NGF treatment potentiates c-fos expression in the rat nucleus basalis upon excitotoxic lesion with quisqualic acid

The induction of the c-fos gene in the rat brain by NGF was studied in a model of acute cholinergic hypofunction, i.e., the lesion of the nucleus basalis magnocellularis (NBM) with quisqualic acid. Choline acetyltransferase and Fos immunoreactivity (IR) in the NBM were analyzed at different times after the excitotoxic lesion. NGF treatment induced a potentiation of Fos expression 4 and 24 h after lesion. The possibility is discussed that c-fos induction is one of the early mechanisms of the neuroprotective action of NGF.     

C-fos expression in the rat nucleus basalis upon excitotoxic lesion with quisqualic acid: a study in adult and aged animals

Summary. A unilateral quisqualic acid lesion was placed in the nucleus basalis magnocellularis of 3- and 24-month-old rats, and the animals were sacrificed at different times post-surgery. The morphology and the number of the cholinergic neurons of the nucleus basalis were analyzed by means of immunohistochemistry for cholineacetyltransferase, in order to evaluate the size and severity of the lesion. Immunohistochemistry for the immediate early gene c-fos was also performed in order to clarify its role in the process of neurodegeneration following the excitotoxin injection. The DNA laddering and TUNEL techniques were used to define the type of cell death involved. At short times (4 hr) the lesion induced alterations in the morphology of cholinergic neurons of the nucleus basalis. Subsequently, a significant decrease in the number of neurons was found in comparison to the contralateral unlesioned side. In the older animals the loss of cholineacetyltransferase immunoreactivity had an earlier onset (4 hr) than in the young (24 hr). C-fos expression was induced by the lesion and not by saline injection in the nucleus basalis and in neighbouring areas of the brain as early as 4 hr after surgery. The c-fos protein was no longer present by 24 hr. Furthermore, the c-fos gene product was consistently absent from the nuclei of cholinergic cells. The aged animals exhibited a slower and smaller increase in c-fos as measured by counting the labelled nuclei in the injected area. Analysis of DNA fragmentation did not provide any evidence for apoptosis as the type of cell death involved in the cholinergic degeneration. These results indicate that the c-fos protein might have a protective role in the response to excitotoxic lesions. Furthermore, we have shown that the aged brain displays a reduced ability to produce a c-fos-mediated plastic response to the lesion. Received December 17, 1997; accepted February 17, 1998     

Effect of nerve growth factor and GM1 ganglioside on the recovery of cholinergic neurons after a lesion of the nucleus basalis in aging rats

Summary A unilateral ibotenic acid lesion was placed in the nucleus basalis magnocellularis of 3- and 18-month-old rats. In the lesioned aging rats, the number of choline acetyltransferase-immunoreactive neurons of the nucleus basalis magnocellularis was markedly reduced in the ipsilateral side and to a lesser extent in the contralateral side. Twenty-one days after the lesion, the activity of choline acetyltransferase in the ipsilateral cortex was reduced by 40% in both groups of rats and by 24% in the contralateral frontal cortex of the aging rats. Intracerebroventricular administration of nerve growth factor (10 g, twice a week) to aging lesioned rats for 3 weeks after surgery resulted in a complete recovery in the number of choline acetyltransferase-immunoreactive neurons in the nucleus basalis of both sides, and choline acetyltransferase activity in the contralateral cortex, with little effect on the ipsilateral cortex. No potentiation was seen after the concurrent administration of GM1 ganglioside and nerve growth factor. Complete recovery in cortical choline acetyltransferase activity was only observed in the lesioned rats treated with nerve growth factor for 1 week before and 3 weeks after lesioning. Nerve growth factor treatment, both after the lesion, and before and after the lesion, improved the passive avoidance performance disrupted by the lesion. In young lesioned rats daily intraperitoneal administration of GM1 (30 mg/kg) for 21 days after surgery promoted both the recovery of choline acetyltransferase activity and passive avoidance performance. In aging rats GM1, even at a dose twice as large, failed to reverse the biochemical and morphological deficits and behavioral impairment induced by the lesion. Only when GM1 administration was started 3 days before the lesion, were a complete recovery in choline acetyltransferase activity in the contralateral cortex and a partial recovery in the ipsilateral cortex obtained.Our results indicate that nerve growth factor and, to some extent, GM1 facilitate the recovery of the cholinergic neurons after a lesion of the nucleus basalis in aging rats, but their efficacy is reduced. The lower efficacy of GM1 as compared to NGF might be due to the different routes of administration used.     

Learning disturbances following excitotoxic lesion of cholinergic pedunculo-pontine nucleus in the rat

Compared to brain anterior cholinergic systems such as the septo-hippocampal and nucleus basalis-cortical pathways, posterior cholinergic groups have received little attention with respect to their involvement in learning and memory. In this study, the effect of lesion of the cholinergic pedunculo-pontine cell bodies (PPN) by the excitotoxin quisqualic acid was investigated on spontaneous locomotor activity and learning in rats. Behavioral tasks designed to test both reference memory (cross maze and water maze) or working memory (radial maze) were used. PPN lesion had no effect on initial nor on nocturnal locomotor activity in a circular corridor. The lesion disrupted learning in the water and radial mazes, but was without influence on acquisition in the cross maze. The difference in results obtained in the two tasks designed to test reference memory (cross maze and water maze) indicated that the disturbance depended on task difficulty rather than on a particular memory component. It is suggested that the PPN is involved in the sustained attention required to perform correctly in water and radial mazes. The PPN cannot therefore be considered as a uniquely extrapyramidal structure. In addition to its descending outputs, the PPN has ascending connections to the neocortex, either directly or indirectly via the thalamus, and so pathological changes in this region may be partly responsible for the cognitive disorders of aging or those observed in various neurodegenerative conditions.     

Exogenous NGF restores endogenous NGF distribution in the brain of the cognitively impaired aged rat

Alzheimer's disease and normal aging may impair retrograde transport of nerve growth factor (NGF) from cortical areas to basal forebrain cholinergic neurons. We demonstrate a relationship between performance in a spatial reference memory task and NGF distribution in the aged rat brain. In addition, exogenous NGF restored endogenous NGF distribution in cognitively impaired aged rats. These data suggest that NGF administration restores utilization of endogenous growth factor in the brain of cognitively impaired aged rats.     

Discriminable excitotoxic effects of ibotenic acid, AMPA, NMDA and quinolinic acid in the rat laterodorsal tegmental nucleus

Excitotoxins are valuable tools in neuroscience research as they can help us to discover the extent to which certain neurones are necessary for different types of behaviour. They have distinctive neurotoxic effects depending on where they are infused, and this study was conducted to delineate the neurotoxic profiles of excitotoxins in the laterodorsal tegmental nucleus (LDTg). Two 0.1 ml infusions of 0.1 M ibotenate, 0.1 M quinolinate, 0.04–0.1 M NMDA, or 0.05–0.015 M AMPA, were made unilaterally into the LDTg under either pentobarbitone or Avertin anaesthesia. The injection needle was oriented at an angle of 24° from vertical in the mediolateral plane. After 23–27 days, sections through the mesopontine tegmentum were processed using standard histological procedures for NADPH-diaphorase histochemistry, tyrosine hydroxylase or 5-hydroxytryptamine immunohistochemistry, and Cresyl violet. Lesions were assessed in terms of the size of the damaged area (identified by reactive gliosis), the extent of cholinergic cell loss in the mesopontine tegmentum (by counting NADPH-diaphorase-positive neurones), and neuronal loss induced in the locus coeruleus and dorsal raphe nucleus. Ibotenate induced compact lesions in the LDTg (more than 80% cholinergic loss) and did little damage to the locus coeruleus and dorsal raphe nucleus. Quinolinate and low doses of AMPA and NMDA made very small lesions with less than 35% cholinergic loss, while at higher doses, AMPA and NMDA induced large areas of reactive gliosis but killed only a proportion of the cholinergic neurones. AMPA appeared to have a particular affinity for noradrenergic neurones in the locus coeruleus, with the 0.015 M dose injected into the LDTg typically destroying the majority of these neurones. The results are discussed in the context of what is known about the mechanisms of excitotoxins and the glutamate receptor profile of mesopontine neurones.     

Induction of c-fos gene expression by spinal cord transection in the rat

Sympathetic nerve activity is maintained after high spinal injury through circuits that remain in question. We evaluated patterns of c-fos gene induction as a monitor of spinal neurons responding to high spinal cord transection in the rat. Rats were anesthetized with isofluorane. Lower cervical or upper thoracic spinal segments were exposed, immersed in warm mineral oil and transected. Spinal cords were exposed but not transected in anesthetized controls. After 2.5 h, spinalized and control rats were perfused for immunocytochemistry. Cervical and thoracolumbar spinal segments and dorsal root ganglia were sectioned coronally. Tissues were incubated in primary, polyclonal antisera raised in rabbit or sheep against a peptide sequence unique to the N-terminal domain of Fos, and processed immunocytochemically. Neurons were induced to express Fos-like immunoreactivity (FLI), bilaterally, in the spinal gray, but not in primary sensory ganglia. Spinal cord transection induced neurons to express FLI in thoracic laminae I, IIo (outer substantia gelatinosa). Vre (lateral reticulated division), VII (lamina intermedia) and X, and the intermediolateral cell column. Lamina VIII was also labeled in spinal-injured but not in control animals. Immunolabeled nuclei were prominent in lumbar segments and were concentrated in the medial third of laminae I and IIo, and in laminae VII and X. Few cells were labeled in upper cervical or sacral segments. FLI was sparse in the spinal gray of controls and expressed mainly within the dorsal root entry zone of upper thoracic segments. Patterns of c-fos gene expression were site-specific and correlated with laminae that respond predominantly to noxious stimulation and that contain sympathetic interneurons. Laminae that are responsive to non-noxious stimuli and activated by walking, IIi, nucleus proprius, medial V and layer VI were not induced to express FLI. We conclude that neurons in specific spinal laminae that process high threshold afferents and that harbor neurons with sympathetic nerve-related activity are activated selectively by spinal cord transections. We hypothesize that peripheral afferents processed by spinal-sympathetic circuit neurons may regulate sympathetic discharge in the absence of supraspinal drive.     

Oxytocin neurons in the rat hypothalamus exhibit c-fos immunoreactivity upon osmotic stress

In order to evaluate the responses to osmotic stress of oxytocinergic neurons in vivo, we have studied oxytocin (OXY) and c-fos protein expression in the brain by means of double-immunostaining. C-fos immunoreactivity was detected in a subset of OXY neurons, as well as in other neurons non-immunoreactive for OXY, as early as 90 min after intraperitoneal injection of a hypertonic saline solution. C-fos expression was found in approx. 70% of OXY-immunoreactive neurons in the supraoptic (SON), lateral subcommisural (LSN) and paraventricular (PVN) nuclei, and not in OXY neurons in other hypothalamic areas. The expression of c-fos may be used as a means to map the circuitry by which osmotic stimulation activates OXY-containing neurons, and thus provide further insights into the functions with which OXY may be associated.     

Induction of c-fos protein by patterned visual stimulation in central visual pathways of the rat

Localized patterned visual stimulation was used in rats to investigate the feasibility of stimulus-dependent induction of the immediate early gene c-fos in neurons of cortical and subcortical visual centers of this mammal. Moving and stationary visual patterns, consisting of gratings and arrays of dark dots, induced Fos-like immunoreactivity in populations of neurons in retinotopically corresponding stimulated regions of the dorsal and ventral lateral geniculate nucleus (dLGN, vLGN), stratum griseum superficiale of the superior colliculus, nucleus of the optic tract, and primary (striate) visual cortex. Only moving stimuli induced Fos-like immunoreactive (FLI) neurons in extrastriate visual areas, particularly in the anterolateral (AL) visual area. This suggests that area AL is equivalent to the motion sensitive areas MT and PMLS of the monkey and cat. Stimulus-induced FLI neurons in the striate cortex were predominantly distributed in layers 4 and 6, while few labeled neurons were present in layers 2–3, and almost none in layer 5. The laminar distribution of stimulus-induced FLI cells in the extrastriate cortical area AL was similar to that of the striate cortex, with the exception that more FLI cells were present in layer 5. Statistical comparison of somata size of the stimulus-induced FLI neurons in dLGN with that of Cresyl violet stained neurons in the same sections revealed that the population of geniculate FLI neurons is composed of relay cells and interneurons.     

Inflammation causes expression of NGF in epithelial cells of the rat colon

Nerve growth factor (NGF) is a neurotrophin implicated in intestinal pathophysiology, such as impaired barrier function, altered motility and a lowered threshold to noxious stimuli in colitis. We evaluated the cellular source of NGF and determined the effect of inflammation on its expression in TNBS-induced colitis in the rat. Receptors for NGF were studied by immunocytochemistry, showing that submucosal neurons expressed both trkA and p75(NTR). NGF presence and activity was assessed by bioassay, ELISA, western blotting and immunocytochemistry. Bioassay of colonic mucosa using the PC12 cell line showed low levels in control tissue but a marked increase in NGF activity with inflammation. Western blotting showed the appearance of 13 kDa NGF in inflamed mucosa by 6 h, declining over time to become similar to control by 35 days. Semi-quantitative PCR showed minimal mRNA for NGF in control mucosa that increased sharply by 6 h post-TNBS. Laser-capture microdissection was used to collect colonic epithelial cells, where mRNA for NGF was markedly increased by 6 h post-TNBS. While the epithelium of the inflamed colon was positive for NGF by immunocytochemistry, other cell types remained negative. A potential precursor form of NGF, but not 13 kDa NGF itself, was detected in several epithelial cell lines and a mucosal mast cell line. We conclude that NGF is principally synthesized by epithelial cells in the inflamed colon, where the presence of specific receptors suggests the potential for wide-spread action.     

Effects of chronic infusion of nerve growth factor (NGF) in rats with nucleus basalis magnocellularis lesion

We attempted to evaluate the effects of bilateral injection of ibotenic acid (IA) into the nucleus basalis magnocellularis (nbm) of rats as well as the potential recovery mediated by the infusion of nerve growth factor (NGF). The lesion caused an impairment of learning and memory processes. Also, a severe depletion of choline acetyl transferase activity was detected in cortical areas. After the NGF administration, a significant reversion of these functional changes was observed. Thus, IA-lesioned rats might serve as a model for the evaluation of neurotrophic factors actions on basal forebrain damaged neurons.     

Induction of c-fos gene expression by spinal cord transection in Sus scrofa

Functional responses of primary sensory afferents and spinal cord were monitored in swine subjected to a high cervical (C1) spinal transection. Two and a half hours after transection, dorsal root ganglia and cervical and thoracolumbar spinal segments were processed immunocytochemically for the c-fos gene product, Fos and related antigens. In spinal-transected animals, Fos-like immunoreactivity (FLI) was induced in spinal laminae I, V, VII and X and the intermediolateral cell column but not in sensory ganglia as compared to controls: spinal-intact age-matched littermates. Spinal laminae expressing FLI harbor sympathetic and somatic interneurons and may aid in maintaining sympathetic outflow.     

Intracerebroventricular administration of β-endorphin increases the expression of c-fos and of corticotropin-releasing factor messenger ribonucleic acid in the paraventricular nucleus of the rat

We evaluated the effects of intracerebroventricular (i.c.v.) administration of β-endorphin and naloxone, an opioid antagonist, on the induction of c-fos and corticotropin-releasing factor (CRF) mRNA to clarify the effects of β-endorphin on cellular activity and CRF gene expression in the paraventricular nucleus (PVN) of the rat using in situ hybridization. A significant induction of c-fos mRNA was noted in the PVN after i.c.v. injection of β-endorphin, compared to control. This induction was inhibited by the administration of naloxone. A significant increase in CRF mRNA levels in the PVN was observed 120 min after the i.c.v. injection of β-endorphin. This increase was partially, but significantly, inhibited by naloxone administration. In addition, i.c.v. administration of β-endorphin increased plasma ACTH concentration in freely moving rats, which was inhibited by intravenous injection of CRF antiserum. These results suggest that the i.c.v. injection of β-endorphin increases the neuronal activity and the biosynthesis of CRF in the PVN, and stimulates the secretion of ACTH by increasing CRF secretion. This effect on the PVN was mediated, at least in part, via the opioid receptor.     

Relationship of calbindin D-28k and cholinergic neurons in the nucleus basalis of Meynert of the monkey and the rat

Double-labeling immunocytochemistry reactions were carried out in the monkey and the rat nucleus basalis of Meynert (NBM) to determine the extent of overlap between cholinergic neurons and neurons immunoreactive for calbindin-D-28k (CaBP), a Vitamin D-dependent calcium binding protein. The results indicate that most, but not all, NBM cholinergic neurons in the monkey are immunoreactive for CaBP. On the other hand, none of the rat NBM cholinergic neurons are immunoreactive for CaBP.     

Environmental context modulates the ability of cocaine and amphetamine to induce c-fos mRNA expression in the neocortex, caudate nucleus, and nucleus accumbens

We reported previously that environmental novelty enhances the acute psychomotor activating effects of amphetamine, its ability to induce behavioral sensitization, and its ability to induce c-fos mRNA in the striatum and other structures, relative to when amphetamine is given in the home cage. The purpose of the present experiment was 2-fold: to determine (1) whether environmental novelty has a similar effect on the ability of cocaine to induce c-fos mRNA, and (2) whether this effect is seen in neurologically-intact rats (in previous experiments we studied the intact hemisphere of rats with a unilateral 6-OHDA lesion). In the dorsal portion of the caudate putamen, core and shell of the nucleus accumbens, and in several cortical regions, both amphetamine (1.5 mg/kg) and cocaine (15 mg/kg) induced higher levels of c-fos mRNA expression when administered in a novel environment, relative to when they were administered in the home cage. The ability of environmental context to modulate psychostimulant drug-induced immediate early gene expression may be related to its ability to modulate forms of drug experience-dependent plasticity, such as behavioral sensitization.     

Fos induction by nerve growth factor in the adult rat brain

The distribution of Fos, the protein product of the immediate early gene c-fos, was studied with immunocytochemistry in the adult male rat brain after nerve growth factor (NGF) administration. NGF was injected in the lateral cerebral ventricle through a previously implanted cannula. The total number of Fos-immunoreactive (ir) neurons in the brain was 2–3 times higher after NGF administration than in control animals (untreated or injected with cytochrome c). With respect to control rats, in the NGF-treated cases Fos-ir cells were more numerous in the anterior olfactory nucleus, in the medial prefrontal and anterior cingulate cortices, in the basal forebrain, in the preoptic and ventromedial nuclei of the hypothalamus, as well as interior hypothalamic area, in the thalamic midline nuclei, and in some brainstem structures, such as the parabrachial nucleus. The relative quantitative increase of Fos-ir neurons varied in the different structures. In addition, Fos-ir neurons were evident after NGF administration in areas devoid of immunopositive cells in control animals. These included: frontoparietal and occipital cortical fields, the hypothalamic arcuate nucleus, and many brainstem structures, such as the dorsal nucleus of the lateral lemniscus, posterodorsal tegmental, medial and lateral vestibular, ventral cochlear, and prepositus hypoglossal nuclei. These findings demonstrate that the intracerebroventricular administration of NGF can induce c-fos expression in neurons in vivo. The distribution of Fos-ir neurons indicates that NGF can induce activation of functionally and chemically hetergeneous neuronal subsets in the brain.