The results showed that degeneration of the dopaminergic terminals in the striatum may lead to cell death of the parent cell bodies in the SN and suggest that the striatum may be the initial site in which the neurodegeneration occurs in Parkinson's disease.

We investigated the quantitative participation of calcitonin gene-related peptide (CGRP), substance P (SP), and leu-enkephalin (ENK) in canine laryngeal sensory innervation by immunohistochemistry in combination with retrograde labeling using the recently introduced retrograde tracer cholera toxin subunit B—conjugated gold (CTBG). In the nodose ganglion, neurons labeled from the internal branch of the superior laryngeal nerve with CTBG were investigated immunohistochemically by means of antisera against CGRP, SP, and ENK. The percentages of neurons immunoreactive to each neuropeptide were as follows: CGRP 81.5%, SP 24.5%, and ENK 7.0%. These results suggest that CGRP is the main sensory neurotransmitter in canine laryngeal sensory innervation.

The influence of serotonergic inputs on the levels of vasoactive intestinal peptide (VIP) mRNA in the rat suprachiasmatic nucleus (SCN) was examined by in situ hybridization combined with morphometrical analysis. Depletion of serotonin by p-chlorophenylalanine methyl ester (300 mg/kg i.p. daily for 3 days) caused a marked decrease in VIP mRNA signal levels in the ventrolateral part of the SCN. This finding suggests that serotonergic input controls VIP production at the mRNA level.

We determined the regional distribution of the dopamine D2 receptor group in the rat central nervous system by quantitative receptor autoradiography with a high-affinity and selective antagonist, [3H]YM-09151-2. Saturation and competition experiments demonstrated that the binding of [3H]YM-09151-2 to striatal sections was saturable (Bmax=37.3 fmol/section), of high affinity (Kd=0.315 nM), and was inhibited selectively by prototypic D2 ligands. The anatomical localization of binding sites was determined by comparison of autoradiograms and the original 3H-ligand-exposed sections stained with cresyl violet. Very high levels of [3H] YM-09151-2 binding were found in the caudate-putamen, nucleus accumbens, tuberculum olfactorium and the insula of Calleja, to each of which midbrain dopaminergic neurons project densely. High levels of binding were also observed in other regiions rich in dopaminergic neurons and fibers including the glomerular layer of the olfactory bulb, the intermediate lobe of the pituitary, lateral septum, substantia nigra pars compacta, interfascicular nucleus, dorsal raphe nucleus, locus coeruleus, and nucleus of the solitary tract. Some regions poor in dopaminergi innervation, however, had high levels of [3H]YM-09151-2 binding including the molecular layer of gyrus dentatus, all layers of CA1 and the nonpyramidal layer of CA4 of hippocampus, and the deeper layer of medial entorhinal cortex. Motor neurons present in brainstem motor nuclei and spinal ventral horn were also strongly labeled. Neocortical, cerebellar, and thalamic regions had low levels of binding, except lobules 9–10 of the cerebellum, the olivary pretectal nucleus, zona incerta and lateral mammillary nucleus, in which moderate to high levels of binding were detected. Our findings concerning the widespread but region-specific localization of [3]YM-09151-2 binding sites in the brain and spinal cord may prove useful for analyzing varoius dopaminergic functions in the central nervous system. © 1994 Wiley-Liss, Inc.

Nitric oxide (NO), which was firstly identified as an endothelium-derived relaxing factor, has recently been demonstrated to be a neurotransmitter in the central and peripheral nervous systems. In the hypothalamus, abundant nitric oxide synthase (NOS) immunoreactivity and its histochemical marker, NADPH-diaphorase activity, have been demonstrated in the hypothalamo-neurohypophyseal system. In the present study, we examined whether NOS is coexpressed with posterior pituitary hormones in the rat hypothalamus by combination of oxytocin and vasopressin immunofluorescence and NADPH-diaphorase histochemistry. Most oxytocin-immunoreactive neurons in the paraventricular and supraoptic nuclei expressed NADPH-diaphorase activity, but virtually no vasopressin-immunoreactive neurons contained NADPH-diaphorase activity. This suggests that oxytocin neurons are the main source of NO production in the hypothalamic-pituitary system.