Intraspinal transplantation of subventricular zone-derived neural progenitor cells improves phrenic motor output after high cervical spinal cord injury.

Following spinal cord injury (SCI), intraspinal transplantation of neural progenitor cells (NPCs) harvested from the forebrain sub-ventricular zone (SVZ) can improve locomotor outcomes. Cervical SCI often results in respiratory-related impairments, and here we used an established model cervical SCI (C2 hemisection, C2Hx) to confirm the feasibility of mid-cervical transplantation of SVZ-derived NPCs and the hypothesis that that this procedure would improve spontaneous respiratory motor recovery. NPCs were isolated from the SVZ of enhanced green fluorescent protein (GFP) expressing neonatal rats, and then intraspinally delivered immediately caudal to an acute C2Hx lesion in adult non-GFP rats. Whole body plethysmography conducted at 4 and 8wks post-transplant demonstrated increased inspiratory tidal volume in SVZ vs. sham transplants during hypoxic (P=0.003) or hypercapnic respiratory challenge (P=0.019). Phrenic nerve output was assessed at 8wks post-transplant; burst amplitude recorded ipsilateral to C2Hx was greater in SVZ vs. sham rats across a wide range of conditions (e.g., quiet breathing through maximal chemoreceptor stimulation; P<0.001). Stereological analyses at 8wks post-injury indicated survival of ~50% of transplanted NPCs with ~90% of cells distributed in ipsilateral white matter at or near the injection site. Peak inspiratory phrenic bursting after NPC transplant was positively correlated with the total number of surviving cells (P<0.001). Immunohistochemistry confirmed an astrocytic phenotype in a subset of the transplanted cells with no evidence for neuronal differentiation. We conclude that intraspinal transplantation of SVZ-derived NPCs can improve respiratory recovery following high cervical SCI.

Sustained AAV-mediated overexpression of CRF in the central amygdala diminishes the depressive-like state associated with nicotine withdrawal.

Smoking cessation leads to a dysphoric state and this increases the risk for relapse. Animal studies indicate that the dysphoric state associated with nicotine withdrawal is at least partly mediated by an increase in corticotropin-releasing factor (CRF) release in the central nucleus of the amygdala (CeA). In the present study, we investigated whether a sustained overexpression of CRF in the CeA affects the dysphoric-like state associated with nicotine withdrawal. To study brain reward function, rats were prepared with intracranial self-stimulation (ICSS) electrodes in the medial forebrain bundle. An adeno-associated virus (AAV, pseudotype 2/5) was used to overexpress CRF or green fluorescent protein (GFP, control) in the CeA and minipumps were used to induce nicotine dependence. The AAV2/5-CRF vector induced a 40% increase in CRF protein and mRNA levels in the CeA. Administration of the nicotinic receptor antagonist mecamylamine (precipitated withdrawal) or nicotine pump removal (spontaneous withdrawal) led to elevations in ICSS thresholds. Elevations in ICSS thresholds are indicative of a dysphoric-like state. The overexpression of CRF did not affect baseline ICSS thresholds but diminished the elevations in ICSS thresholds associated with precipitated and spontaneous nicotine withdrawal. The real-time reverse transcriptase (RT)-PCR analysis showed that the overexpression of CRF led to a decrease in CRF1 mRNA levels and an increase in CRF2 mRNA levels in the CeA. In conclusion, the overexpression of CRF in the CeA diminishes the dysphoric-like state associated with nicotine withdrawal and this might be driven by neuroadaptive changes in CRF1 and CRF2 receptor gene expression.

Estradiol enhances neurogenesis in the dentate gyri of adult male meadow voles by increasing the survival of young granule neurons.

This study investigated whether estradiol influenced the survival of new granule neurons, independent of altering cell proliferation, in the adult rodent dentate gyrus and whether estradiol-induced changes in new neuron number relate to any observed changes in hippocampus-dependent behavior. To test whether estradiol specifically promotes the survival of new neurons we injected castrated adult male meadow voles with the cell synthesis marker bromodeoxyuridine (BrdU; 50 mg/kg) twice on day 0 and then injected either estradiol (10 microg) or vehicle for 5 consecutive days either over days 1-5, days 6-10 or days 11-15 and perfused them on day 16. Estradiol doubled the number of hippocampal BrdU-labeled neurons but only when administered during a discrete period (days 6-10; P< or =0.01) when most new neurons extend their axons [J Comp Neurol 413 (1999) 146]. To test whether the estradiol-induced increase in new neuron number was related to hippocampus-dependent behavior, males were injected with BrdU twice on day 0 and with estradiol or vehicle over days 6-10 before standard Morris water maze training commenced on day 16, 5 days after the final hormone injection. As in the first study, estradiol-treated males had more BrdU-labeled cells than vehicle-treated males. On a probe trial, estradiol-treated males spent significantly more time in the training quadrant than vehicle-treated males despite similar performance between groups during acquisition and reversal training trials. Thus estradiol enhanced the survival of young neurons but only when administered during their 'axon extension' phase and this effect was related to better spatial memory in male voles.

N-methyl-D-aspartate receptor activity and estradiol: separate regulation of cell proliferation in the dentate gyrus of adult female meadow vole.

We have previously found that estradiol increases (within 4 h) but then decreases (within 48 h) cell proliferation in the dentate gyrus of adult female ovariectomized (OVX) rats and of intact meadow voles and that estradiol partially stimulates adrenal activity to suppress cell proliferation in rats. Estradiol enhances N-methyl-D-aspartate receptor (NMDAr) activity and NMDAr activation suppresses cell proliferation in the adult rodent dentate gyrus. Therefore, we tested whether estradiol alters cell proliferation in the dentate gyrus of adult OVX female meadow voles by stimulating NMDAr activity. In experiment 1, OVX females were injected with estradiol (10 micro g) or oil and then with NMDA (30 mg/kg) or vehicle 3 h later and bromodeoxyuridine 4 h later (BrdU; 50 mg/kg). Voles were perfused 1 h after BrdU injection. Relative to oil vehicle, estradiol increased (P

Reproductive status influences the survival of new cells in the dentate gyrus of adult male meadow voles.

Reproductive status influences cell proliferation and the survival of new cells in the dentate gyrus of adult laboratory-reared and wild female meadow voles; reproductively inactive (RI) females have more proliferating cells and more labeled cells that survive 5 weeks vs. 2 h than reproductively active (RA) females. However, the effect of season has only been studied in a wild sample of male meadow voles and factors such as age and experience that have been shown to influence neurogenesis in the dentate gyrus of adult mammals cannot be controlled in a wild sample. Therefore, we investigated whether reproductive status regulates neurogenesis (cell proliferation and/or the survival of new cells) in the dentate gyrus of laboratory-reared adult male meadow voles so that confounding variables could be controlled. Males were acclimated to a short- or a long-photoperiod to simulate the non-breeding or breeding season, respectively, and reproductive status was verified by testes mass. The density of labeled cells was similar in RI and RA males (P=0.20) 2 h after an injection of bromodeoxyuridine (50 mg/kg) but the density of labeled cells was elevated in the RA males relative to the RI males 5 weeks after an injection of [(3)H]thymidine (5 microCi/g; P

Estradiol initially enhances but subsequently suppresses (via adrenal steroids) granule cell proliferation in the dentate gyrus of adult female rats.

In the dentate gyrus of adult female meadow voles, a high dose of estradiol benzoate (EB) increases (within 4 h) then decreases (within 48) the number of dividing progenitor cells (Ormerod BK, Galea LAM. 2001. Reproductive status regulates cell proliferation within the dentate gyrus of the adult female meadow vole: A possible regulatory role for estradiol. Neurosci 2:169-179). We investigated whether time-dependent EB exposure differentially influences the number of new granule cells produced in the adult female rat dentate gyrus and whether EB-stimulated adrenal activity mediates the decrease in cell proliferation. Ovariectomized rats received either an EB (10 microg in 0.1 mL) or vehicle (0.1 mL) injection either 4 or 48 h (Experiment 1) before a BrdU injection (200 mg/kg) and were perfused 24 h later to assess the number of new cells. Relative to vehicle, the number of new cells increased following a 4 h exposure (p < or = 0.04) but decreased following a 48 h exposure (p < or = 0.006) to EB. In Experiment 2, the number of new cells within the dentate gyrus of ovariectomized and adrenalectomized females did not significantly differ between groups exposed to EB versus vehicle for 48 h prior to BrdU administration, suggesting the decreased number of new cells observed within the dentate gyrus of adrenal-intact adult female rats is mediated by EB-stimulated adrenal activity. We conclude that estradiol dynamically regulates cell proliferation within the dentate gyrus of adult female rats in the time-dependent manner observed previously in voles and suppresses cell proliferation by influencing adrenal steroids. Investigating how estradiol dynamically regulates neurogenesis could provide insight into the mechanisms by which the proliferation of progenitor cells is controlled within the adult rodent hippocampus.

High levels of estradiol disrupt conditioned place preference learning, stimulus response learning and reference memory but have limited effects on working memory.

The present study investigated the effects of high levels of estradiol in female rats on four different radial arm maze tasks: the hippocampus-dependent spatial working-reference memory task; the prefrontal cortex-hippocampus dependent delayed win-shift task; the striatum-dependent cued win-stay task; and the amygdala-dependent conditioned place preference task. Ovariectomized female rats were injected daily with either 10 microg of estradiol benzoate or sesame oil vehicle approximately 4 h prior to testing. In Experiment 1, treatment with estradiol disrupted learning on the spatial working-reference memory task by increasing the number of reference memory errors to reach criterion. In Experiment 2, treatment with estradiol had no significant effect on the delayed win-shift task. In Experiment 3, treatment with estradiol resulted in impaired performance on a striatum-dependent cued win-stay task. In Experiment 4, treatment with estradiol impaired the acquisition of a conditioned place-preference task. Taken together these findings suggest that high levels of estradiol inhibit reference memory, stimulus response learning, and amygdala-dependent appetitive conditioning while having little effect on working memory.

Reproductive status influences cell proliferation and cell survival in the dentate gyrus of adult female meadow voles: a possible regulatory role for estradiol.

Galea and McEwen [Galea and McEwan (1999) Neuroscience 89, 955-964] found that cell proliferation was suppressed in female meadow voles trapped during the breeding season relative to females trapped during the non-breeding season. We investigated the effect of reproductive status and estradiol level on cell proliferation and cell survival in adult laboratory-reared female meadow voles to control for the variables of age, experience and pregnancy that could confound the results derived from a wild sample. Voles were housed in either a long- or short-photoperiod to simulate season and a male or female cage partner was introduced to influence reproductive status. Because females are reflex ovulators, exposure to a male rapidly induces behavioural estrous and high levels of estradiol. Forty-eight hours after introducing a cage partner, we injected either bromodeoxyuridine or [3H]thymidine to mark cell synthesis and then examined labelled cells 2h (cell proliferation) or five weeks (cell survival) later, respectively. To determine whether estradiol mimicked the effect of reproductive status, groups of reproductively inactive females were given a single injection of estradiol benzoate (10 microg) either four or 48h prior to bromodeoxyuridine labelling. The density of proliferating cells in the granule cell layer and the hilus was elevated in reproductively inactive females compared to reproductively active females and was correlated negatively with serum estradiol level. Exposure to estradiol benzoate initially increased cell proliferation (within 4h) but subsequently suppressed cell proliferation (within 48h). In addition, the density of surviving cells was greater in reproductively inactive females relative to reproductively active females but reproductively active females had a greater rate of cell survival than did reproductively inactive females. Reproductive status did not influence the number of pyknotic cells in the dentate gyrus (at either 2h or five weeks).We conclude that reproductive status regulates cell proliferation in adult female meadow voles, possibly via an estradiol-regulated mechanism. The results from the present study showed that reproductively active female meadow voles have suppressed rates of cell proliferation in the dentate gyrus relative compared with reproductively inactive female meadow voles. Administering estradiol initially (within 4h) elevates the cell proliferation within the dentate gyrus of adult females but subsequently (within 48h) suppresses cell proliferation. However, more new cells survived in females with high endogenous levels of estradiol (reproductively active females). In conclusion, reproductive status regulates the level of cell proliferation and survival through a complex estradiol regulated mechanism(s).

Neurosteroids and reward: allopregnanolone produces a conditioned place aversion in rats.

The neurosteroid 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone) has been reported to have rewarding properties in mice tested for place conditioning. Another study found that allopregnanolone reduced dopamine (DA) output in the nucleus accumbens (NAc) of rats. As many rewarding stimuli increase accumbens DA, these results may appear contradictory. Thus, the present study examined the rewarding properties of allopregnanolone in rats tested for place conditioning using an unbiased conditioning procedure. In control studies, a place preference was observed following conditioning with intraperitoneal (2.0 mg/kg) or intracerebroventricular (i.c.v.) (100 microg/0.5 microl) amphetamine. Conditioning with i.c.v. allopregnanolone produced a significant aversion at a dose of 5.0 microg (in 5.0 microl) and a near aversion at 25.0 microg (in 8.3 microl); doses of 0 microg (i.e., vehicle alone, in 10 microl) or 30.0 microg (in 10 microl) produced little effect on place preference. During conditioning, locomotor activity was stimulated by amphetamine using either route of administration, but allopregnanolone had no significant main effect on locomotor activity. Thus, there was a dissociation between the effects of drugs on locomotor activity vs. place conditioning. Results show that i.c.v. amphetamine produces a place preference, whereas allopregnanolone produces either no effect or an aversion, depending on the dose.

Spatial working memory and hippocampal size across pregnancy in rats.

The present experiments investigated the effects of pregnancy on performance in the Morris water maze and on hippocampal volume. In the first study, pregnant rats (in between the first and second trimester) outperformed nonpregnant rats on the Morris water maze on 1 day of testing. In the second study, rats were tested in a working memory variation of the maze in which the spatial location of the platform varied. Pregnant females traveled shorter distances than nonpregnant females during the first two trimesters, but performed worse than nonpregnant females during the third trimester. Latency measures showed a similar profile. Group differences in performance were not related to changes in swim speed. However, changes in performance in pregnant females may be related to estrogen, progesterone, and/or corticosterone levels during pregnancy, with low levels of estradiol and high levels of progesterone being associated with better performance. There were no significant differences between pregnant and nonpregnant animals on any of the brain measures, although pregnant animals tended to have a smaller hippocampus than nonpregnant animals. These results indicate that pregnancy can affect performance, possibly related to the hormonal changes that accompany pregnancy.