Neuronal degeneration and regeneration in the olfactory epithelium of pigeon following transection of the first cranial nerve.

The pigeon olfactory nerve has been sectioned to explore the course of retrograde degeneration of the sensory neurons' perikarya, which are located in the olfactory neuroepithelium. Both light- and electron-microscopic observations have shown that from 3 to 8 days after axotomy the sensory neurons undergo retrograde, irreversible degeneration. Following disappearance of the mature neurons, the basal cells of the neuroepithelium actively divide and differentiate into mature olfactory sensory neurons. Consequently, the basal cells represent true stem cells of the olfactory sensory neurons. The olfactory mucosa regains a structural organization close to normal in a period of 30-50 days after axotomy. These observations indicate that, when the primary olfactory neurons degenerate as a consequence of the experimental section of their axons, restitutio ad integrum of the sensory olfactory connections can be reestablished by new elements which differentiate from basal cells of the olfactory neuroepithelium.20     

Über die Glia im Hypoglossuskern der Ratte nach Axotomie

Zusammenfassung Es wird unter besonderer Berücksichtigung der Glia über den Feinbau des Hypoglossuskerns normaler Albinoratten und nach Axotomie berichtet. Untersucht wurden Tiere vom 12.–90. Lebenstag.Folgende Ergebnisse sind neu: Weder bei jungen noch bei erwachsenen Ratten kommt im Hypoglossuskern Mikroglia vor. Etwa ab. 3. Tag nach Axotomie kann bei erwachsenen Tieren Mikroglia nachgewiesen werden. Sie entsteht aus aktivierten und proliferierten Oligodendrocyten und pericytenähnlichen Bindegewebszellen. Die Mikroglia umhüllt die Nervenzellen. Eine Aktivierung und Hypertrophie von Astrocyten erfolgt etwa ab 8. Tag nach Axotomie. Von diesem Tage an drängen sich Astrocytenfortsätze zwischen Mikroglia und Nervenzelloberfläche. Die Mikroglia beginnt zu degenerieren. Bei Jungtieren bewirkt die Axotomie nur geringgradige Veränderungen. Schon beim Normaltier findet man aktive Oligodendroglia und Astrocyten sowie häufig pericytenähnliche Bindegewebszellen.

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Glia in the hypoglossal nucleus of the rat following axotomy

Summary The ultrastructure of the Nucleus hypoglossi in normal albino rats and following axotomy is described with special reference to the glia. Animals 12 to 90 days old were investigated. The following results are new: Normally in young and adult rats microglia is absent in the nucleus. Following axotomy from the 3rd day no microglia can be demonstrated in adult rats. It originates from activated and proliferated oligodendrocytes and pericyte-like cells of the vascular connective tissue. These cells surround the neurons. An activation and hypertrophy of astrocytes is visible from the 8th postoperative day on. Astrocyte processes make contacts with the perikarya and separate the microglia from the neurons. Microglia cells then show signs of degeneration. In young animals alterations following axotomy are only sparse. Already normally activated oligodendrocytes and astrocytes as well as pericyte-like connective tissue cells can be seen.

Herrn Prof. Schiebler zum 50. Geburtstag gewidmet.     

A Histochemical Examination of the Staining of Kainate-Induced Neuronal Degeneration by Anionic Dyes

Anionic dyes, notably acid fuchsine, strongly stain the nuclei and cytoplasm of neurons severely damaged by injury or disease. We provide detailed instructions for staining nervous tissue with toluidine blue and acid fuchsine for optimal demonstration of injured neurons. Degeneration was induced in the hippocampus of the mouse by systemic administration of kainic acid, and the resulting acidophilia was investigated using paraffin sections of the Carnoy-or Bouin-fixed brains. The affected cells were bright red with the toluidine blue-acid fuchsine sequence. Their nuclei were stainable also with alkaline Biebrich scarlet and with the 1,2-naphthoquinone-4-sulfonic acid-Ba(OH)₂ method; all staining was blocked by benzil but was relatively refractory to deamination by HNO₂. These properties indicated an arginine-rich protein. The nuclei were strongly acidophilic in the presence of a high concentration of DNA (strong Feulgen reaction), and acidophilia could not be induced in normal neuronal nuclei by chemical extraction of nucleic acids. The cytoplasmic acidophilia of degenerating hippocampal neurons was due to a protein rich in lysine (extinguished by alkalinity, easily prevented by deamination, and unaffected by benzil). Stainable RNA was absent from the perikarya of the affected cells, but normal neuronal cytoplasm did not become acidophilic after extraction of nucleic acids. We suggest that kainate-induced cell death is preceded by increased production of basic proteins, which become concentrated in the nucleus and perikaryon. Groups of small, darkly staining neurons were seen in the cerebral cortex in control and kainite-treated mice. These shrunken cells were purple with the toluidine blue-acid fuchsine stain, and were attributed to local injury incurred during removal of the unfixed brain.     

Variations in the axon reaction after different types of nerve lesion. Light and electron microscopic studies on the facial nucleus of the rat

The retrograde nerve cell reaction was studied after evulsion, transection and crush lesion of the facial nerve in rats. Crush lesion caused barely discernible light and electron microscopic changes. The Nissl bodies became slightly smaller than normal and the arrangement of the granular endoplasmic reticulum (rER) somewhat more irregular. The crush lesions were followed by complete functional and morphologic recovery. After nerve evulsion, the cells showed severe chromatolysis, nuclear caps, nuclear eccentricity, and folding of the nuclear membrane. Ultrastructurally there was a dispersion of the rER and formation of laminated dense bodies. Lager, the rER was partly degranulated and some of the polyribosomes dissociated. These neurons ultimately disappeared. Transection of the nerve caused an intermediate axon reaction and a moderate loss of neurons. It is concluded that certain neurons may regenerate after axotomy in spite of minimal light and electron microscopic changes in the nerve cell bodies, and that the same neurons may show the typical axon reaction after more severe nerve injuries. Mechanisms which may be involved in the regulation of the retrograde nerve cell reaction after axotomy are briefly discussed.     

NGF receptor-mediated reduction in axonal NGF uptake and retrograde transport following sciatic nerve injury and during regeneration.

Injury to the rat sciatic nerve leads to the induction of nerve growth factor (NGF) receptors on the denervated Schwann cells and their disappearance on the regenerating axons of the axotomized, normally NGF-sensitive sensory and sympathetic neurons. This disappearance in the axonal expression and retrograde transport of NGF receptors is associated with a similarly dramatic reduction in the axonal uptake and retrograde transport of NGF following axotomy and during regeneration. In view of the massive NGF synthesis occurring in the injured nerve, these results suggest that, while sensory and sympathetic neurons are the primary targets of NGF in the normal peripheral nervous system, the denervated Schwann cells may become its primary target in the aftermath of nerve injury.     

Motor neuron degeneration after sciatic nerve avulsion in adult rat evolves with oxidative stress and is apoptosis.

The mechanisms for motor neuron degeneration and regeneration in adult spinal cord following axotomy and target deprivation are not fully understood. We used a unilateral sciatic nerve avulsion model in adult rats to test the hypothesis that retrograde degeneration of motor neurons resembles apoptosis. By 21 days postlesion, the number of large motor neurons in lumbar spinal cord was reduced by approximately 30%. The death of motor neurons was confirmed using the terminal transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling method for detecting fragmentation of nuclear DNA. Motor neuron degeneration was characterized by aberrant accumulation of perikaryal phosphorylated neurofilaments. Structurally, motor neuron death was apoptosis. Apoptotic motor neurons undergo chromatolysis followed by progressive cytoplasmic and nuclear condensation with chromatin compaction into uniformly large round clumps. Prior to apoptosis, functionally active mitochondria accumulate within chromatolytic motor neurons, as determined by cytochrome c oxidase activity. These dying motor neurons sustain oxidative damage to proteins and nucleic acids within the first 7 days after injury during the progression of apoptosis, as identified by immunodetection of nitrotyrosine and hydroxyl-modified deoxyguanosine and guanosine. We conclude that the retrograde death of motor neurons in the adult spinal cord after sciatic nerve avulsion is apoptosis. Accumulation of active mitochondria within the perikaryon and oxidative damage to nucleic acids and proteins may contribute to the mechanisms for apoptosis of motor neurons in the adult spinal cord.     

Motor neuron degeneration after sciatic nerve avulsion in adult rat evolves with oxidative stress and is apoptosis

The mechanisms for motor neuron degeneration and regeneration in adult spinal cord following axotomy and target deprivation are not fully understood. We used a unilateral sciatic nerve avulsion model in adult rats to test the hypothesis that retrograde degeneration of motor neurons resembles apoptosis. By 21 days postlesion, the number of large motor neurons in lumbar spinal cord was reduced by ∼30%. The death of motor neurons was confirmed using the terminal transferase‐mediated deoxyuridine triphosphate‐biotin nick‐end labeling method for detecting fragmentation of nuclear DNA. Motor neuron degeneration was characterized by aberrant accumulation of perikaryal phosphorylated neurofilaments. Structurally, motor neuron death was apoptosis. Apoptotic motor neurons undergo chromatolysis followed by progressive cytoplasmic and nuclear condensation with chromatin compaction into uniformly large round clumps. Prior to apoptosis, functionally active mitochondria accumulate within chromatolytic motor neurons, as determined by cytochrome c oxidase activity. These dying motor neurons sustain oxidative damage to proteins and nucleic acids within the first 7 days after injury during the progression of apoptosis, as identified by immunodetection of nitrotyrosine and hydroxyl‐modified deoxyguanosine and guanosine. We conclude that the retrograde death of motor neurons in the adult spinal cord after sciatic nerve avulsion is apoptosis. Accumulation of active mitochondria within the perikaryon and oxidative damage to nucleic acids and proteins may contribute to the mechanisms for apoptosis of motor neurons in the adult spinal cord. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 185–201, 1999     

Structural changes in spinal ganglion neurons of lizards after cold-exposure

We studied structural changes in spinal ganglion neurons that occur in lizards exposed to the cold, both at the light and electron microscope levels. Two types of perikaryal changes were found in the cold-exposed animals: (a) In 25% of all ganglion neurons, the central region of the perikaryon was devoid of Nissl bodies and a narrow peripheral zone stained deeply basophilic. Electron microscopic examination of these cells showed that mitochondria, Golgi complexes and other organelles were assembled in the central region of the perikaryon, while most cisternae of granular endoplasmic reticulum and free polysomes were confined to the periphery of the perikaryon. These changes seem to take place mainly in dark neurons. (b) In 8.6% of all ganglion neurons, Nissl bodies were present throughout the perikaryon, but separated by large, clear spaces. Under the electron microscope, these clear spaces were filled with large numbers of densely packed filaments. It seems that mainly light neurons undergo this type of structural change. The degree of nuclear eccentricity was significantly greater in the neurons of cold-exposed animals than in controls. The nucleolar volume was significantly increased and both the percentages of nuclei with two nucleoli and of nuclei with 'vacuolated' nucleoli were significantly greater in neurons displaying structural changes than in the other neurons. The structural modifications observed in spinal ganglion neurons of cold-exposed lizards closely resemble those seen in the same lizard neurons following axonal section. They could be due to a) metabolic changes induced by low temperature and fasting, b) alterations in the flow of nerve impulses from the periphery, or c) impaired retrograde transport of trophic substances from the periphery to the cell body.     

Neurofilaments and microtubules in sensory neurons after peripheral nerve section

Summary Sensory neurons were examined in spinal ganglia of the rat 1 to 55 days after section of the plexus brachialis nerves. Only light neurons of the type A were investigated. Maximal reaction to axotomy was found 7 to 14 days after the operation. The majority of the axotomized perikarya developed central chromatolysis. In such neurons, Nissl bodies virtually disappeared from the central area of the neuron and formed a more or less continuous zone at the cell circumference. The cytocentrum became filled with large numbers of mitochondria, dense bodies and other organelles. Neurofilaments and microtubules were disarranged and ran at random among the accumulated particles. Microtubules were often more prominent in chromatolytic areas than neurofilaments. Both these organelles were rare in the peripheral areas filled with massed Nissl substance.Part of the neurons that did not show typical chromatolysis contained increased numbers of neurofilaments among Nissl bodies dispersed throughout the cytoplasm. Neurofilaments were roughly arrayed in bundles up to several microns wide; they were linked by cross-bridges and separated by distances of about 500 Å. Microtubules were rarely found in the filamentous areas. However, they were numerous in the axon hillock and in the initial segment where they formed fascicles similar to those described in normal neurons of other types.During the period from 14 to 55 days after axotomy, many perikarya recovering from chromatolysis contained enlarged bundles of neurofilaments with occasional microtubules among the restored Nissl bodies.Mean diameters of sensory neurons, measured 7 to 55 days after axotomy, in no instance exceeded those of contralateral control neurons. It thus appears that sensory perikarya do not increase in size either during the chromatolytic process or during the period of recovery.This project was supported by a grant from the Muscular Dystrophy Association of America, Inc. The main part of this study was done while the author was a Research Fellow in Anatomy at the Harvard Medical School, Boston. The author wishes to thank prof. S. L. Palay for his valuable advice and help received during her stay at the Department of Anatomy at the Harvard Medical School, under NIH training grant NBO5591.     

Transneuronal uptake of horseradish peroxidase in the central nervous system of dipterous insects

Summary Horseradish peroxidase (HRP) applied to lesioned neurons in the retina and thoracic ganglia of the flies Musca, Calliphora and Drosophila labeled axon terminals, dendrites and perikarya of the severed neurons after anterograde or retrograde passage. In addition, HRP reaction product secondarily labeled intact neurons that are contiguous with injured nerve cells. In many cases labeling of optic lobe neurons remote from primarily filled ones was also seen (here called tertiary labeling). HRP labeling was extensive and both primarily and transneuronally filled neurons could be resolved in almost as much detail as Golgi-impregnated or cobalt-silver-labeled cells. Electron microscopy showed that in both primarily and secondarily filled neurons, reaction product was distributed diffusely in the cytoplasm.Transneuronal uptake of HRP was specific to certain types of neurons in the brain and thus displayed certain pathways. The pathways resolved by transneuronal labeling with HRP extend from the optic lobes to the thoracic ganglia and include visual neurons previously identified electrophysiologically and anatomically.Transneuronal HRP uptake, although believed to occur in vivo, could not be shown to be dependent on synaptic activity. Three other heme peptides tested were taken up by injured neurons, but showed no transneuronal labeling: lactoperoxidase, cytochrome c, and microperoxidase.     

Somato-dendritic synapses in the nucleus reticularis thalami of the rat

In the reticular nucleus of the rat thalamus, about 30% of the synapses are brought about by the perikarya of parvalbumin-immunopositive neurons, which establish somato-dendritic synapses with large dendrites of nerve cells of specific thalamic nuclei. Although the parvalbumin-immunopositive presynaptic structures bear resemblance to goblet-like or calyciform axonal endings, electron microscopic immunocytochemistry and in situ hybridization revealed that these structures are parts of the perikaryal cytoplasm studded with synaptic vesicles. In about 15% of the somato-dendritic synapses, axons are seen to be in synaptic contact with the parvalbumin-immunoreactive perikaryon. Double immunohistochemical staining revealed that the parvalbumin immunoreactive presynaptic perikarya and dendrites contained GABA. It is assumed that the peculiar somato-dendritic synaptic complexes subserve the goal of filtration of impulses arriving at the reticular nucleus from various thalamic nuclei, thus processing them for further sampling.     

Cellular reactions to axotomy in rat superior cervical ganglia includes apoptotic cell death

The cellular response to axonal injury in the superior cervical ganglion was examined by immunofluoresence at intervals from 6 h to 14 days after transection of the internal and external carotid nerves. GAP-43-immunoreactivity (IR) appeared in some neurons in the ganglia 1 day after axotomy, while neurons in control ganglia were GAP-43 negative. In 3 days axotomized ganglia GAP-43-IR structures were increased in number and intensity in nerve fiber bundles, while GAP-43-positive perikarya were restricted to the middle and caudal parts of the ganglia and showed an intensity that was stronger than at 1 day after axotomy. These GAP-43-positive neurons were also galanin positive. In the cranial part of the ganglia, S100-IR in satellite cells was weak at 18 h after axotomy. Peripheral to this area, S100-IR was stronger and co-localized with HSP-72-IR, preferentially located in satellite cells. HSP-72-IR was, however, occasionally observed also in principal neurons at 1 and 3 days after axotomy. In eosin-stained sections, neurons and satellite cells in the cranial part of 1 day axotomized ganglia were reduced in number, and a further loss was noted at 3 days. At 12 h some satellite cells in the cranial part of the ganglia were labelled by the in situ DNA 3'-end labelling method, indicating apoptosis, and at 18 h many cells were labelled. Some neuronal perikarya were also labelled in this region. Labelling was not observed at 1 day or later after axotomy, nor in control ganglia. The results may imply that not only neurons but also satellite cells react to neuronal axonal injury with apoptosis. Neurons in the middle and caudal part of the ganglia survived and showed increased content of GAP-43 and galanin, possibly a sign of regeneration/neuronal plasticity.     

Neither axotomy nor target-tissue inflammation changes the NOS- or VIP-synthesis rate in distal bowel-projecting neurons of the porcine inferior mesenteric ganglion (IMG)

The present study was aimed at disclosing axotomy- and inflammation-induced changes in the chemical coding of retrogradely labelled distal bowel-projecting neurons in the porcine IMG. Particular attention was paid to the changes in the expression pattern of vasoactive intestinal polypeptide and nitric oxide synthase (as a marker of nitric oxide) in affected cells, as these substances are thought to play a crucial role in the regeneration of injured sympathetic neurons. However, while both pathological processes failed to induce an increase in the number of sympathetic bowel-projecting neurons exhibiting vasoactive intestinal polypeptide or nitric oxide synthase, axotomy, but not target-tissue inflammation, led to the upregulation in the expression pattern of galanin, pituitary adenylate cyclase-activating peptide and/or Leu5-enkephalin in the affected perikarya. On the other hand, axotomy resulted in a diminished density of vasoactive intestinal polypeptide-immunoreactive intraganglionic nerve fibres, whilst target-tissue inflammation evoked a distinct increase in the number of visible vasoactive intestinal polypeptide-immunoreactive terminals, especially in those regions where bowel-projecting neurons were located. Thus, the data obtained in the present study run counter to the results of the injury-related responses observed in neurons of the sympathetic chain ganglia, suggesting the existence of either species- or target tissue-dependent differences in the injury-induced responses of the affected sympathetic neurons.     

Co-existence of CRF and vasopressin immunoreactivity in parvocellular paraventricular neurons of rat hypothalamus

New dual immunocytochemical staining procedures were used in the same tissue section to elucidate the distribution and co-existence of CRF and vasopressin in parvocellular neuronal perikarya in the paraventricular nucleus (PVN) of rat hypothalamus. CRF immunostained cells were for the most part concentrated in the medial parvocellular component of PVN. Few vasopressin-immunoreactive (ir) neurons were seen in this area in the normal and colchicine-treated animals. Vasopressin-containing neurons predominated in the magnocellular component of PVN. In the adrenalectomized and adrenalectomized-colchicine-treated animals, a dense accumulation of vasopressin-ir cells were observed in the medial parvocellular area of PVN; this region is normally vasopressin-ir poor and CRF-ir rich. The vasopressin immunostained cells appeared to have an anatomical distribution similar to that seen for CRF-containing cell bodies. Results of this study unequivocally establish the co-existence of vasopressin and CRF in the same parvocellular perikarya of PVN following pertubation of the pituitary-adrenal axis.     

Light and electron microscopic histochemistry of the monoamines in the human foetal sympathetic ganglion in culture

Synopsis Sympathetic ganglia of 13 to 19-week-old human foetuses were cultured in small pieces with and without nerve growth factor for up to 5 weeksin vitro. The cultures were studied using phase-contrast, fluorescence and electron microscopy. Monoamines were demonstrated with the formaldehyde-induced fluorescence method, with and without pretreatment of the cultures with catecholamines or monoamine oxidase inhibitor.In the long-term cultures, primitive sympathetic cells, sympathicoblasts of types I and II, and young sympathetic neurons showed a fine structure identical to that described earlierin vivo. There were virtually no satellite or Schwann cells in the cultures. The neurons showed a considerable capacity to grow new nerve fibres in culture, even without nerve growth factor. Nerve terminals with accumulations of other nervous structures. Large granular vesicles were regularly found in the sympathicoblasts after glutaraldehyde-osmium tetroxide fixation. After permanganate fixation, dense-cored vesicles typical of adrenergic neurons were not seen, either in the perikarya, or in the processes, although it was possible to demonstrate specific fluorescence. No small intensely fluorescent (SIF) cells were observed.Variable formaldehyde-induced fluorescence was observed in the nerve cell perikarya and nerve fibres. The intensity of the fluorescence increased after treatment of the cultures with monoamine oxidase inhibitor and after incubation with catecholamines.     

Golgi morphology of the neurons in frontal sections of human interthalamic adhesion

The Golgi morphology of the neurons in the human interthalamic adhesion (IA, which is not present in every human brain) is very variable. Four types of Golgi-impregnated neurons were found in the adult human IA: (1) fusiform neurons (most characteristic of the human IA); (2) neurons with an oval perikaryon; (3) triangular neurons (rarely found), and (4) multipolar neurons (polygonal perikaryon and at least 4 primary dendrites). Fusiform neurons, as well as triangular and multipolar ones, belong to the isodendritic type, but the neurons with oval perikarya do not.     

PK (‘peripheral benzodiazepine’) – binding sites in the CNS indicate early and discrete brain lesions: microautoradiographic detection of [3H]PK 11195 binding to activated microglia

The isoquinoline PK 11195 has been suggested as a marker of glial pathology in the lesioned brain. The aim of the present study is to clarify the precise cellular location of its binding site in the central nervous system. Here, we report that in the facial nucleus after facial nerve axotomy–a lesion causing a retrograde neuronal reaction without nerve cell death while keeping the blood–brain barrier intact–activated microglia are the predominant source of lesion-induced increases of PK 11195 binding. Likewise, increased PK 11195 binding is seen in the gracile nucleus after anterograde neuronal injury following sciatic nerve transection. The peak of PK 11195 binding, using the single isomer R-PK 11195, was observed 4 days after the peripheral nerve lesion, consistent with the well-known time course of microglial activation. Photoemulsion microautoradiography confirmed the restriction of PK 11195 binding to activated microglia. The increase of PK 11195 binding in the facial nucleus seen after selective cell death of facial motoneurons by retrograde suicide transport of toxic ricin, a lesion that is accompanied by the rapid transformation of microglia into phagocytes, was no higher than that seen following axotomy. This suggests that the full transformation of microglia into parenchymal phagocytes is not necessary to reach maximal levels of PK 11195 binding. PK 11195, therefore, is a well-suited marker to detect microglial activation in areas of subtle brain pathology, where neither a disturbance of the blood–brain barrier function nor the presence of macrophages and inflammatory cells indicate an on-going disease process.     

Distribution and morphology of descending brain neurons in the cricket Gryllus bimaculatus

The number and distribution of descending brain neurons have been investigated in the cricket. The results are based on retrograde labeling of these cells with either Lucifer yellow or Neurobiotin via whole or small split portions of the cervical connectives. Various groups of cells and single neurons have been identified, and the morphology of more than 40 cells is described. Nearly 200 descending brain neurons can be stained via one cervical connective. Their perikarya are concentrated in clusters that occur ipsi- and contralateral to the filled connective and that lie dorsal and ventral in the brain. Descending cells only arborize in the nonglomerular neuropils of the brain and never branch in the optic lobe. Cells descending ipsilaterally never arborize in the contralateral hemisphere, whereas contralateral descending neurons often branch in both hemispheres. Irrespective of soma position, cells can arborize in the ventral and/or dorsal neuropils of the brain. Neurons with somata in the protocerebrum often have branches in the deutocerebrum and vice versa. The main arborizations of the cells from the prominent ventral i5 group are found in the same part of the protocerebrum. In contrast, various cells arborize in the ventral posterior deutocerebrum, but their somata are not located in different clusters. Thus, neurons from the same cluster may, but need not necessarily, arborize in the same brain area.     

Immunocytochemical studies on the central nervous system of the earthworm, Lumbricus terrestris

There are numerous aldehyde fuchsin (AF)-positive, neurosecretory cells of medium size (A cells) and a small number of large, AF-negative neurons (B cells) in the cortical layer of the cerebral ganglion. In the subesophageal ganglion, symmetrical groups of AF-positive cells lie ventrally. The peroxidase--antiperoxidase (PAP) method was used for the immunocytochemical study of substance P and ACTH in these ganglia. In addition, the presence of L-enkephalin and alpha endorphin could be confirmed. Using rabbit antibodies to substance P we found small immunoreactive neurons among negative A and B cells in the cerebral ganglion. The processes of these immunoreactive cells could be traced to the subcortical synaptic neuropil. With antibodies to ACTH, activity was visible in perikarya similar in size to A neurons. A part of the nerve terminals of the synaptic zone, some of the B neurons and further several nerve cells of the subesophageal ganglion reacted positively. Successive demonstration of substance P and ACTH on the same section showed that the two materials occurred in different cell types. Using antiopsin antibody in an indirect immunocytochemical test we observed strong reaction in numerous medium-sized perikarya and in nerve fibres of the synaptic zone of the cerebral ganglion, further in some neurons of the subesophageal and abdominal ganglia. In contrast to this result, the photoreceptor cells of the prostomium and cerebral ganglion were negative. Presumably, substance P is present in a perikaryon type hitherto unrecognized while ACTH and antiopsin reactions seem to be located first of all in A cells.     

Distribution of lamprey gonadotropin-releasing hormone-III (GnRH-III) in brains of larval lampreys (Petromyzon marinus)

Two immunoreactive forms of gonadotropinreleasing hormone (GnRH), lamprey GnRH-I and lamprey GnRH-III, were found in neurons in larval sea lampreys (Petromyzon marinus). Using antisera preferentially directed against either lamprey GnRH-I or-III, dense reaction product was seen in cell bodies in the rostral hypothalamus and preoptic area. Reaction product was also dense in fibers to and within the neurohypophysis, in addition to numerous fibers which projected caudally, beyond the neurohypophysis through the mesencephalon. The majority of immunoreactive GnRH was lamprey GnRH-III, and when lamprey GnRH-I was seen, it was in cells that appeared to contain both forms of GnRH. A small number of cells found in the caudal hypothalamus contained only immunoreactive lamprey GnRH-III, and these may constitute a functional subgroup within the population of GnRH neurons. In animals undergoing metamorphosis there was a large increase in reaction product in all GnRH-containing cells and fibers. A striking change within the distribution of GnRH cells was localized to a distinct group of GnRH-immunoreactive cells (GnRH-I and-III) in the ventral anterior hypothalamic area. These cells were minimally detectable in larvae, but during metamorphosis became densely filled with immunoreactive product in perikarya and distal processes. The results are consistent with the hypothesis that lamprey GnRH-III is an important form of GnRH during the maturation of GnRH cells and fibers, and further indicates that these cells have attained their normal positions in the preoptic area and hypothalamus before metamorphosis.