Lesion environments direct transplanted neural progenitors towards a wound repair astroglial phenotype in mice.

Neural progenitor cells (NPC) represent potential cell transplantation therapies for CNS injuries. To understand how lesion environments influence transplanted NPC fate in vivo, we derived NPC expressing a ribosomal protein-hemagglutinin tag (RiboTag) for transcriptional profiling of transplanted NPC. Here, we show that NPC grafted into uninjured mouse CNS generate cells that are transcriptionally similar to healthy astrocytes and oligodendrocyte lineages. In striking contrast, NPC transplanted into subacute CNS lesions after stroke or spinal cord injury in mice generate cells that share transcriptional, morphological and functional features with newly proliferated host astroglia that restrict inflammation and fibrosis and isolate lesions from adjacent viable neural tissue. Our findings reveal overlapping differentiation potentials of grafted NPC and proliferating host astrocytes; and show that in the absence of other interventions, non-cell autonomous cues in subacute CNS lesions direct the differentiation of grafted NPC towards a naturally occurring wound repair astroglial phenotype.

Survival of adult basal forebrain cholinergic neurons after loss of target neurons.

Target cells are thought to regulate the survival of afferent neurons during development by supplying limiting amounts of neurotrophic factors, but the degree to which afferent neurons remain dependent on target-derived support in the adult is uncertain. In this study, uninjured basal forebrain cholinergic neurons did not die after excitotoxic ablation of their target neurons in young adult rats, indicating that they are either not dependent on neurotrophic factors for survival or can obtain trophic support from other sources after target neurons are lost. This finding suggests that cholinergic cell death in neurodegenerative conditions such as Alzheimer's disease is not due solely to a loss of target neurons or factors provided by them.

Leukocyte infiltration, neuronal degeneration, and neurite outgrowth after ablation of scar-forming, reactive astrocytes in adult transgenic mice.

Reactive astrocytes adjacent to a forebrain stab injury were selectively ablated in adult mice expressing HSV-TK from the Gfap promoter by treatment with ganciclovir. Injured tissue that was depleted of GFAP-positive astrocytes exhibited (1) a prolonged 25-fold increase in infiltration of CD45-positive leukocytes, including ultrastructurally identified monocytes, macrophages, neutrophils, and lymphocytes, (2) failure of blood-brain barrier (BBB) repair, (3) substantial neuronal degeneration that could be attenuated by chronic glutamate receptor blockade, and (4) a pronounced increase in local neurite outgrowth. These findings show that genetic targeting can be used to ablate scar-forming astrocytes and demonstrate roles for astrocytes in regulating leukocyte trafficking, repairing the BBB, protecting neurons, and restricting nerve fiber growth after injury in the adult central nervous system.

Injectable polypeptide hydrogels via methionine modification for neural stem cell delivery.

Injectable hydrogels with tunable physiochemical and biological properties are potential tools for improving neural stem/progenitor cell (NSPC) transplantation to treat central nervous system (CNS) injury and disease. Here, we developed injectable diblock copolypeptide hydrogels (DCH) for NSPC transplantation that contain hydrophilic segments of modified l-methionine (Met). Multiple Met-based DCH were fabricated by post-polymerization modification of Met to various functional derivatives, and incorporation of different amino acid comonomers into hydrophilic segments. Met-based DCH assembled into self-healing hydrogels with concentration and composition dependent mechanical properties. Mechanical properties of non-ionic Met-sulfoxide formulations (DCHMO) were stable across diverse aqueous media while cationic formulations showed salt ion dependent stiffness reduction. Murine NSPC survival in DCHMO was equivalent to that of standard culture conditions, and sulfoxide functionality imparted cell non-fouling character. Within serum rich environments in vitro, DCHMO was superior at preserving NSPC stemness and multipotency compared to cell adhesive materials. NSPC in DCHMO injected into uninjured forebrain remained local and, after 4 weeks, exhibited an immature astroglial phenotype that integrated with host neural tissue and acted as cellular substrates that supported growth of host-derived axons. These findings demonstrate that Met-based DCH are suitable vehicles for further study of NSPC transplantation in CNS injury and disease models.

Essential protective roles of reactive astrocytes in traumatic brain injury.

Astrocytes respond to traumatic brain injury (TBI) by altered gene expression, hypertrophy and proliferation that occur in a gradated fashion in relation to the severity of the injury. Both beneficial and detrimental effects have been attributed to reactive astrocytes, but their roles after brain injury are not well understood. To investigate these roles, we determined the effects on cortical tissue of ablating reactive astrocytes after contusion injury generated by controlled cortical impact (CCI) of different severities in transgenic mice that express a glial fibrillary acidic protein-herpes simplex virus-thymidine kinase transgene. Treatment of these mice with the antiviral agent, ganciclovir, conditionally ablates proliferating reactive astrocytes. Moderate or severe CCI were generated with a precisely regulated pneumatic piston, and forebrain tissue was evaluated using immunohistochemistry and quantitative morphometry. Moderate CCI in control mice triggered extensive and persisting reactive astrogliosis, with most neurons being preserved, little inflammation and an 18% loss of cortical tissue beneath the impact site. Ablation of reactive astrocytes after moderate CCI in transgenic mice caused substantial neuronal degeneration and inflammation, with a significantly greater 60% loss of cortical tissue. Severe CCI in control mice caused pronounced neuronal degeneration and loss of about 88% of cortical tissue that was not significantly altered by ablating reactive astrocytes in transgenic mice. Thus, ablation of dividing reactive astrocytes exacerbated cortical degeneration after moderate CCI, but did not alter cortical degeneration after severe CCI. These findings indicate that the reactive astrocytes play essential roles in preserving neural tissue and restricting inflammation after moderate focal brain injury.

PBK/TOPK, a proliferating neural progenitor-specific mitogen-activated protein kinase kinase.

We performed genomic subtraction coupled to microarray-based gene expression profiling and identified the PDZ (postsynaptic density-95/Discs large/zona occludens-1)-binding kinase/T-LAK (lymphokine-activated killer T cell) cell originating protein kinase (PBK/TOPK) as a gene highly enriched in neural stem cell cultures. Previous studies have identified PBK/TOPK as a mitogen-activated protein kinase (MAPK) kinase that phosphorylated P38 MAPK but with no known expression or function in the nervous system. First, using a novel, bioinformatics-based approach to assess cross-correlation in large microarray datasets, we generated the hypothesis of a cell-cycle-related role for PBK/TOPK in neural cells. We then demonstrated that both PBK/TOPK and P38 are activated in a cell-cycle-dependent manner in neuronal progenitor cells in vitro, and inhibition of this pathway disrupts progenitor proliferation and self-renewal, a core feature of progenitors. In vivo, PBK/TOPK is expressed in rapidly proliferating cells in the adult subependymal zone (SEZ) and early postnatal cerebellar external granular layer. Using an approach based on transgenically targeted ablation and lineage tracing in mice, we show that PBK/TOPK-positive cells in the SEZ are GFAP negative but arise from GFAP-positive neural stem cells during adult neurogenesis. Furthermore, ablation of the adult stem cell population leads to concomitant loss of PBK/TOPK-positive cells in the SEZ. Together, these studies demonstrate that PBK/TOPK is a marker for transiently amplifying neural progenitors in the SEZ. Additionally, they suggest that PBK/TOPK plays an important role in these progenitors, and further implicates the P38 MAPK pathway in general, as an important regulator of progenitor proliferation and self-renewal.

Developmentally regulated and tissue specific expression of mRNAs encoding the two alternative forms of the LIM domain oncogene rhombotin: evidence for thymus expression.

The T-cell oncogene rhombotin was first identified as a gene near a chromosomal translocation breakpoint in a human T-cell tumour and represents the first example of an oncogene carrying the duplicated cysteine-rich regions (CRR or LIM domains). Transgenic expression of a reporter gene under the control of one of the rhombotin gene promoters subsequently showed high levels of expression in the developing brain. These disparate sites of transcriptional activity suggested that the gene may have been activated de novo specifically in the T cell tumour via the translocation. Here, we assess this possibility by analysing rhombotin gene expression in mouse development by in situ hybridization of whole embryos, Northern filter hybridization, and a sensitive semiquantitative PCR method. The results show that the central nervous system is the major site of rhombotin mRNA production. Low level expression does, however, occur in other tissues including thymus. Furthermore, both promoters are active and differentially regulated during mouse embryogenesis in both brain and thymus. In subregions of the adult brain, different levels of rhombotin activity can be observed, with evidence for regional variation in promoter usage. A detailed analysis of mouse and human T-cell differentiation suggests that fluctuating promoter activities are related to a general T-cell differentiation process rather than to the differentiation of functionally distinct subsets of T-cells. These data suggest that the transforming activity of rhombotin in the T-cell with the chromosomal translocation was not due to de novo transcriptional activation, but rather to a quantitative or qualitative change in expression levels of this CRR-containing oncogene after chromosomal translocation.

Extrahypothalamic neurophysin-containing perikarya, fiber pathways and fiber clusters in the rat brain.

Immunoreactive neurophysin (Np) is demonstrated by the immunoperoxidase technique not only within neurones of the classical hypothalamo-neurohypophyseal system, but also in extrahypothalamic perikarya and fibers. Np-positive perikarya are found in the triangular nucleus of the septum. Np fibers join the stria terminalis (ST) at the level of the anterior hypothalamus, and course medially in the ST to the central nucleus of the amygdala. Np fibers are found in the brain stem and spinal cord. Fine caliber Np fibers from the suprachiasmatic nucleus ascend to the medial dorsal thalamus and lateral septum. The presence of Np within neurones implies the presence of either vasopressin or oxytocin. Np-positive fibers in extrahypothalamic sites may interact with non-neurosecretory neurones involved in neuroendocrine regulation, or may serve as yet unknown functions.

Radioimmunoassay of arginine vasopressin in Rhesus monkey plasma.

Using a new antiserum, an enzymatic radioiodination of arginine vasopressin (AVP), and the methodology of Robertson et al. (1,2), we have developed a sensitive and specific radioimmunoassay for plasma AVP in the monkey. The sensitivity of the assay is 0.5 muU/ml, the cross reaction with oxytocin (OT), minimal. We used this assay to study the effects that variations in blood osmolality have in regulating AVP secretion in unanesthetized, chair-restrained, chamber-isolated, adult female rhesus monkeys. Under water ad lib conditions, plasma AVP and osmolality were relatively constant, averaging 1.7 +/- 0.6 (SD) muU/ml and 298 +/- 3 mosmol/kg, respectively. Water loading decreased plasma AVP and osmolality to 0.6 +/- 0.2 muU/ml and 282 +/- 6 mosmol/kg, respectively. When fluid restriction increased osmolality, plasma AVP rose progressively to twice the baseline after 1 day, and to 6 times the baseline after 3 days. The rise in plasma AVP was linearly correlated with the rise in osmolality (r = 0.93; P less than 0.001). Intravenous infusions of hypertonic saline produced significant rises in plasma osmolality and plasma AVP. There was a dose-related rise in plasma AVP that declined later at the expected rate with the infusion of physiological amounts of synthetic AVP.

Targeting expression of hsp70i to discrete neuronal populations using the Lmo-1 promoter: assessment of the neuroprotective effects of hsp70i in vivo and in vitro.

Transgenic technology provides a powerful means of studying gene regulation and specific gene function with complex mammalian systems. In this study, the authors exploited the specific and discrete neuronal expression pattern mediated by promoter 1 of the Lmo-1 gene to study the neuroprotective effects of the inducible form of heat shock protein 70kD (hsp70i) in primary hippocampal cultures in a mouse model of global cerebral ischemia. Targeting expression of hsp70i to hippocampal neurons protected these cells significantly from toxic levels of glutamate and oxidative stress (for example, exposure to 10 micromol/L free iron produced a 26% increase in lactate dehydrogenase release from neurons cultured from wild-type mice, but a 7% increase in neurons cultured from hsp70i transgenic mice). Bilateral carotid occlusion (25 minutes) produced significantly less neuronal damage in the caudate nucleus and posterior thalamus in hsp70i transgenic mice than in wild-type littermates (for example, 21% +/- 9.3% and 12.5% +/- 9.0% neuronal damage in lateral caudate nucleus of wild-type and hsp70i transgenic mice, respectively, P < 0.05). The current study highlights the utility of targeted expression of transgenes of interest in cerebral ischemia and demonstrates that expression of hsp70i alone is sufficient to mediate the protection of primary neurons from denaturing stress and that expression of human hsp70i in vivo plays crucial role in determining the fate of neurons after ischemic challenge.

Can activity modulate the susceptibility of neurons to degeneration?

The provocative suggestion that maintaining an appropriate level of continuous neuronal activation, either by training or pharmacological intervention, might make neurons more robust and able to withstand degenerative insults during aging is fascinating and clearly warrants further investigation. Two cautionary points: 1) it is far from certain that Alzheimer's disease represents accelerated aging, and 2) excessive activation could be harmful to neurons in a context other than that of normally occurring "wear and tear."

Transfection-mediated expression of human Hsp70i protects rat dorsal root ganglian neurones and glia from severe heat stress.

Considerable evidence suggests that the expression of heat shock proteins prior to a toxic insult (e.g. ischaemia, excitoxins, heat) can confer protection to neurones and glia. It is not certain which hsp(s) are involved in conveying these neuroprotective effects. Here we show that calcium phosphate-mediated transfection of dorsal root ganglia with an EF-1 alpha promoter-hsp70i expression vector significantly increased the survival of neurones and glia exposed to a severe heat stress. These data suggest that overexpression of hsp70i plays an important role in protecting neurones and glia from the denaturing effects of severe thermal stress. Inducing the expression of specific hsps may lead to the development of novel treatment strategies for CNS diseases.

Direct reciprocal connections between the bed nucleus of the stria terminalis and dorsomedial medulla oblongata: evidence from immunohistochemical detection of tracer proteins.

Connections between the bed nucleus of the stria terminalis and the dorsomedial medulla oblongata have been examined by immunohistochemical detection of the tracer proteins horseradish peroxidase (HRP) or wheat germ agglutinin (WGA). Two sets of four rats received 0.2 or 0.4 microliter of either tracer injected into the dorsomedial medulla oblongata, were fixed by perfusion 48 hours later, and were processed for immunohistochemical detection of the tracers. Rats receiving HRP showed only a few single neurons retrogradely labelled in the ipsilateral bed nucleus, and some anterogradely labelled fibers. Rats receiving WGA showed a large number of retrogradely labelled neurons in the ipsilateral bed nucleus. Labelled neurons were most concentrated in a group in the central, dorsal, and lateral aspects of the nucleus at the level of the anterior commissure and just caudal to this. Just ventral to this group was a dense cluster of anterogradely labelled fibers. The retrogradely labelled neurons ranged from 12 to 20 microns in size and were multipolar. These findings indicate that there are direct reciprocal connections between the bed nucleus of the stria terminalis and the autonomic centers of the dorsomedial medulla oblongata and strengthen the concept that this nucleus is involved in forebrain integration of autonomic functions.

Identification of parvocellular vasopressin and neurophysin neurons in the suprachiasmatic nucleus of a variety of mammals including primates.

The presence of parvocellular vasopressin- and neurophysin-containing neurons in the suprachiasmatic nucleus (SCN) was investigated in 13 mammalian species representing six mammalian orders (marsupials, rodents, lagomorphs, artiodactyls, carnivores, and primates), using specific antisera to vasopressin and neurophysin in the unlabelled antibody=enzyme immunoperoxidase method. In all mammals examined, including man, parvocellular vasopressin and neurophysin neurons were found in the SCN. Only a portion of SCN neurons contain vasopressin and neurophysin, the number varying with species. Cell counts comparing the number of immunoreactive to Nissl-stained neurons showed averages of 17% immunopositive neurons in the rat SCN, and 31% in the human SCN. No oxytocin-containing SCN neurons were observed. These findings suggest that parvocellular vasopressin and neurophysin neurons are widely represented in mammals.

Evidence for subdivisions in the human suprachiasmatic nucleus.

The human suprachiasmatic nucleus was analysed by immunohistochemical demonstration of various substances in combination with 3-dimensional computerized reconstruction and video overlay facilities. In the human, the suprachiasmatic nucleus is not as compact as in the rodent. Its boundaries are not easily delineated using conventional stains, and it shows no obvious cytoarchitectonic structure. However, based on its chemoarchitecture, the human suprachiasmatic nucleus can be apportioned into five major subdivisions: Dorsal, comprising a crescent shaped mass of densely packed neurophysin/vasopressin-neurons as well as neurotensin-neurons, and also containing 3-fucosyl-N-acetyl-lactosamine (FAL)-positive neurons in its medial part. Central, occupying the core of the nucleus and consisting precisely of a region devoid of neurophysin/vasopressin neurons but demarcated by calbindin, synaptophysin, and a circumscribed cluster of vasoactive intestinal polypeptide-neurons and containing neurotensin neurons as well. Anteroventrally this division also contains some intermingled neurons positive for neurotensin, neuropeptide Y, somatostatin, and FAL. Ventral, extending from the anterior extreme of the preoptic recess caudolaterally to a field between the optic chiasm and the anteroventral margin of the supraoptic nucleus. This subdivision is specified by synaptophysin, calbindin, and substance P immunoreactivity and is almost free of glial fibrillary acidic protein. From its rostral portion, fibers immunoreactive for calbindin, vasoactive intestinal polypeptide, synaptophysin, and substance P protrude deeply into the optic chiasm. Medial, comprising a thin band between the subependymal zone and the dorsal subdivision, containing scattered somatostatin neurons. External, extending as a band around the dorsal and lateral borders of the nucleus, containing astrocytes expressing the FAL-epitope and scattered neurophysin/vasopressin and neurotensin neurons. These findings indicate that the human suprachiasmatic nucleus contains well-defined subdivisions with different, chemically specific, connections and provides a basis for comparing these subdivisions with the structure and function of subdivisions previously described for the suprachiasmatic nucleus in experimental animals. In addition, the findings strengthen the concept that the human suprachiasmatic nucleus generates and expresses circadian rhythms in a manner similar to that documented for the suprachiasmatic nucleus in experimental animals, and suggest that different subdivisions may subserve specific functional roles.

Developmental organization of neurophysin neurons in the human brain.

Neurophysin (NPH) was detected immunohistochemically in 34 human brains ranging in age from 10 weeks of gestation (wg) to 3 months postnatal. Weakly-stained NPH-immunoreactive (NPH-IR) cells were already aggregated in the lateral hypothalamus in the supraoptic nucleus at 10 wg, the first time point examined. From this time, there was a clear and consistent chronology in the first appearance of NPH-immunoreactivity in different cell groups progressing from the supraoptic nucleus at 10 wg to cells in the accessory NPH cell group at 13 wg, paraventricular nucleus at 14 wg, suprachiasmatic nucleus at 18 wg and various other well defined clusters in the basal forebrain at 18-20 wg. NPH-IR fibers were present in the hypothalamo-hypophyseal tract from 10 wg, and together with other available evidence, our findings suggest the presence of a potentially functional hypothalamohypophyseal system by the end of the first trimester. NPH staining patterns and orientations of cells suggest that NPH-IR cells originate from the region of the hypothalamic sulcus in a manner consistent with animal studies, and migrate to their settling areas before expressing NPH-immunoreactivity. In spite of the likelihood that most NPH-IR cells (with the probable exception of those in the suprachiasmatic nucleus) derive from a single primordium, the final organization of NPH-IR cells consists of many scattered groups, as seen in the late fetal period and mature brain. Developmental analysis provides further evidence that there is a high degree of conservation in the topographic organization of the numerous diverse NPH-IR cell groups in humans and other mammals, suggesting that the separation and organization of these groups may be of functional importance.

Naloxone reversible inhibition of reticular neurones in the rat caudal medulla produced by electrical stimulation of the periaqueductal grey matter.

Chronic dorsal periaqueductal grey matter electrodes were implanted into adult rats under pentobarbitone anaesthesia. Stimulating these electrodes (25-300 microA) produced behavioural analgesia in 23 of 44 rats tested. In rats given the opiate antagonist naloxone attenuation of this analgesia was seen. In 14 rats displaying behavioural analgesia to periaqueductal grey matter stimulation acute electrophysiological experiments were performed under urethane anaesthesia. Microelectrode recordings were made from neurones, excited by noxious heat or pinch applied to the limbs and tail, and located in the reticular formation of the caudal medulla. Stimulation of the periaqueductal grey matter at an intensity sufficient to produce analgesia in the conscious animal produced direct inhibition of the firing of 62% of neurones tested, excited 23%, had no effect on 14% and attenuated the nociceptive responses of 66%. The inhibitions were characteristically long. Local application of naloxone by microiontophoresis attenuated these long inhibitions in 11 out of 16 neurons tested. Immunohistochemical localization of beta-endorphin containing structures in the vicinity of stimulating and recording sites suggested that the naloxone sensitive inhibition of nociceptive neuronal responses in caudal medulla reticular formation may be due to activation of beta-endorphin fibres descending through the periaqueductal area to the caudal medulla.

Vasopressin- and neurophysin-immunoreactive neurons in the septal region, medial amygdala and locus coeruleus in colchicine-treated rats.

The distribution and morphology of neurons containing vasopressin, oxytocin and their associated neurophysins were examined immunohistochemically in rats given intracerebroventricular injections of colchicine. Under these conditions, numerous neurons containing vasopressin and neurophysin were found in several brain areas in addition to those previously described in the hypothalamus. Individual parvocellular vasopressin neurons were scattered in the medial and lateral septum and vertical limb of the nucleus of the diagonal band, while a large number of such neurons were found throughout both the bed nucleus of the stria terminals and the dorsal portion of the medial amygdala. In addition a small cluster of parvocellular vasopressin neurons was present adjacent to the top of the third ventricle in the posterior dorsal hypothalamic area and a number of such neurons were found in the ventral locus coeruleus and sub coeruleus. The mean diameters of these parvocellular vasopressin neurons ranged from 16.6 to 19.8 micron in the different regions, in contrast to the 25.4 micron mean diameter of hypothalamic magnocellular vasopressin neurons, or the 13.7 micron mean diameter of parvocellular vasopressin neurons in the suprachiasmatic nucleus. No vasopressin neurons were found in other brain and spinal cord regions under the conditions used in this study, although all regions were examined. No oxytocin neurons other than those previously described in the hypothalamus and immediately contiguous regions were found. Measurement of the mean diameter of oxytocin neurons showed that neurons in the caudal paraventricular nucleus were clearly smaller (18.9 micron) than magnocellular oxytocin neurons (24.8 micron) in other parts of the hypothalamus. These parvocellular oxytocin neurons with experimentally documented central connections were similar in both size and appearance to the parvocellular vasopressin neurons seen after colchicine treatment, which are potential sources of certain central vasopressin pathways. These findings indicate that there are at least two types of oxytocin neurons in the hypothalamus and several types of vasopressin neurons in a variety of different areas in the brain, many of which are outside of the hypothalamus.

Ciliary neurotrophic factor supports p75NGFR-immunoreactive non-cholinergic, but not cholinergic, developing septal neurons in vitro.

Ciliary neurotrophic factor is known to exert both survival and differentiative actions on a number of neuronal populations of the peripheral and central nervous systems. In this study we have compared the trophic effects of ciliary neurotrophic factor and nerve growth factor on developing septal neurons of the rat in vitro. Fetal septal neurons were grown in vitro under glass coverslips in sandwich culture. Septal cultures grown for 14 days in the continual presence of nerve growth factor contain a population of cholinergic neurons that stain intensely for the low-affinity nerve growth factor receptor (p75NGFR), choline acetyltransferase and acetylcholinesterase. Without added nerve growth factor, few neurons stain for these markers. Ciliary neurotrophic factor addition for 14 days from plating in the absence of exogenous nerve growth factor results in the appearance of a population of neurons that stains for p75NGFR. This population is similar in number to that seen in nerve growth factor-treated cultures but is not immunoreactive for choline acetyltransferase and is significantly smaller in mean cross-sectional area. Delayed addition of nerve growth factor to ciliary neurotrophic factor-supported cultures at 14 days for a further seven days fails to induce choline acetyltransferase immunoreactivity in these p75NGFR-positive septal neurons. In cultures grown in the continual presence of nerve growth factor from plating, removal of nerve growth factor and addition of nerve growth factor antibodies at 14 days results in the death of over 80% of the cholinergic neurons after a further four days. Addition of ciliary neurotrophic factor during the period of nerve growth factor withdrawal appears to preserve a p75NGFR-positive, choline acetyltransferase-negative neuronal population. However, seven day re-addition of nerve growth factor to ciliary neurotrophic factor-treated, nerve growth factor-withdrawn cultures fails to induce choline acetyltransferase immunoreactivity in the ciliary neurotrophic factor-supported p75NGFR-positive septal neurons. Simultaneous treatment of cultures with both ciliary neurotrophic factor and nerve growth factor for 14 days from plating approximately doubles the number of p75NGFR-positive neurons relative to cultures treated with either ciliary neurotrophic factor or nerve growth factor alone, but the number of choline acetyltransferase-positive neurons in these cultures is not significantly greater than that found in cultures treated solely with nerve growth factor. These results suggest that ciliary neurotrophic factor does not support the survival and differentiation of developing septal cholinergic neurons in vitro, but can support the development of a p75NGFR-immunoreactive population of non-cholinergic septal neurons.

Increased vulnerability of septal cholinergic neurons to partial loss of target neurons in aged rats.

To investigate whether the ageing process might affect neuron-target interactions which influence the phenotype of septal cholinergic neurons, we compared the response of these neurons to partial loss of target tissue in young adult and aged animals. Groups of young adult (four to six months) or aged (24-33 months) male Sprague-Dawley rats received unilateral infusions into the hippocampus of either the excitotoxic amino acid N-methyl-D-aspartate, or vehicle. The resulting excitotoxic lesions reduced the mean cross-sectional area of the hippocampus by 55-60%. Ipsilateral septal cholinergic neurons immunohistochemically stained for either choline acetyltransferase or low-affinity neurotrophin receptor (p75NTR) were morphometrically evaluated. In young adult rats with partial hippocampal lesions, the number and staining intensity of ipsilateral septal cholinergic neurons were not significantly different from age-matched control values, but these cholinergic neurons exhibited a significant 12% reduction in cross-sectional area. In aged rats with hippocampal lesions of equivalent size, ipsilateral cholinergic neurons showed a significant 29% reduction in cross-sectional area, a significant 19% reduction in choline acetyltransferase staining intensity as measured by densitometry, and a significant 21% reduction in the number of choline acetyltransferase- but not p75NTR-stained septal neurons, as compared with age-matched control animals. These findings show that in aged rats, septal cholinergic neurons atrophy more severely in response to the partial loss of their target neurons than in young adult rats, in the form of pronounced cell shrinkage and down-regulation of intracellular levels of the transmitter-synthesizing enzyme, choline acetyltransferase, in some cases to the point of the absence of detectable staining for this marker in some cells. The continued detection of p75NTR indicates that significant neuronal cell death did not take place. These findings suggest that basal forebrain cholinergic neurons have an increased vulnerability to disturbances of neuron-target interactions in aged animals, which may contribute to the degenerative changes exhibited by these cholinergic neurons in ageing and age-related conditions such as Alzheimer's disease.