[Neuropsychiatric symptoms and caregivers' distress in anti-N-methyl-D-aspartate receptor encephalitis].

Objective: To explore the characteristics of psychiatric symptoms and caregivers' distress in anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis. Methods: Seventy-two patients with anti-NMDAR encephalitis were investigated in Peking Union Medical College Hospital from 2011 to 2018. The Chinese version of the Neuropsychiatric Inventory-Questionnaire (NPI-Q) was used to assess the severity of neuropsychiatric symptoms and caregivers' distress around the treatment. Results: A highly positive correlation existed between severity scores of neuropsychiatric symptoms and caregiver distress, and pre- and post-treatment correlation coefficients was 0.872 and 0.947, respectively (all P<0.001). The frequency of 12 symptom domains was higher than 45% before treatment, however, it significantly reduced to below 45% after treatment. Irritability/lability, agitation/aggression were the most common neuropsychiatric symptoms. Irritability/lability, agitation/aggression, and nighttime behavioral disturbances occurred concurrently. Patients with severe symptoms tended to be administrated antipsychotics (Z=-2.581, P=0.01). Neuropsychiatric symptoms significantly improved after immunotherapy (Z=-6.611, P<0.001). There was no significant difference in the symptom severity and distress subscale scores between the first episode and relapse around the therapy (P>0.05). Conclusion: Patients with anti NMDAR encephalitis often present severe neuropsychiatric symptoms, which aggravate the burden on caregivers. Immunotherapy significantly improves neuropsychiatric symptoms, and thus reduces the distress of caregivers.

Tocilizumab in the treatment of a critical COVID-19 patient: a case report.

In December 2019, Coronavirus disease 2019 (COVID-19) emerged in Wuhan and rapidly spread throughout China and the rest of the world. COVID-19 is currently a global pandemic. There are cytokine storms in severe COVID-19 patients. Interleukin-6 plays an important role in cytokine storm. Tocilizumab is a blocker of interleukin-6 receptor, which is likely to become an effective drug for patients with severe COVID-19. Here, we reported a case in which tocilizumab was effective for a critical COVID-19 patient.

Characterization of the promoter region of the viral interferon regulatory factor encoded by Kaposi's sarcoma-associated herpesvirus.

Viral interferon regulatory factor (vIRF) encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) inhibits the expression of interferon-responsive genes, causes cellular transformation and transactivates KSHV genes. In the present study, we characterized the mRNA expression pattern of the vIRF gene and its promoter. A vIRF transcript of 1.7 kb in size was detected in low level in uninduced KSHV-infected cells and its expression was inducible by 12-O-tetradecanoylphorbol-13-acetate (TPA), sensitive to cycloheximide and resistant to phosphonoacetic acid. The transcription start site was mapped to 79 nt upstream of the ATG initiation site by 5'-RACE. Mutagenesis analysis identified a region between -56 and the transcription start site (+1) as the minimal promoter region that contains a functional TATA box at -27. A region between -337 and -125 contains a repressor domain negated by sequence from -991 to -499 in BCBL-1 cells, a region which was also identified to be responsive to TPA induction. These results demonstrate vIRF as a KSHV early gene, identify its promoter and define the promoter regions that contain regulatory elements controlling vIRF transcription.

Differential polarization of serotonin transporters in axons versus soma-dendrites: an immunogold electron microscopy study.

In spite of the conventional belief that neurotransmitter uptake occurs at the synapses, we demonstrated previously that serotonin transporters and the high-affinity uptake of serotonin were not confined to the terminals but rather occurred throughout the axons [Zhou F. C. et al. (1998) Brain Res. 805, 241-254]. In the present study, the detailed distribution of serotonin transporters over various parts of the neuron was illustrated and analysed morphometrically using a pre-embedding immunogold method with a characterized serotonin transporter antibody at the electron microscopic level. Our findings reveal a highly polarized distribution of serotonin transporters between axons and soma-dendrites in two aspects. (1) On the plasma membrane, serotonin transporter-immunogold is extremely low on soma-dendrites and synaptic junctions, but consistently dense along the axons and perisynaptic area. (2) In contrast, serotonin transporter labeling in the cytoplasm is concentrated in soma and dendrites, particularly on the membranes of rough endoplasmic reticulum, Golgi complexes and tubulovesicular structures, but low in the axoplasm. The extensive distribution of serotonin transporter along the axolemma suggests a broad range of uptake sites beyond synaptic junctions, and is consistent with the notion that the major mode of transmission for serotonin neurons is through volume (extrasynaptic) transmission. The highly polarized distribution also indicates that the major serotonin uptake sites are on axons and not on soma-dendrites.

Neuronal and glial elements of fetal neostriatal grafts in the adult neostriatum.

The cellular components of striatal grafts into the host striatum of rats were studied using [3H]thymidine autoradiography, histochemistry, immunocytochemistry and Golgi-staining. Autoradiography revealed that a layer of glial cells, somas smaller than 8 microns in diameter, stained positive with glial fibrillary acidic protein, and demarcating transplant from host, is derived mainly from the donor. Golgi studies revealed that many neuronal fibers fail to cross the glial layer to reach the host striatum. Migration of transplanted striatal cells into the host milieu was evident. The density of migrated cells decreased linearly as a function of distance from the transplant. Most of the far-migrated cells were glial cells. Neuronal migration was limited. In the transplant, donor cells marked by [3H]thymidine constituted at least 70% of the population. Neurons which stained positively for GABA, substance P, and acetylcholinesterase were identified in the transplant. Fibers of two of these three neuronal types, substance P and acetylcholinesterase, formed patchy patterns in the transplant. Detailed morphology on GABAergic fiber is not available to date, because of the limited antibodies or the method used. GABA is the highest population in the striatal transplant. Two types of GABA-positive cells were clearly distinguishable according to cell size. A majority resembled the medium-sized cell commonly found in striatum, while those of the other type resembled the larger GABA cells usually found in the globus pallidus.

Serotonin transporter antibodies: production, characterization, and localization in the brain.

Serotonin (5-HT) transporter, the mechanism for 5-HT high affinity uptake, is the essential component for the termination of 5-HT transmission. In order to identify transporter sites on 5-HT neurons or on other 5-HT uptaking cells, three rabbit antisera against cocaine sensitive-serotonin transporter (5-HTT) were produced. Antisera 5-HTT55 (against amino acid sequence 55-68 in cytoplasmic N-terminal) and 5-HTT315 (against amino acid sequence 315-325, a 3rd external loop peptide) were produced against synthetic multiple-antigenic peptides (MAP). Antiserum 5-HTTN was produced against a fusion protein of the first 71 amino acids of N-terminal peptide expressed in recombinant DNA transformed bacteria. SDS-PAGE/Western blots indicate that 5-HTT55 and 5-HTT315 recognized bands of 74 and 64 kDa in rat brains with densities in the order of cortex > or = hippocampus > cerebellum, but not in liver, or muscle. The 5-HTTN recognized the fusion protein expressed in the bacteria, and the 64 kDa band with a similar density profile in the rat brain regions, and negative in liver and muscle. The immunocytochemistry of all three antisera revealed 5-HTT-immunostaining (5-HTT-im) in a pattern similar to 5-HT fiber distribution. 5-HTT55 and 5-HTT315 stainings were punctate in appearance, while 5-HTTN outlined the fibers in the 5-HT fiber areas, and neurons in raphe but not in substantia nigra or locus ceruleus. The preimmune serum and immune serum preabsorbed with 5-HTTN showed negative or diminished staining. Specific neurotoxin, 5,7-dihydroxytryptamine lesion removed all of the 5-HTTN fibers from the injection site, indicating, that 5-HTTN-im fibers are 5-HT fibers in nature. Our study indicates that the three antibodies we produced recognize various domains of the 5-HTT. Our 5-HTT antibodies could be used as new markers of 5-HT fibers, and are particularly useful for the study of the plasticity of 5-HT fibers free of the complications involved with 5-HT content.

Production and characterization of an anti-serotonin 1A receptor antibody which detects functional 5-HT1A binding sites.

We describe the production and characterization of a specific anti-5-HT1A receptor antibody made against a fusion protein consisting of glutathione-S-transferase (GST) coupled to a 75-amino acid sequence from the middle portion of the third intracellular loop (5-HT1A-m3i, serine253-arginine327) of the rat 5-HT1A receptor protein. This region was chosen to avoid putative phosphorylation and glycosylation sites and regions of known homology with other 5-HT receptors. Western blot analysis indicated that the polyclonal anti-5-HT1A-m3i antibody accurately recognized the fusion protein expressed in bacteria and labeled a prominent 67 kDa protein band in the hippocampus, cortex, brainstem, cerebellum and kidney with a density profile corresponding to the relative abundance of the 5-HT1A receptor in these tissues. No protein was detected in liver or muscle tissue preparations, and no protein bands were labeled in any of the above tissues following preabsorption of the antibody with the 5-HT1A-m3i fusion protein. Immunohistochemistry revealed prominent labeling in limbic structures including the hippocampus, amygdala, entorhinal cortex, and septum as well as in raphe nuclei. In the hippocampus, 5-HT1A-m3i labeling revealed a characteristic laminar pattern that coincided with that seen by autoradiographic binding of the 5-HT1A agonist [3H]-8-OH-DPAT in all strata of the hippocampal formation. In the dorsal and medial raphe nuclei, anti-5-HT1A-m3i antibodies labeled the somatodendritic membranes of 5-HT neurons, consistent with its role as an autoreceptor. The detailed matching of the anti-5-HT1A-m3i antibody with [3H]-8-OH-DPAT binding suggests that the antibody recognizes a functionally active form of the 5-HT1A receptor protein capable of binding 5-HT1A agonist ligands. These anti-5-HT1A antibodies may therefore be useful tools in localizing functional 5-HT1A receptors in specific regions of the brain as well as in studying the plasticity and ontogeny of the 5-HT1A receptor at the cellular and subcellular level.

Laminin facilitates and guides fiber growth of transplanted neurons in adult brain.

Laminin has been shown in vitro to act as a surface adhesive molecule for neuronal process elongation. To test whether laminin has a similar role in the brain, we sequentially injected laminin and transplanted fetal neurons into various brain regions to determine if the fetal neurons would preferentially grow along a laminin injection tract. In the fetal brain, the raphe area of the rostral rhombencephalon is rich in serotonergic (5-HT) neurons; the rostral ventral mesencephalon is rich in dopamine (DA) neurons, while the lateral rhombencephalon is rich in norepinephrinergic (NE) neurons. These three areas were transplanted to the motor cortex, neostriatum or hippocampus of adult animals. The tract used for microinjection of cell suspension was then immediately filled with laminin in a suspension media or a laminin-collagen (type IV) mixture. In other animals, laminin or control solution was injected in a separate needle tract displaced 0.3-1 mm from the transplant injection tract. Straight and thick 5-HT, DA or NE immunoreactive (IR) fibers (stained with anti-5-HT or anti-tyrosine hydroxylase antiserum) were predominant within the laminin-treated tracts, or were directed toward the laminin-treated parallel tracts when it was positioned less than 0.5 mm from the transplant site. The density of 5-HT-, DA- and NE-IR fibers in the injection tracts in all three brain areas was much higher for laminin and laminin-collagen mixture than control media. Thin axonal fibers of fetal 5-HT and NE neurons were observed surrounding the laminin-treated tracts, but not around vehicle-injected tracts. In addition, a number of transplanted 5-HT, DA and NE neuronal cell bodies were seen within the laminin-treated tracts, but not in vehicle-treated tracts. Finally, laminin injection to the hippocampus, motor cortex or neostriatum of the adult brain did not stimulate sprouting of undamaged adult 5-HT or NE fibers. These results suggest that purified laminin can facilitate and guide process outgrowth of 5-HT, DA and NE neurons during early developmental stage, but does not induce sprouting on these same fiber types in the adult brain.

Nimodipine-enhanced survival of suboptimal neural grafts.

The present experiment was conducted to see whether nimodipine, a calcium channel blocker with the potential to protect vulnerable neurons, could improve the survival rate of striatal grafts under suboptimal conditions. The drug markedly improved graft viability of rats with near-term (E21) striatal implants. Untreated rats showed no surviving neurons while all treated rats showed surviving grafts rich in neuron. It did not affect the survival of striatal grafts from E14/15 donors into the striatum, in which the high rates of survival are normally obtained.

DNA microarray analysis of differential gene expression of 6-year-old rat neural striatal progenitor cells during early differentiation.

EGF-responsive striatal progenitor cells from rat brain have been maintained in culture in the form of neurospheres for six years without exhausting their renewal capacity. The events surrounding differentiation of stem cells in the brain after a long progenitorship remain a mystery. Using DNA microarray analysis we investigated differential gene expression, comparing progenitor cells in their neurosphere state with the cells 24 hours after induction of differentiation. Eighty-one genes showed increased expression in the differentiated condition. Genes associated with cellular growth, neurite outgrowth, and synaptogenesis were activated, including both anti-apoptotic and pro-apoptotic genes. Two transmitter- related genes, acetylcholine receptor-beta and glutamate receptor-beta-unit were also elevated-, these genes not only fit the profile of early neural development, but also reflect the characteristics of striatal neurons. In addition, there are approximately 30 expressed sequence tags (ES7) increased during neural differentiation. Forty-seven genes showed decreased expression; half of them are known genes related to the cell cycle, cell adhesion, transcription, and signaling. Tbe signaling and cell cycle genes may be responsible for the life-long self-renewal. These data demonstrate for the first time that life-long quiescent stem cells retain the potential to become activated and develop into specific types of brain cells. The six-year long-term neural stem cells are an excellent model for studying developmental neurobiological processes and aging.

Quantitative immunocytochemical evaluation of serotonergic innervation in alcoholic rat brain.

Neurotoxicity can be divided into three levels: depletion, degeneration and denervation. The first level is determined by the transmitter content, and the second and third levels, which require anatomical evaluation, can be analyzed by quantitative immunocytochemistry on a specific neurotransmitter system. An antibody specific to serotonin (5-HT) can reveal detailed normal as well as degenerative morphology of 5-HT neurons. Quantitative alterations of 5-HT fibers in a particular brain region indicate degenerative or plastic changes. This study demonstrates quantitative immunocytochemistry by using image analysis of immunostained 5-HT fibers in selectively-bred, alcohol-preferring and nonpreferring rats, which are known to have divergent drinking behaviors, and 5-HT contents in specific brain regions. The method of the image analysis is described in detail and the advantages and disadvantages of using this method to detect the degeneration of a particular fiber system are discussed. The 'area density' traditionally measured in image analysis was converted (with Zhou-Tam formula) into 'volume density' to correct the mismeasurement of fibers through the optical depth. Quantitative immunostaining shows that the difference in 5-HT fiber density in particular brain regions between the two rat lines is consistent with changes in content of the 5-HT/5-HIAA and hypersensitivity of 5-HT1 a receptor. This result indicates either (a) the 5-HT content is too low to be detected in the nerve terminals; (b) degeneration of 5-HT fibers occurs in P rats sometime during development; or (c) a smaller number of 5-HT fibers was preprogrammed in the brain regions of P than NP rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of serotonergic neuronal maturation after fetal mesencephalic raphe transplantation into the 5,7-DHT-lesioned hippocampus of the adult rat.

Neurotoxin lesioning of 5-HT fibers selectively induced the homotypic collateral sprouting of spared 5-HT fibers in the hippocampus. We have used this model to investigate the possibility that the neurotoxin-primed hippocampus will enhance the development of transplanted fetal serotonergic neurons in the brain. The neurotoxin 5,7-DHT, when microinjected into the FF, produced a specific and partial depletion of 5-HT in the hippocampus of adult rats. The ability of the 5,7-DHT-primed hippocampus to selectively support the neurochemical maturation of fetal serotonergic cells was tested by assaying the transplanted fetal raphe or LC 1 month after neuronal transplantation. The neurochemical maturation of fetal 5-HT and NE neurons was dramatically different when they were transplanted in the 5,7-DHT-FF-lesioned hippocampus as compared to the normal hippocampus. The transplanted 5-HT neurons had 480% more SHAU of [3H]5-HT and had a 250% greater content of 5-HT in the partially denervated hippocampus than in the normal hippocampus after 1 month. Furthermore, extracts obtained from lesioned hippocampus enhanced the 5-HT content of 5-HT neurons transplanted in the normal hippocampus, to a level similar to that seen in neurons transplanted in the lesioned hippocampus. In contrast, the implanted NE neurons of fetal LC had a lower NE level in the 5-HT partially denervated hippocampus than in normal hippocampus after 1 month in the host site. The growth of the NE transplants was not facilitated by the vacant postsynaptic space produced by the 5,7-DHT lesion. These results suggest that the 5-HT denervation triggered a trophic signal selectively enhancing the development of the 5-HT neurons but not the NE neurons. Our results are consistent with previous studies showing homotypic collateral sprouting in 5,7-DHT-primed hippocampus.

NGF-response of EGF-dependent progenitor cells obtained from human sympathetic ganglia.

Signalling molecules are thought to play a significant role in determining the fate of neural crest progenitor cells. The human sympathetic chain was identified at 6.5, 7.5, 8.2, 10.2 and 11.4 postconception (PC) weeks demonstrating low affinity nerve growth factor (NGF) receptors, and was processed for tissue culture. In the presence of epidermal growth factor (EGF), floating spheres of proliferating progenitor cells were developed in vitro. In the absence of EGF progenitor cells differentiated into tyrosine hydroxylase (TH)-immunoreactive neuronal and TH-negative flat cells. NGF treatment significantly increased neurite outgrowth and survival of TH-immunoreactive cells. The multipotent cells we isolated differ from previously reported sympathoadrenal progenitors in that they give rise to TH immunoreactive neurones precociously sensitive to NGF.

Immunomagnetic isolation of human developing motor neurons.

Human motor neuron (MN) isolation provides a critical tool to study neurophysiological properties and the effects of molecules of clinical relevance on isolated neurons. We developed an immunomagnetic separation technique based on specific MN antigen recognition for nerve growth factor receptor (p75-NGFR). We cultured an average of 250,000 cells from the anterior horns of a single cord (four specimens at postconception Weeks 6.0, 7.2, 8.0, and 8.3). At day 7 in vitro (DIV), choline acetyltransferase (ChAT) and/or p75-NGFR-expressing cells (MNs) represented 72 +/- 2% of the total growing cells. MNs survived for at least 4 weeks in biochemically defined medium. The immunomagnetic separation method has been demonstrated to be effective, reproducible, and quantitative for separation of MNs.

Excitochemical-induced trophic bridging directs axonal growth of transplanted neurons to distal target.

Brain grafts directly placed in their target regions often lack proper connections and normal regulation. When brain grafts are placed in their ontogenetic normal area, the axonal outgrowth of grafts to distal target regions is a major obstacle. We previously demonstrated that directional axonal growth of grafts can be facilitated by laminin. In this study we demonstrated that excitochemicals induced trophic environment can be strategically used as a bridge to guide transplanted neurons to innervate a distal target of millimeter distance, and has a unique effect on fiber expansion in the target region. To distinguish dopamine (DA) growth from that of transplants, 6-hydroxydopamine was unilaterally injected into medial forebrain bundle to remove dopamine innervation to the striatum. Ibotenic acid (IB), kainic acid (KA), or phosphate buffer (vehicle) were microinjected through a glass-pipet to make a 7 mm long tract between the substantia nigra and the striatum in Sprague-Dawley rats. Gestational 14-15 days brainstem slurry was transplanted either in the same or a separate tract into nigra at the bottom of the bridge. All transplants survived with abundant serotonin (5-HT) and DA neurons. Traceable DA fibers formed distinct bundles from grafts along the length of IB/KA tracts. Upon arrival at their major target regions, fibers from bundles were capable of leaving the tracts, and reinnervating the DA-vacated striatum. The 5-HT fibers formed bundles similar to DA fibers but left the tract earlier upon arrival at their major target the globus pallidus and when arriving at the striatum and cortex. Fibers from grafts with vehicle tracts grew randomly and not into the tract. These results indicate that the trophic effects of chemical lesioning serves as a remarkably effective axonal guidance for more than one type of fetal neuron to innervate distal brain regions. This method has great potential in directing fibers in many transplant models.

Morphological differentiation of astroglial progenitor cells from EGF-responsive neurospheres in response to fetal calf serum, basic fibroblast growth factor, and retinol.

Procurement of multipotential neuroglial stem cells is possible with the addition of epidermal growth factor (EGF). Stem cells will differentiate into neurons and glia upon the removal of EGF from the culture medium. We have previously characterized the neuronal differentiation of stem cells derived from long-term cultured nonpassage neurospheres. In the current study, we (1) characterize the morphological differentiation of the astroglial progenitor cell from 3-mo-old neurospheres, (2) examine whether the astroglial progenitor cells from neurospheres of different brain areas exhibit different differentiation responses to the same exogenous signals, and (3) test the effects of basic fibroblast growth factor (bFGF) and retinol on differentiation. Cerebral cortex, striatum, and mesencephalon cells were obtained from Embryonic Day 14 (E-14) rat fetuses and were dissociated for the procurement of neurospheres in chemically defined medium supplemented with EGF. After 3 mo in culture, the neurospheres, derived from each of the three brain areas, were subcultured into three groups on chamber slides: (1) basal medium, (2) the basal medium plus 20 ng/mL bFGF, and (3) the basal medium plus 10 muM retinol. Phenotypic expression of astroglial cells was examined after 14 days subculture. Our findings indicate that the 3-mo-old cultured nonpassage neurospheres contained numerous multipotential stem cells that stained positive with nestin, and that environmental factors played an important role in influencing the differentiation of astroglial progenitor cells. As detected by glial fibrillary acid protein (GFAP), astroglial progenitor cells turned into protoplasmic astrocytes in the FCS-containing basal medium, fibrous astrocytes in the presence of bFGF, and spindle-shaped astrocytes in the presence of retinol. There were no noticeable differences in differentiation among astroglial progenitor cells of the various brain region-derived neurospheres in any of the three medium conditions. Peculiar varicosity-and growth cone-like structures on the long slender GFAP-positive processes suggest that neuroblasts and glioblast may share common morphologies, features, or common progenitor cells during initial differentiation in vitro.

Fetal tissue derived from spontaneous pregnancy losses is insufficient for human transplantation.

OBJECTIVE: To determine if sufficient fetal tissue with desirable transplant characteristics can be obtained from spontaneous abortions. METHODS: A survey of fetal tissues collected from newly diagnosed spontaneous pregnancy losses from three Indianapolis hospitals was conducted from December 1992 to September 1993. Forty-nine of 356 mothers (13.8%) with spontaneous abortions or ectopic pregnancies consented to the evaluation of their products of conception by gross and microscopic pathologic examination, bacterial culture, cytogenetic analysis, cell culture, and maternal serologic tests. RESULTS: Forty-nine pregnancies (gestational age range 5-30 weeks) provided four identifiable embryos, 12 second-trimester fetuses, and one third-trimester fetus. Nine samples (18.4%) were of excellent or good quality on pathologic grading. Twenty-five of 38 samples tested (66%) grew pathogenic bacteria. Maternal serologic tests were negative for antibodies to human immunodeficiency virus, human T-cell lymphotropic virus, syphilis, and hepatitis B in all cases. One of 43 sera was reactive for hepatitis C, and 33 (77%) were positive for cytomegalovirus. Cytogenetic abnormalities were found in 25% of cultured samples. Five fetal brain samples had cell viabilities of 50% or more. Few viable fetal hepatocytes were found. Only two fetal brain samples (4.1%) were potential candidates for human transplantation. CONCLUSION: Spontaneous pregnancy losses yield minimal usable tissue for human transplantation because of a lack of embryonic or fetal tissues, delayed collection, decomposition, genetic abnormality, and bacterial contamination.

Increased 5-HT1A receptor immunoreactivity in the rat hippocampus following 5,7-dihydroxytryptamine lesions in the cingulum bundle and fimbria-fornix.

Serotonin (5-HT) projections from the ascending raphe nuclei reach the dorsal hippocampus via the cingulum bundle (CB) and fimbria-fornix (FF). Microinjection of the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) into the CB and FF produces a significant decrease in the density of 5-HT immunoreactive fibers in the hippocampus as early as 3 days postlesion (Zhou, F.C. and Azmitia, E.C. (1983) Brain Res. Bull., 373, 337-348). In the present study we used an anti-peptide antibody against the second extracellular loop of the 5-HT1A receptor and employed immunocytochemistry to examine changes in the expression and distribution of the 5-HT1A receptor in the hippocampus 14 days following administration of 5,7-DHT into the CB and FF. The density of 5-HT immunoreactive fibers was greatly reduced 14 days following the lesions. 5-HT1A immunoreactivity (IR) was localized to the proximal axon near the axon hillock of cells in the pyramidal cell layer of the cornu Ammonus and in the granule cell layer of the dentate gyrus. The intensity of 5-HT1A-IR was increased in the CA1 and dentate gyrus following 5,7-DHT lesions. Intensity in the CA3 also increased but not to a significant level. These findings demonstrate that 5-HT denervation in the hippocampus is followed by increased expression of the 5-HT1A receptor protein. These changes in receptor expression 14 days postlesion may represent adaptive changes by postsynaptic cells following reduced 5-HT innervation and may be the molecular basis for 5-HT1A receptor supersensitivity.

Induced homotypic sprouting of serotonergic fibers in hippocampus. II. An immunocytochemistry study.

The dorsal hippocampus of the rat normally receives its 5-HT innervation from two homologous groups of cells in the median raphe nucleus via the cingulum bundle-induseum griseum (CB-IG) and the fornix-fimbria (FF) (J. Comp. Neurol., 179 (1978) 641-667 and Brain Res. Bull., 10 (1983) 445-451). 5-HT immunoreactive (IR) fibers are distributed in a laminar pattern in the hippocampus. These fibers have large varicosities and are densely distributed in the infragranular layer of dentate gyrus, in the stratum lacunosum-moleculare of the cornu Ammonis and in the area fasciola cinerea (FC). The present study provides evidence that the density of the 5-HT-IR fibers in the dorsal hippocampus is greatly decreased but maintained a similar laminar pattern 3 days after lesioning by microinjection of 4 micrograms of 5,7-dihydroxytryptamine (5,7-DHT) into the CB-IG. An apparently normal density and distribution pattern of the 5-HT-IR fiber is seen by 42 days postlesion. The FC in the hippocampus is among the first regions reinnervated by 5-HT-IR fibers with very dense and large varicosities. The restitution of 5-HT-IR fibers in the dorsal hippocampus after the 5,7-DHT lesion in the CB-IG is accompanied by a marked increase in the number and intensity of 5-HT-IR fibers in the FF. No evidence of a regeneration of 5-HT-IR fibers is seen distal to the injection site in the CB-IG. These observations provide direct evidence for homotypic collateral sprouting in the CNS induced by removal of a single fiber type.

Neurotrophic factor for serotonergic neurons prevents degeneration of grafted raphe neurons in the cerebellum.

Earlier studies in this laboratory have shown that partial serotonergic (5-HT) deafferentation from the hippocampus induced a homotypic sprouting. The 5-HT-denervated hippocampus was further found to be a favorable environment for the growth of grafted 5-HT, but not for norepinephrinergic neurons. In the current study, we have extracted a trophic factor in the denervated hippocampus with hypotonic solution in high-speed supernatant fraction. The trophic extract was assayed in a unique experimental paradigm--test the growth of grafted 5-HT neurons in the cerebellum, which receives the least 5-HT innervation, and where grafted 5-HT neurons have seldom survived. The diluted trophic extract (1:200) produced a dramatic increase in (a) the survival rate, (b) the 5-HT fiber-density, and (c) the 5-HT and 5-hydroxyindoleacetic acid levels of grafted 5-HT neurons in the cerebellum. In addition, Nissl-counterstaining showed that the size of the grafted raphe also apparently increased. The concentrated trophic extract (1:1) did not increase the survival rate of the grafted raphe neurons. These results indicate that (a) the supernatant in the 5-HT-denervated hippocampus contains a potent neurotrophic factor to 5-HT neurons (b) which can either directly or indirectly also support non-5-HT raphe neurons, and (c) possibly an inhibitory factor is present which is detrimental to 5-HT neurons in high concentration.