Anna Itzik

Neural progenitors from human embryonic stem cells

The derivation of neural progenitor cells from human embryonic stem (ES) cells is of value both in the study of early human neurogenesis and in the creation of an unlimited source of donor cells for neural transplantation therapy. Here we report the generation of enriched and expandable preparations of proliferating neural progenitors from human ES cells. The neural progenitors could differentiate in vitro into the three neural lineages—astrocytes, oligodendrocytes, and mature neurons. When human neural progenitors were transplanted into the ventricles of newborn mouse brains, they incorporated in large numbers into the host brain parenchyma, demonstrated widespread distribution, and differentiated into progeny of the three neural lineages. The transplanted cells migrated along established brain migratory tracks in the host brain and differentiated in a region-specific manner, indicating that they could respond to local cues and participate in the processes of host brain development. Our observations set the stage for future developments that may allow the use of human ES cells for the treatment of neurological disorders.

Transplantation of Human Embryonic Stem Cell–Derived Neural Progenitors Improves Behavioral Deficit in Parkinsonian Rats

Human embryonic stem cells (hESCs) may potentially serve as a renewable source of cells for transplantation. In Parkinsons disease, hESC‐derived dopaminergic (DA) neurons may replace the degenerated neurons in the brain. Here, we generated highly enriched cultures of neural progenitors from hESCs and grafted the progenitors into the striatum of Parkinsonian rats. The grafts survived for at least 12 weeks, the transplanted cells stopped proliferating, and teratomas were not observed. The grafted cells differentiated in vivo into DA neurons, though at a low prevalence similar to that observed following spontaneous differentiation in vitro. Transplanted rats exhibited a significant partial correction of D‐amphetamine and apomorphine‐induced rotational behavior, along with a significant improvement in stepping and placing non‐pharmacological behavioral tests. While transplantation of uncommitted hESC‐derived neural progenitors induced partial behavioral recovery, our data indicate that the host‐lesioned striatum could not direct the transplanted neural progenitors to acquire a dopaminergic fate. Hence, induction of their differentiation toward a midbrain fate prior to transplantation is probably required for complete correction of behavioral deficit. Our observations encourage further developments for the potential use of hESCs in the treatment of Parkinsons disease.

Differential effect of amygdaloid lesions on CRF-41, ACTH and corticosterone responses following neural stimuli

The effect of amygdaloid (AMG) lesions on changes in median eminence (ME) CRF-41 and serum ACTH and corticosterone (CS) levels following neural stimuli were investigated in rats. In intact animals photic or acoustic stimuli caused CRF-41 depletion from the ME and a rise in serum ACTH and CS levels. In rats with medial or central AMG nuclei lesions, these responses were blocked. Basal AMG lesions were not effective. Also, all groups of animals responded normally to ether stress. These results indicate a differential effect of AMG nuclei on the hypothalamo-pituitary-adrenocortical (HPA) axis and demonstrate that the facilitatory effect of the AMG nuclei on the HPA axis responses involves the release of ME CRF-41, which stimulates ACTH and consequently CS secretion.

The Amygdala Regulates the Pituitary-Adrenocortical Response and Release of Hypothalamic Serotonin following Electrical Stimulation of the Dorsal Raphe Nucleus in the Rat

The amygdala is known to modulate the function of the hypothalamo-pituitary-adrenocortical (HPA) axis, but the mechanism of this effect is still not clear. In the present study we examined the specific role of the serotonin (5-HT) system in mediating the effect of the amygdala on the activity of the HPA axis. Bilateral lesions of the amygdala in rats reduced the adrenocorticotropin (ACTH) and corticosterone responses to electrical stimulation of the dorsal raphe nucleus, where the cell bodies of serotonergic neurons are located. Amygdala lesions had no effect on the ACTH and corticosterone responses to administration of a 5-HT1A receptor agonist directly into the paraventricular nucleus (PVN) of the hypothalamus, indicating that there was no impairment in the activity of postsynaptic 5-HT1A receptors in the hypothalamus. In vivo microdialysis showed that amygdala lesions markedly attenuated the effect of electrical stimulation of the dorsal raphe to increase extracellular secretion of 5-HT in the PVN. This is the first demonstration that the amygdala has a facilitatory effect on the function of dorsal raphe 5-HT neurons which project to the PVN, and suggests a mechanism by which the amygdala may modulate the function of the HPA axis.

A novel permissive role for glucocorticoids in induction of febrile and behavioral signs of experimental herpes simplex virus encephalitis

Herpes simplex virus type 1 (HSV-1) encephalitis may present with fever and behavioral changes, to the extent of a psychotic state and psychomotor agitation. We developed a clinically relevant experimental model of HSV-1 encephalitis and investigated host brain responses associated with its clinical signs and whether these responses depend on the presence of circulating glucocorticoids. Intracerebral inoculation of HSV-1 in rats induced fever, motor hyperactivity and aggressive behavior. In adrenalectomized rats HSV-1 failed to induce these signs, although mortality rate was identical to sham-operated rats. Hypophysectomy or blocking glucocorticoid receptors also prevented HSV-1-induced fever. Dexamethasone replacement therapy to adrenalectomized rats restored the HSV-1-induced fever and behavioral abnormalities. HSV-1 inoculation produced hyperproduction of prostaglandin E(2) by brain slices. This effect was abolished in adrenalectomized rats and was restored by dexamethasone treatment. In intact rats HSV-1 induced brain interleukin-1beta (IL-1beta) gene expression. Adrenalectomy alone caused brain IL-1beta expression, which did not increase after HSV-1 infection. Similarly, HSV-1 induced IL-1beta expression in astrocyte cultures. Removal of cortisol from the culture medium caused basal IL-1beta mRNA expression which was not increased by infection. In conclusion, fever, motor hyperactivity and aggressive behavior during experimental HSV-1 encephalitis are dependent on brain responses, including prostaglandin E(2) and IL-1beta synthesis. Circulating glucocorticoids play an essential permissive role in the induction of these host brain responses.

Effects of HSV-1, a neurotropic virus, on the hypothalamic-pituitary-adrenocortical axis in rats

It is well established that the hypothalamic-pituitary-adrenocortical (HPA) axis is activated during systemic viral diseases. In this study we examined the effects of a neurotropic virus, herpes simplex virus type 1 (HSV-1), on the HPA axis in male rats. Following corneal inoculation with HSV-1, the virus invaded the nervous system and replicated in the brainstem without clinical signs of disease. During this asymptomatic brainstem infection with HSV-1, significant changes were found in the function of the HPA axis: On days 3, 7 and 14 post-infection (p.i.) basal ACTH and corticosterone (CS) levels were markedly elevated, and photic stressful stimulation failed to further increase the levels of these hormones. In addition, the elevated basal serum levels of ACTH and CS could not be suppressed by pretreatment with dexamethasone. The content of CRF-41 in the paraventricular nucleus of the hypothalamus and in the median eminence measured at 6 days p.i. was similar to that of vehicle inoculated rats. By 4 weeks p.i. the basal levels of ACTH and CS returned to normal and these animals responded to photic stimulation and dexamethasone similar to vehicle inoculated rats. Systemic (intraperitoneal) inoculation of HSV-1 did not induce any changes in the HPA axis responses. We therefore suggest that asymptomatic acute infection of the brainstem with HSV-1 may affect brain regions involved in the regulation of the HPA axis, and that those effects are mediated centrally and not by a systemic mechanism.

The Role of Limbic Structures in the Modulation of ACTH Responses following Adrenalectomya

The role of the HIPP, DF, and the lateral SPT as well as of the central AMG nucleus in ACTH hypersecretion following Adex, was studied in male rats. In animals with bilateral dorsal hippocampectomy, DF section, or SPT lesions there was a much greater increase in ACTH hypersecretion when compared to Adex alone. Implants of CS in the paraventricular nucleus of the hypothalamus prevented the rise in serum ACTH following Adex, and this effect was reversed by hippocampectomy. Bilateral lesions in the AMG prevented the Adex-induced rise of serum ACTH. These results indicate that the dorsal HIPP and its efferent pathways to the hypothalamus have normally an inhibitory effect on Adex-induced ACTH secretion. Their removal permits an elevated ACTH hypersecretion as well as attenuation of the CS feedback effect. The central AMG nucleus, which has a facilitatory effect on the hypothalamopituitary-adrenocortical axis, can also modulate ACTH secretion following Adex.

Evidence for a mutual interaction between noradrenergic and serotonergic agonists in stimulation of ACTH and corticosterone secretion in the rat.

We investigated the mutual interactions between hypothalamic norepinephrine (NE) and serotonin (5-HT) in mediating the ACTH and corticosterone responses to direct stimulation of the paraventricular nucleus (PVN) with adrenergic and serotonergic agonists. The hormone responses to the intrahypothalamic injection of the alpha1-adrenergic agonist phenylephrine (20 nmol/2 microl) were significantly reduced by prior depletion of hypothalamic 5-HT with intra-PVN injection of the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT), but not after depletion of hypothalamic NE by intra-PVN injection of the noradrenergic neurotoxin 6-hydroxydopamine (6-OHDA). The ACTH and corticosterone responses to intrahypothalamic injection of the 5-HT(1A) receptor agonist 8-OH-DPAT (20 n mol/2 microl) were significantly reduced by depletion of hypothalamic NE with 6-OHDA, but not after depletion of hypothalamic 5-HT with 5,7-DHT. These mutual interactions between the NE and 5-HT neuronal systems, which innervate the PVN, may explain previous findings of equivalent reductions in the hypothalamic-pituitary-adrenal axis responses to neural stimulation after neurotoxic lesioning of either the NE or 5-HT systems.

Glucocorticoid Resistance following Herpes Simplex-1 Infection: Role of Hippocampal Glucocorticoid Receptors

Herpes simplex-1 (HSV-1) is a sporadic cause of viral encephalitis. We have previously demonstrated that corneal HSV inoculation markedly activates the hypothalamo-pituitary-adrenal (HPA) axis. This activation depends on host derived brain interleukine-1 and was resistant to pretreatment with dexamethasone (dex), possibly because immune factors such as pro-inflammatory cytokines can modify the binding capacity of glucocorticoids in the hippocampus. In the present study, we examined whether resistance of the HPA axis activation following intracerebral HSV-1 infection to dex-induced suppression is associated with modifications in hippocampal or pituitary glucocorticoids (GC) receptors or GC receptors in cultured astrocytes. Male rats were injected intracerebroventricularly with purified HSV-1 or vehicle. 48 h later, dex or vehicle was injected intraperitoneally. Rats were sacrificed 3.5 h later. ACTH and corticosterone (CS) were measured in the serum. Specific binding of 3H-dex was measured in the cytosolic fraction of the hippocampus and the pituitary. Dex failed to reduce ACTH and CS responses to HSV-1 infection. In contrast, dex significantly reduced ACTH and CS responses to acoustic neural stimuli. Infection with HSV-1 markedly reduced the hippocampal maximal specific binding of dex with no effect on the dissociation constant (Kd) values. HSV-1 had no effect on the binding of dex in the pituitary. Infection of cultured astrocytes with HSV-1 also reduced the maximal specific binding of dex, but increased the Kd value. The results suggest that HSV-1 induced GC resistance may be mediated by downregulation of GC receptors in hippocampal tissue. These results may clarify a mechanism responsible for GC resistance following immune challenges.

Acute Effects of Purified and UV-Inactivated Herpes simplex Virus Type 1 on the Hypothalamo-Pituitary-Adrenocortical Axis

Herpes simplex virus type 1 (HSV-1) is a common cause of viral encephalitis, manifested by neuroendocrine and behavioral changes. We have previously demonstrated that HSV-1 induces marked hypothalamo-pituitary-adrenocortical (HPA) axis activation. In this study we characterized the acute effects of HSV-1 on the HPA axis occurring before viral replication and appearance of clinical signs of encephalitis. Since in previous studies we used crude virus preparations which may contain immune factors produced by the infected cells, we tested here the effects of purified HSV-1 virions. HSV-1 was propagated on Vero cells and virions were purified by centrifugation in sucrose gradients. Inactivation of viral infectivity was achieved by UV-irradiation, which caused a million-fold decrease in virus titer, as determined by plaque assay. Intracerebroventricular (ICV) inoculation of crude or purified virions induced a dose dependent increase in serum corticosterone and corticotropin (ACTH). This effect was maximal within 3.5 h postinfection and lasted for 72 h. ICV inoculation of UV-inactivated purified virions caused a marked increase in serum corticosterone and ACTH at 3.5 h, but in contrast to the effect of the active virus, the hormone levels gradually decreased at 24 h, and returned to basal levels at 72 h postinfection. HSV-1-induced HPA axis activation at 3.5 h was completely abolished by pretreatment with interleukin-1 receptor antagonist, injected ICV. Adrenalectomized rats failed to respond to ICV inoculation of purified HSV-1 by increase in ACTH. In contrast, these rats responded to ICV injection of LPS. In conclusion: (1) HSV-1 can acutely activate the HPA axis before and independently of any viral replication; (2) HSV-1-induced HPA axis activation depends on a permissive action of circulating glucocorticoids and on host derived brain interleukin-1.