CNTF receptor alpha is expressed by magnocellular neurons and expression is upregulated in the rat supraoptic nucleus during axonal sprouting.

Ciliary neurotrophic factor (CNTF) is expressed by glial cells at multiple levels of the magnocellular neurosecretory system (MNS). CNTF is present in astrocytes in the hypothalamic supraoptic nucleus (SON) as well as in perivascular cells in the neurohypophysis, and a several fold increase in CNTF immunoreactivity occurs in the SON following either axotomy of magnocellular neurons or during axonal sprouting by intact magnocellular neurons. CNTF also promotes survival and stimulates process outgrowth from magnocellular neurons in vitro. While these findings suggest that CNTF may act as a growth factor in support of neuronal plasticity in the MNS, little is known regarding possible expression of receptors for CNTF in the MNS. We have therefore used immunocytochemistry and in situ hybridization to examine the expression of CNTF receptor alpha (CNTFRalpha) in the rat MNS. Robust immunoreactivity for CNTFRalpha was observed associated with oxytocinergic and vasopressinergic neurons distributed throughout the SON. Astrocytes located within the ventral glial lamina (VGL) of the SON were also immunoreactive for CNTFRalpha. Robust hybridization of an anti-sense [(35)S]-cRNA probe to CNTFRalpha mRNA was observed throughout the SON, while binding of a control sense probe was much lower. Grains were found clustered predominantly over neuronal somata, indicative of expression by magnocellular neurons within the SON. We next examined changes in expression of CNTFRalpha mRNA by magnocellular neurons 7 days following unilateral transection of the hypothalamo-neurohypophysial tract. The level of CNTFRalpha mRNA was increased 32% (compared to age-matched intact controls; p<0.05) in magnocellular neurons in the SON contralateral to the lesion, which are undergoing extensive collateral axonal sprouting, but was unchanged in axotomized magnocellular neurons in the SON ipsilateral to the lesion. These findings suggest that CNTF produced by MNS glia and acting via CNTFRalpha may exert neurotrophic effects on magnocellular neurons.

Watt JA ，Lo D ，Cranston HJ ，Paden CM ... -  《-》

Neuronal activity and axonal sprouting differentially regulate CNTF and CNTF receptor complex in the rat supraoptic nucleus.

We demonstrated previously that the hypothalamic supraoptic nucleus (SON) undergoes a robust axonal sprouting response following unilateral transection of the hypothalamo-neurohypophysial tract. Concomitant with this response is an increase in ciliary neurotrophic factor (CNTF) and CNTF receptor alpha (CNTFRα) expression in the contralateral non-uninjured SON from which the axonal outgrowth occurs. While these findings suggest that CNTF may act as a growth factor in support of neuronal plasticity in the SON, it remained to be determined if the observed increase in neurotrophin expression was related to the sprouting response per se or more generally to the increased neurosecretory activity associated with the post-lesion response. Therefore we used immunocytochemistry and Western blot analysis to examine the expression of CNTF and the components of the CNTF receptor complex in sprouting versus osmotically-stimulated SON. Western blot analysis revealed a significant increase in CNTF, CNTFRα, and gp130, but not LIFRß, protein levels in the sprouting SON at 10days post lesion in the absence of neuronal loss. In contrast, osmotic stimulation of neurosecretory activity in the absence of injury resulted in a significant decrease in CNTF protein levels with no change in CNTFRα, gp130, or LIFRß protein levels. Immunocytochemical analysis further demonstrated gp130 localization on magnocellular neurons and astrocytes while the LIFRß receptor was found only on astrocytes in the SON. These results are consistent with the hypothesis that increased CNTF and CNTFR complex in the sprouting, metabolically active SON are related directly to the sprouting response and not the increase in neurosecretory activity.

Askvig JM ，Leiphon LJ ，Watt JA 《-》

Ciliary neurotrophic factor is expressed in the magnocellular neurosecretory system of the rat in vivo: evidence for injury- and activity-induced upregulation.

Although ciliary neurotrophic factor (CNTF) has been shown to promote the survival of magnocellular neurons when applied exogenously to explants of the paraventricular and supraoptic nuclei (SON) in vitro, little is known regarding its expression or regulation in the adult magnocellular neurosecretory system (MNS) following injury in vivo. Therefore, we utilized in situ hybridization and immunocytochemical analysis in conjunction with quantitative optical densitometric analysis to identify the cellular source of CNTF and examine the temporal pattern of its expression, following unilateral transection of the hypothalamo-neurohypophysial tract in the adult rat. In intact rats, CNTF immunoreactivity (CNTF-ir) was predominantly localized within identified astrocytes within the ventral glial limitans subjacent to the SON. Quantitative optical densitometric analysis of CNTF-ir levels in the axotomized SON demonstrated that the proportional area of CNTF-ir was significantly elevated between 3 and 30 days following injury. A significant but more limited increase was also observed in the non-injured contralateral SON. In situ hybridization confirmed the expression and upregulation of CNTF in the axotomized SON. These results demonstrate the expression of CNTF in the adult rodent MNS in vivo and provide evidence that levels of CNTF are upregulated in response to both direct injury, and heightened metabolic activity, within the lesioned and sprouting SON, respectively.

Watt JA ，Bone S ，Pressler M ，Cranston HJ ，Paden CM ... -  《EXPERIMENTAL NEUROLOGY》

Absence of axonal sprouting following unilateral lesion in 125-day-old rat supraoptic nucleus may be due to age-dependent decrease in protein levels of ciliary neurotrophic factor receptor alpha.

Within the supraoptic nucleus (SON) of a 35-day-old rat, we previously demonstrated a collateral sprouting response that reinnervates the partially denervated neural lobe (NL) after unilateral lesion of the hypothalamo-neurohypophysial tract. Others have shown a decreased propensity for axonal sprouting in an aged brain; therefore, to see if the SON exhibits a decreased propensity for axonal sprouting as the animal ages, we performed a unilateral lesion in the 125-day-old rat SON. Ultrastructural analysis of axon profiles in the NL of the 125-day-old rat demonstrated an absence of axonal sprouting following injury. We previously demonstrated that ciliary neurotrophic factor (CNTF) promotes process outgrowth from injured magnocellular neuron axons in vitro. Thus, we hypothesized that the lack of axonal sprouting in the 125-day-old rat SON may be due to a reduction in CNTF or the CNTF receptor components. To this point, we found that as the rat ages there is significantly less CNTF receptor alpha (CNTFRα) protein in the uninjured, 125-day-old rat compared to the uninjured, 35-day-old rat. We also observed that protein levels of CNTF and the CNTF receptor components were increased in the SON and NL following injury in the 35-day-old rat, but there was no difference in the protein levels in the 125-day-old rat. Altogether, the results presented herein demonstrate that the plasticity within the SON is highly dependent on the age of the rat, and that a decrease in CNTFRα protein levels in the 125-day-old rat may contribute to the loss of axonal sprouting following axotomy.

Askvig JM ，Watt JA 《-》

Inhibition of the Jak-STAT pathway prevents CNTF-mediated survival of axotomized oxytocinergic magnocellular neurons in organotypic cultures of the rat supraoptic nucleus.

Previous studies have demonstrated that ciliary neurotrophic factor (CNTF) enhances survival and process outgrowth from magnocellular neurons in the paraventricular (PVN) and the supraoptic (SON) nuclei. However, the mechanisms by which CNTF facilitates these processes remain to be determined. Therefore, the aim of this study was to identify the immediate signal transduction events that occur within the rat SON following administration of exogenous rat recombinant CNTF (rrCNTF) and to determine the contribution of those intracellular signaling pathway(s) to neuronal survival and process outgrowth, respectively. Immunohistochemical and Western blot analyses demonstrated that axonal injury and acute unilateral pressure injection of 100 ng/μl of rrCNTF directly over the rat SON resulted in a rapid and transient increase in phosphorylated-STAT3 (pSTAT3) in astrocytes but not neurons in the SON in vivo. Utilizing rat hypothalamic organotypic explant cultures, we then demonstrated that administration of 25 ng/ml rrCNTF for 14days significantly increased the survival and process outgrowth of OT magnocellular neurons. In addition, pharmacological inhibition of the Jak-STAT pathway via AG490 and cucurbitacin I significantly reduced the survival of OT magnocellular neurons in the SON and PVN; however, the contribution of the Jak-STAT pathway to CNTF-mediated process outgrowth remains to be determined. Together, these data indicate that CNTF-induced survival of OT magnocellular neurons is mediated indirectly through astrocytes via the Jak-STAT signaling pathway.

Askvig JM ，Lo DY ，Sudbeck AW ，Behm KE ，Leiphon LJ ，Watt JA ... -  《-》