Neonatal Hosts Research Articles

Host circuit engagement of human cortical organoids transplanted in rodents.

Human neural organoids represent promising models for studying neural function; however, organoids grown in vitro lack certain microenvironments and sensory inputs that are thought to be essential for maturation. The transplantation of patient-derived neural organoids into animal hosts helps overcome some of these limitations and offers an approach for neural organoid maturation and circuit integration. Here, we describe a method for transplanting human stem cell-derived cortical organoids (hCOs) into the somatosensory cortex of newborn rats. The differentiation of human induced pluripotent stem cells into hCOs occurs over 30-60 days, and the transplantation procedure itself requires ~0.5-1 hours per animal. The use of neonatal hosts provides a developmentally appropriate stage for circuit integration and allows the generation and experimental manipulation of a unit of human neural tissue within the cortex of a living animal host. After transplantation, animals can be maintained for hundreds of days, and transplanted hCO growth can be monitored by using brain magnetic resonance imaging. We describe the assessment of human neural circuit function in vivo by monitoring genetically encoded calcium responses and extracellular activity. To demonstrate human neuron-host functional integration, we also describe a procedure for engaging host neural circuits and for modulating animal behavior by using an optogenetic behavioral training paradigm. The transplanted human neurons can then undergo ex vivo characterization across modalities including dendritic morphology reconstruction, single-nucleus transcriptomics, optogenetic manipulation and electrophysiology. This approach may enable the discovery of cellular phenotypes from patient-derived cells and uncover mechanisms that contribute to human brain evolution from previously inaccessible developmental stages.

The microbiota-gut-brain axis: A crucial immunomodulatory pathway for Bifidobacterium animalis subsp. lactis' resilience against LPS treatment in neonatal rats

An imbalanced gut microflora may contribute to immune disorders in neonates due to an immature gut barrier. Bacterial toxins, particularly, can trigger the immune system, potentially resulting in uncontrolled gut and systemic inflammation. Previous research has revealed that Bifidobacterium animalis subsp. lactis (B. lactis) could protect against early-life pathogen infections by enhancing the gut barrier. However, the effects of B. lactis on a compromised immune system remain uncertain. Hence, this study concentrated on the immunomodulatory effects and mechanisms of B. lactis in neonatal rats intraperitoneally injected with lipopolysaccharide (LPS), a bacterial toxin and inflammatory mediator. First, B. lactis significantly alleviated the adverse effects induced by LPS on the growth, development, and body temperature of neonatal rats. Second, B. lactis significantly reduced the immune responses and damage induced by LPS, affecting both systemic and local immune responses in the peripheral blood, gut, and brain. Notably, B. lactis exhibited extra potent neuroprotective and neurorepair effects. Our research found that pre-treatment with B. lactis shaped the diverse gut microecology by altering both microbial populations and metabolic biomolecules, closely linked to immunomodulation. Overall, this study elucidated the multifaceted roles of B. lactis in neonatal hosts against pathogenic infection and immune disorder, revealing the existence of the microbiota-gut-brain axis.

Differentiation and functional connectivity of fetal tectal transplants.

Data from studies analyzing the differentiation and functional connectivity of embryonic neural tissue grafted into the mammalian nervous system has led to the clinical testing of the fetal graft approach in patients with neurodegenerative disease. While some success has been achieved, ethical concerns have led to a search for alternative therapeutic strategies, mostly exploring the use of neural precursors or neurons derived from pluripotent stem cells to replace damaged host neurons and restore lost circuitries. These more recent studies address questions of graft viability, differentiation, and connectivity similar to those posed by researchers in earlier fetal transplant work, thus reviews of the fetal graft literature may inform and help guide ongoing research in the stem cell/organoid field. This brief review describes some key observations from research into the transplantation of neural tissue into the rat visual system, focusing on grafts of the fetal superior colliculus (tectal grafts) into neonatal or adult hosts. In neonate hosts, grafts quickly develop connections with the underlying host midbrain and attain a morphology typical of mature grafts by about 2 weeks. Grafts consistently contain numerous localized regions which, based on neurofibrillar staining, neuronal morphology (Golgi), neurochemistry, receptor expression, and glial architecture, are homologous to the stratum griseum superficiale of normal superior colliculus. These localized "patches" are also seen after explant culture and when donor tectal tissue is dissociated and reaggregated prior to transplantation. In almost all circumstances, host retinal innervation is restricted to these localized patches, but only those that are located adjacent to the graft surface. Synapses are formed and there is evidence of functional drive. The only exception occurs when Schwann cells are added to dissociated tecta prior to reaggregation. In these co-grafts, the peripheral glia appear to compete with local target factors and host retinal ingrowth is more widespread. Other afferent systems (e.g., host cortex, serotonin) show different patterns of innervation. The host cortical input originates more from extrastriate regions and establishes functional excitatory synapses with grafted neurons. Finally, when grafted into optic tract lesions in adult rat hosts, spontaneously regrowing host retinal axons retain the capacity to selectively innervate the localized patches in embryonic tectal grafts, showing that the specific affinities between adult retinal axons and their targets are not lost during regeneration. While the research described here provides some pertinent information about development and plasticity in visual pathways, a more general aim is to highlight how the review of the extensive fetal graft literature may aid in an appreciation of the positive (and negative) factors that influence survival, differentiation, connectivity and functionality of engineered cells and organoids transplanted into the central nervous system.

Sophoridine Suppresses Herpes Simplex Virus Type 1 Infection by Blocking the Activation of Cellular PI3K/Akt and p38 MAPK Pathways.

Herpes simplex virus type 1 (HSV-1) is a ubiquitous and important human pathogen capable of causing significant clinical diseases ranging from skin damage to encephalitis, particularly in immunocompromised and neonatal hosts. Currently, widely used nucleoside analogs, including acyclovir and penciclovir, have some limitations in their use due to side effects and drug resistance. Herein, we report sophoridine's (SRI) dramatic inhibition of HSV-1 replication in vitro. SRI exhibited a remarkable inhibitory influence on HSV-1 virus-induced cytopathic effect and plaque formation, as well as on progeny viruses in Vero and HeLa cells, with selection indexes (SI) of 38.96 and 22.62, respectively. Moreover, SRI also considerably suppressed HSV-1 replication by hindering the expression of viral immediate-early (ICP0 and ICP22), early (ICP8 and TK), and late (gB and gD) genes and the expression of viral proteins ICP0, gB, and gD. We suggest that SRI can directly inactivate viral particles and block some stages in the life cycle of HSV-1 after adsorption. Further experiments showed that SRI downregulated the cellular PI3K/Akt signaling pathway and obstructed HSV-1 replication even more. Most importantly, SRI markedly repressed HSV-1-induced p38 MAPK pathway activation. Collectively, this natural bioactive alkaloid could be a promising therapeutic candidate against HSV-1 via the modulation of cellular PI3K/Akt and p38 MAPK pathways.

Neonatal Transplant in Hypoxic Injury.

Even with therapeutic hypothermia as standard of care, hypoxic-ischemic encephalopathy in neonates often causes long-term disabilities. Stem cell therapy may be a successful treatment for HIE both in the acute and chronic time periods post-injury. Neurogenic astrocytes with characteristics of neural stem cells can be cultured as adherent monolayers and be induced to generate neuronal progeny in vitro and in vivo following reintroduction into the neural stem cell niche of both neonatal and adult hosts. Thus, these neurogenic astrocytes represent promising candidates for cell replacement therapy in HIE. Such an approach requires optimized cell cultivation protocols as well as extensive testing of donor cells to assess their capacity for engraftment, survival, and integration in the HIE animal models. In this chapter, we will describe methods of generating the HIE model, generating and culturing monolayer neurogenic astrocytes, and transplanting these cells into HIE animal models.

Case Report: Neonatal Varicella Acquired From Maternal Zoster

The incidence of neonatal varicella has decreased dramatically since the introduction of the varicella vaccination. Although the varicella zoster virus is often associated with a mild infection, it may cause severe morbidity and mortality, particularly in the neonatal period and immunocompromised hosts. We report a case of neonatal varicella acquired from maternal zoster in a mother on biological immunosuppressive therapy. Following the diagnosis, the baby improved on antiviral therapy without any neurological sequelae. This case highlights the limited published data on neonatal varicella following herpes zoster reactivation to inform practice. This includes the role of varicella zoster immunoglobulin in neonates exposed to maternal zoster, the degree of trans-placental immunity and optimum antiviral dosing and duration.

Rapid Evolution of the CD8+ TCR Repertoire in Neonatal Mice.

Currently, there is little consensus regarding the most appropriate animal model to study acute infection and the virus-specific CD8(+) T cell (CTL) responses in neonates. TCRβ high-throughput sequencing in naive CTL of differently aged neonatal mice was performed, which demonstrated differential Vβ family gene usage. Using an acute influenza infection model, we examined the TCR repertoire of the CTL response in neonatal and adult mice infected with influenza type A virus. Three-day-old mice mounted a greatly reduced primary NP(366-374)-specific CTL response when compared with 7-d-old and adult mice, whereas secondary CTL responses were normal. Analysis of NP(366-374)-specific CTL TCR repertoire revealed different Vβ gene usage and greatly reduced public clonotypes in 3-d-old neonates. This could underlie the impaired CTL response in these neonates. To directly test this, we examined whether controlling the TCR would restore neonatal CTL responses. We performed adoptive transfers of both nontransgenic and TCR-transgenic OVA(257-264)-specific (OT-I) CD8(+) T cells into influenza-infected hosts, which revealed that naive neonatal and adult OT-I cells expand equally well in neonatal and adult hosts. In contrast, nontransgenic neonatal CD8(+) T cells when transferred into adults failed to expand. We further demonstrate that differences in TCR avidity may contribute to decreased expansion of the endogenous neonatal CTL. These studies highlight the rapid evolution of the neonatal TCR repertoire during the first week of life and show that impaired neonatal CTL immunity results from an immature TCR repertoire, rather than intrinsic signaling defects or a suppressive environment.

Impaired CD8+ T cell responses to influenza infection in neonatal mice are due to the immature T cell receptor repertoire (VIR2P.1020)

Abstract To understand the role of virus antigen-specific CD8+ T cell responses in neonates during respiratory viral infection, we developed a model where 3-day old C57Bl/6 mice were infected intranasally with influenza type A virus strain A PR/8/34 (H1N1). When these neonatal mice are infected with influenza virus they mount a greatly reduced and delayed lung NP(366-374)-specific primary CD8+ T cell response. To investigate what was responsible for the reduced response, we performed adoptive transfers of TCR-transgenic OVA(257-264)-specific (OT-I) CD8+ T cells into hosts infected with recombinant influenza virus WSN-OVA, which expresses the OVA(257-264) peptide. These studies revealed that naïve adult OT-I cells expand equally well in neonatal and adult hosts, which excludes the neonatal environment as an inhibitor of the CD8+ T cell response. When naïve neonatal OT-I cells were transferred into neonatal and adult hosts, these neonatal OT-I cells expanded equally as well as adult OT-I cells, excluding an intrinsic defect in the neonatal CD8+ T cells. These studies demonstrate that the TCR repertoire of neonates is responsible for reduced CD8+ T cell immunity in neonates, not a suppressive environment or intrinsic signaling defects. Finally, despite impaired primary response, animals primed as neonates exhibited normal secondary responses to influenza virus. These findings raise important issues that need to be taken into consideration for vaccination of neonates.

Molecular epidemiology of group B streptococcal meningitis in children beyond the neonatal period from Angola

Streptococcus agalactiae is a major pathogen of neonates and immunocompromised adults. Prior studies have demonstrated that, beyond the neonatal period, S. agalactiae rarely causes invasive infections in children. However, during 2004-2005, S. agalactiae was the causative agent of 60 meningitis episodes in children aged 3 months to 12 years from Angola. To identify and study the specific causative genetic lineages of S. agalactiae childhood meningitis, which lack characterization to date, we conducted an extensive molecular analysis of the recovered isolates (n = 21). This constitutes what we believe to be the first molecular study of the population structure of invasive S. agalactiae isolates from Africa. A low genetic diversity was observed among the isolates, where the majority belonged to clonal complex (CC) 17 presenting the capsular subtype III-2 (86 % of cases) and marked by the intron group II GBSi1, which has previously been observed to be associated with neonatal hosts. The predominance of single-locus variants of sequence type (ST) 17 suggested the local diversification of this hypervirulent clone, which displayed novel alleles of the fbsB and sip virulence genes. The absence of the scpB-lmb region in two S. agalactiae isolates with the Ia/ST23 genotype is more typical of cattle than human isolates. Globally, these data provide novel information about the enhanced invasiveness of the CC17 genetic lineage in older children and suggest the local diversification of this clone, which may be related to the future emergence of a novel epidemic clone in Angola.

Leptomeningeal metastases in the MRI era

Innervation of specific peptidergic and cholinergic compartments of the interpeduncular nucleus (IPN) was investigated using embryonic cell suspension transplants immunoreactive for substance P (SP) and ChAT. In both neonatal and adult host rats, the IPN was first denervated of its normal SP and cholinergic input from the medial habenula by bilateral lesions of the fasciculi retroflexi (FR). In adult hosts, transplants of embryonic habenular cells placed near the denervated IPN mediated a return of the normal pattern of SP staining restricted to habenula- target subnuclei, plus an increase in staining intensity of SP cells intrinsic to the IPN. There was no recovery of ChAT staining. A similar pattern of SP staining resulted following habenular transplants into neonatal hosts, but in addition there was a partial recovery of normal ChAT staining in cholinergic subnuclei and anomalous ChAT staining in normally peptidergic subnuclei. Control transplants of embryonic thalamus cells placed into adult hosts produced a surprising pattern of ChAT staining in the IPN identical to that seen with habenula transplants placed into neonatal hosts; the adult IPN was thus able to support reinnervation mediated by an aberrant cholinergic source while being refractory to its normal habenular cholinergic afferents. This pattern of results implies regulation by the IPN of habenular SP and cholinergic innervation, and some interaction between the maturing normal cholinergic afferents and their targets that is missing when these afferent sources are abnormal.

Abstract 5036: NF-κB pathway inhibition delays the onset of MYC-induced liver cancer

Abstract Liver cancer is one of the most lethal forms of cancer worldwide. Hence, it is critical to identify pathways that may be essential for the genesis of liver cancer and can thus be considered as targets for therapeutic intervention. Previously, our laboratory developed a conditional transgenic murine model of MYC-induced liver cancer that is dependent upon the expression of this oncogene and regresses following its inactivation. Here, we present evidence of an important role for the nuclear factor-kappa B (NF-κB) pathway in MYC-induced hepatocarcinogenesis. In order to examine whether NF-κB activation is necessary for MYC-induced liver cancer, we established a transgenic system in which MYC can be activated in hepatocytes concomitantly with a mutant form of IκB, thereby inhibiting the NF-κB pathway. Our approach has revealed that suppression of NF-κB activation can significantly inhibit MYC-induced hepatocellular carcinoma (HCC) in adult hosts. Indeed, animals with hepatocyte-inhibited NF-κB remain tumor free up to 8 months after oncogene activation compared to mortality in 100% within 6 months in animals expressing MYC alone. Moreover, inhibition of NF-κB reduces disease penetrance from 100% to 75%. In hosts that do develop liver tumors, there is evidence for activation of the NF-κB pathway, suggesting a necessity for NF-κB activation in MYC-induced HCC. Importantly, suppression of NF-κB activation in neonatal hosts is not only able to similarly suppress MYC-induced tumor formation, but animals that develop liver cancer in this context demonstrate a strikingly altered tumor phenotype. In addition, MYC inactivation results in a concomitant decrease in expression of NF-κB target genes, suggesting a direct role of MYC in the activation of NF-κB in hepatocytes. The inhibition of NF-κB did not result in increased apoptosis upon MYC activation. Rather, NF-κB activation may be important for MYC to evade oncogene-induced senescence, thereby facilitating tumorigenesis. Our data suggests that the inhibition of the NF-κB pathway may be sufficient to abrogate MYC's ability to induce tumorigenesis in the liver and thereby establish NF-κB activation as an attractive therapeutic target for liver cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5036.

Oral tolerance induction is defective during the neonatal period in the mouse (39.42)

Abstract Congenital T cell (T)-deficient mice, such as RAG-1- or SCID mice, succumb to inflammatory bowel disease (IBD) upon adoptive transfer of syngenic naive T cells as a consequence of chronic inflammation in the intestine from unfettered response to commensal bacterial antigens. Although the prevailing view for the onset of IBD is believed to be the absence of regulatory T cells (Tregs), the finding that donor T cells undergo considerable chronic activation even in the presence of Tregs suggests an abnormality in the presentation of enteric antigens also appears to be involved. To study if such a defect is also evident during the lymphopenic neonatal period, we compared the induction of oral tolerance between B6 neonates and adults to host commensal or exogenously provided nominal Ags by adoptively transferring polylconal or Ag-specific donor T cells. Strikingly, donor T cells in neonatal hosts proliferated much faster and more readily acquired effector function than donor T cells in adults. These finding suggests that oral tolerance in normal mice is acquired later with the development of T immunity.

Characterization of an extended-spectrum beta-lactamase Enterobacter hormaechei nosocomial outbreak, and other Enterobacter hormaechei misidentified as Cronobacter (Enterobacter) sakazakii

Enterobacter hormaechei is a Gram-negative bacterium within the Enterobacter cloacae complex, and has been shown to be of clinical significance by causing nosocomial infections, including sepsis. Ent. hormaechei is spread via horizontal transfer and is often associated with extended-spectrum beta-lactamase production, which increases the challenges associated with treatment by limiting therapeutic options. This report considers 10 strains of Ent. hormaechei (identified by 16S rDNA sequencing) that had originally been identified by phenotyping as Cronobacter (Enterobacter) sakazakii. Seven strains were from different neonates during a nosocomial outbreak in a California hospital. PFGE analysis revealed a clonal relationship among six of the seven isolates and therefore a previously unrecognized Ent. hormaechei outbreak had occurred over a three-month period. Antibiotic-resistance profiles were determined and extended-spectrum beta-lactamase activity was detected. The association of the organism with powdered infant formula, neonatal hosts and Cr. sakazakii suggested that the virulence of these organisms may be similar. Virulence traits were tested and all strains were shown to invade both gut epithelial (Caco-2) and blood-brain barrier endothelial cells (rBCEC4), and to persist in macrophages (U937). Due to misidentification we suggest that Ent. hormaechei may be an under-reported cause of bacterial infection, especially in neonates. Also, its isolation from various sources, including powdered infant milk formula, makes it a cause for concern and merits further investigation.

Are Neonatal Stem Cells as Effective as Adult Stem Cells in Providing Ischemic Protection?

Bone marrow stem cells (BMSCs) may be a novel treatment modality for organ ischemia, possibly through beneficial paracrine mechanisms. However, stem cells from older hosts exhibit decreased function during stress. We therefore hypothesized that (1) BMSCs derived from neonatal hosts would provide protection to ischemic myocardium, and (2) neonatal stem cells would enhance postischemic myocardial recovery above that seen with adult stem cell therapy. Female adult Sprague Dawley rat hearts were subjected to an ischemia/reperfusion protocol via Langendorff isolated heart preparation (15 min equilibration, 25 min ischemia, and 60 min reperfusion). BMSCs were harvested from adult and neonatal mice and cultured through several passages under normal conditions (37 degrees C, 5% CO(2)/air). Immediately prior to ischemia, 1 million adult or neonatal BMSCs were infused into the coronary circulation. Cardiac functional parameters were continuously recorded. Pretreatment with adult BMSCs significantly increased postischemic myocardial recovery as noted by improved left ventricular developed pressure, end diastolic pressure, contractility, and rate of relaxation. Neonatal stem cells, however, did not cause any noticeable improvement in myocardial functional parameters following ischemia. Neonatal and adult BMSCs are distinctly different in the degree of beneficial tissue protection that they can provide. The data herein suggests that a critical age exists as to when stem cells become fully activated to provide their beneficial protective properties. Defining the genes that initiate these protective properties may allow for genetic amplification of beneficial signals, and the generation of "super stem cells" that provide maximum protection to ischemic tissues.

A feline immunodeficiency virus vif-deletion mutant remains attenuated upon infection of newborn kittens

This report characterizes lentivirus attenuation associated with a vif mutation by inoculation of newborn kittens with a vif-deleted feline immunodeficiency virus provirus plasmid (FIV-pPPRDeltavif). Virus in peripheral blood, antiviral antibody or CD4 T-cell count alterations were not detected in kittens inoculated with FIV-pPPRDeltavif plasmid, with the exception of one kitten that demonstrated FIV Gag antibody production at 42 weeks after inoculation. In contrast, wild-type FIV-pPPR-infected kittens were viraemic, seropositive and exhibited a decrease in the CD4 T-cell subset in peripheral blood. Interestingly, FIV-specific T-cell proliferative responses detected at 32 and 36 weeks after infection were comparable for both FIV-pPPRDeltavif- and wild-type FIV-pPPR-inoculated kittens and suggested the possibility of a discreet tissue reservoir supporting sustained FIV-pPPRDeltavif expression or replication. Overall, these findings confirmed that the severe virus attenuation for both replication and pathogenicity exhibited by a vif-deleted FIV mutant is similar for both neonatal and adult hosts.

Two Oncogenic Hits Are Required To Initiate Lymphomagenesis in Adult, but Not Neonatal Hosts.

The hallmark (8:14) translocation of Burkitt's lymphoma causes MYC overexpression, a primary event in the development of Burkitt's lymphoma. While Burkitt's lymphoma comprises nearly one third of childhood malignancies, it is rare in adults. Among many explanations is the possibility that the capacity for a lymphocyte to undergo malignant transformation by MYC activation is influenced by factors related to the developmental state of the host. To address this possibility, we used transgenic mice in which the MYC proto-oncogene is conditionally regulated via the Tetracycline Regulatory System (Tet system). When MYC was activated in cohorts of mice less than 3 weeks of age, mice universally succumbed to lymphoma within 23 weeks. However, when MYC was activated in adult mice (over 7 weeks of age) the mice failed to develop lymphoma within 50 weeks. We were not able to detect differences in the effects of MYC on the proliferation or apoptosis in lymphocytes from adult versus neonatal hosts. We next explored whether the concomitant activation of MYC with a second oncogene could induce lymphomagenesis in adult mice. When we conditionally induced both MYC and BCL2 or MYC and RAS in adult hosts, mice succumbed to lymphoma with a mean latency of 24–27 weeks. Our results suggest that in adult hosts, concurrent activation of at least two oncogenes is required for MYC to initiate tumorigenesis.

Simian immunodeficiency virus infection in neonatal macaques.

Children with human immunodeficiency virus infection often have higher viral loads and progress to AIDS more rapidly than adults. Since the intestinal tract is a major site of early viral replication and CD4(+) T-cell depletion in adults, we examined the effects of simian immunodeficiency virus (SIV) on both peripheral and intestinal lymphocytes from 13 neonatal macaques infected with SIVmac239. Normal neonates had more CD4(+) T cells and fewer CD8(+) T cells in all tissues than adults. Surprisingly, neonates had substantial percentages of CD4(+) T cells with an activated, memory phenotype (effector CD4(+) T cells) in the lamina propria of the intestine compared to peripheral lymphoid tissues, even when examined on the day of birth. Moreover, profound and selective depletion of jejunum lamina propria CD4(+) T cells occurred in neonatal macaques within 21 days of infection, which was preceded by large numbers of SIV-infected cells in this compartment. Furthermore, neonates with less CD4(+) T-cell depletion in tissues tended to have higher viral loads. The persistence of intestinal lamina propria CD4(+) T cells in some neonates with high viral loads suggests that increased turnover and/or resistance to CD4(+) T-cell loss may contribute to the higher viral loads and increased severity of disease in neonatal hosts.

Differences in Host Serotonin Innervation of Intrastriatal Grafts Are Not Determined by a Glial Scar or Chondroitin Sulfate Proteoglycans

Serotoninergic (5-HT) neurons of adult recipients provide a much denser innervation of striatal than ventral mesencephalic grafts implanted into the neostriatum of the rat. Moreover, grafts from both brain regions are more innervated by host 5-HT axons after implantation in neonatal than adult hosts. To test the hypothesis that differences in glial scarring or expression of the growth inhibitory molecules, chondroitin sulfate proteoglycans (CSPG), be responsible for these differences in 5-HT innervation of neural grafts, we examined the 5-HT innervation, the astroglial reaction and the expression of CSPG in ventral mesencephalic grafts implanted into newborn (1–5 days old), juvenile (15 days old), or adult rats and in striatal grafts implanted in adult rats, using immunohistochemistry against 5-HT, glial fibrillary acidic protein (GFAP) and CSPG. Immunostaining for GFAP showed a stronger initial gliosis (1–10 days after grafting) in neonatal than adult recipients of mesencephalic grafts, but this gliosis subsided gradually at later time points. Nevertheless, a glial scar formed at the graft-host interface in both neonatal and adult recipients, 5–10 days after transplantation, although it decreased over a longer time course—up to 60 days—in adults. Immunostained astrocytes appeared first in the host brain tissue around the graft and then immunoreactive processes and perikarya gradually invaded the graft. Immunoreactivity for CSPG was similar in neonatal and adult hosts: it was strongly expressed inside the graft early after transplantation, and almost completely down-regulated at 60 days. The reaction of adult hosts to striatal and mesencephalic grafts was similar, although GFAP was more heterogeneously distributed and CSPG immunoreactivity remained in patches inside striatal grafts, even after 60 days. The 5-HT innervation of mesencephalic grafts was much denser after implantation in newborns than in adults. It was also stronger in striatal than in mesencephalic grafts implanted in adults. Thus, the presence of a glial scar or the expression of CSPG cannot totally account for the different degrees of 5-HT innervation in the various types of neural grafts.

Control of postganglionic neurone phenotype by the rat pineal gland

As neurones develop they are faced with choices as to which genes to express, to match their final phenotype to their role in the nervous system. A number of processes can guide these decisions. Within the autonomic and sensory nervous systems, there are a handful of examples that suggest that one mechanism that may match phenotype to function is the presence of target-derived differentiation factors. We tested whether the rat pineal gland controls the expression of a neuropeptide (neuropeptide Y) and a calcium-binding protein (calbindin) in sympathetic postganglionic neurones that innervate it. We first showed that the chemical phenotype of sympathetic neurones innervating the rat pineal includes the expression of both neuropeptide Y and the calcium-binding protein, calbindin. After transplanting the pineal gland of neonatal rats into the submandibular salivary gland of neonatal hosts, it was innervated by sympathetic axons from the surrounding salivary gland tissue, which do not normally express neuropeptide Y and calbindin. The presence of the pineal gland led to the appearance of neuropeptide Y and calbindin in many of the postganglionic neurones that innervated the graft. From these findings we suggest that, like the rodent sweat gland, the pineal gland generates a signal that can direct the neurochemical phenotype of innervating sympathetic neurones.

Altered expression of apolipoprotein E, amyloid precursor protein and presenilin-1 is associated with chronic reactive gliosis in rat cortical tissue

A major characteristic feature of Alzheimer’s disease is the formation of compact, extracellular deposits of β-amyloid (senile plaques). These deposits are surrounded by reactive astrocytes, microglia and dystrophic neurites. Mutations in three genes have been implicated in early-onset familial Alzheimer’s disease. However, inflammatory changes and astrogliosis are also believed to play a role in Alzheimer’s pathology. What is unclear is the extent to which these factors initiate or contribute to the disease progression. Previous rat studies demonstrated that heterotopic transplantation of foetal cortical tissue onto the midbrain of neonatal hosts resulted in sustained glial reactivity for many months. Similar changes were not seen in cortex-to-cortex grafts. Using this model of chronic cortical gliosis, we have now measured reactive changes in the levels of the key Alzheimer’s disease proteins, namely the amyloid precursor protein, apolipoprotein E and presenilin-1. These changes were visualised immunohistochemically and were quantified by western blot analysis. We report here that chronic cortical gliosis in the rat results in a sustained increase in the levels of apolipoprotein E and total amyloid precursor protein. Reactive astrocytes in heterotopic cortical grafts were immunopositive for both of these proteins. Using a panel of amyloid precursor protein antibodies we demonstrate that chronic reactive gliosis is associated with alternative cleavage of the peptide. No significant changes in apolipoprotein E or amyloid precursor protein expression were seen in non-gliotic cortex-to-cortex transplants. Compared to host cortex, the levels of both N-terminal and C-terminal fragments of presenilin-1 were significantly lower in gliotic heterotopic grafts. The changes described here largely mirror those seen in the cerebral cortex of humans with Alzheimer’s disease and are consistent with the proposal that astrogliosis may be an important factor in the pathogenesis of this disease.