Kim B. Seroogy

Dietary restriction enhances neurotrophin expression and neurogenesis in the hippocampus of adult mice

The adult brain contains small populations of neural precursor cells (NPC) that can give rise to new neurons and glia, and may play important roles in learning and memory, and recovery from injury. Growth factors can influence the proliferation, differentiation and survival of NPC, and may mediate responses of NPC to injury and environmental stimuli such as enriched environments and physical activity. We now report that neurotrophin expression and neurogenesis can be modified by a change in diet. When adult mice are maintained on a dietary restriction (DR) feeding regimen, numbers of newly generated cells in the dentate gyrus of the hippocampus are increased, apparently as the result of increased cell survival. The new cells exhibit phenotypes of neurons and astrocytes. Levels of expression of brain‐derived neurotrophic factor (BDNF) and neurotrophin‐3 (NT‐3) are increased by DR, while levels of expression of high‐affinity receptors for these neurotrophins (trkB and trkC) are unchanged. In addition, DR increases the ratio of full‐length trkB to truncated trkB in the hippocampus. The ability of a change in diet to stimulate neurotrophin expression and enhance neurogenesis has important implications for dietary modification of neuroplasticity and responses of the brain to injury and disease.

Neurotensin and cholecystokinin coexistence within neurons of the ventral mesencephalon: projections to forebrain.

SummaryThe colocalization of neurotensin- and cholecystokinin-like immunoreactivities was demonstrated in neurons of the ventral mesencephalon of the rat by using a double-labeling indirect immunofluorescence procedure for the simultaneous detection of two antigens in the same tissue section. Greater than 90% of the neurotensin-positive perikarya distributed throughout the ventral mid-brain (primarily located in the ventral tegmental area, medial substantia nigra, and rostral and caudal linear raphe nuclei) were found to also contain cholecystokinin immunoreactivity. Neurons single-labeled for either peptide were also present, with those immunoreactive for cholecystokinin alone far outnumbering those containing only neurotensin. By combining the double-labeling colocalization technique with fluorescence retrograde tracing, some of the forebrain projections of these neurons were determined. Ventral mesencephalic neurons containing both neurotensin and cholecystokinin were found to project to the nucleus accumbens, prefrontal cortex, or amygdala. The present results, combined with those of previous studies, suggest that there are complex heterogeneous subpopulations of presumed dopaminergic ventral mesencephalic neurons which give rise to the ascending mesotelencephalic systems and which may contain both neurotensin and cholecystokinin, either peptide alone, or neither of these two peptides.

Alterations in BDNF and NT-3 mRNAs in rat hippocampus after experimental brain trauma

Previous studies have suggested that the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are neuroprotective or neurotrophic for certain subpopulations of hippocampal neurons following various brain insults. In the present study, the expression of BDNF and NT-3 mRNAs in rat hippocampus was examined after traumatic brain injury. Following lateral fluid percussion (FP) brain injury of moderate severity (2.0-2.1 atm) or sham injury, the hippocampi from adult rats were processed for the in situ hybridization localization of BDNF and NT-3 mRNAs using 35S-labeled cRNA probes at post-injury survival times of 1, 3, 6, 24 and 72 h. Unilateral FP injury markedly increased hybridization for BDNF mRNA in the dentate gyrus bilaterally which peaked at 3 h and remained above control levels for up to 72 h post-injury. A moderate increase in BDNF mRNA expression was also observed bilaterally in the CA3 region of the hippocampus at 1, 3, and 6 h after FP injury, but expression declined to control levels by 24 h. Conversely, NT-3 mRNA was significantly decreased in the dentate gyrus following FP injury at the 6 and 24 h survival times. These results demonstrate that FP brain injury differentially modulates expression of BDNF and NT-3 mRNAs in the hippocampus, and suggest that neurotrophin plasticity is a functional response of hippocampal neurons to brain trauma.

Expression of trkB and trkC mRNAs by adult midbrain dopamine neurons: A double-label in situ hybridization study

The documented trophic actions of the neurotrophins brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3), and neurotrophin‐4/5 (NT‐4/5) upon ventral mesencephalic dopamine neurons in vitro and in vivo are presumed to be mediated through interactions with their high‐affinity receptors TrkB (for BDNF and NT‐4/5) and TrkC (for NT‐3). Although both neurotrophin receptor mRNAs have been detected within the rat ventral midbrain, their specific association with mesencephalic dopaminergic cell bodies remains to be elucidated. The present study was performed to determine the precise organization of trkB and trkC mRNAs within rat ventral midbrain and to discern whether the neurotrophin receptor mRNAs are expressed specifically by dopaminergic neurons. In situ hybridization with isotopically labeled cRNA probes showed that trkB and trkC mRNAs were expressed in all mesencephalic dopamine cell groups, including all subdivisions of the substantia nigra and ventral tegmental area, and in the retrorubral field, rostral and caudal linear raphe nuclei, interfascicular nucleus, and supramammillary region. Combined isotopic/nonisotopic double‐labeling in situ hybridization demonstrated that virtually all of the tyrosine hydroxylase (the catecholamine biosynthetic enzyme) mRNA‐containing neurons in the ventral midbrain also expressed trkB or trkC mRNAs. Additional perikarya within these regions expressed the neurotrophin receptor mRNAs but were not dopaminergic. The present results demonstrate that essentially all mesencephalic dopaminergic neurons synthesize the neurotrophin receptors TrkB and TrkC and thus exhibit the capacity to respond directly to BDNF and NT‐3 in the adult midbrain in vivo. Moreover, because BDNF and NT‐3 are produced locally by subpopulations of the dopaminergic cells, the present data support the notion that the neurotrophins can influence the dopaminergic neurons through autocrine or paracrine mechanisms. J. Comp. Neurol. 403:295–308, 1999.

Brain-derived neurotrophic factor and neurotrophin-3 mRNAs are expressed in ventral midbrain regions containing dopaminergic neurons

Recent evidence suggests that brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) enhance the survival of ventral mesencephalic dopaminergic neurons. In this study, cellular distributions of mRNAs for the nerve growth factor (NGF) family of neurotrophins (NGF, BDNF, and NT-3) and the catecholamine biosynthetic enzyme tyrosine hydroxylase (TH) were evaluated in the ventral mesencephalon of adult rat to determine if the neurotrophins are synthesized in regions of the responsive dopaminergic cells. Messenger RNAs were localized by in situ hybridization of (35)S-labeled cRNA probes and emulsion autoradiography. Neurotrophin-3 cRNA labeled neurons in the ventral tegmental area, medial substantia nigra pars compacta, and retrorubral field. The distributions of NT-3 mRNA-containing and TH mRNA-containing neurons corresponded very well in these areas. Hybridization of the BDNF cRNA labeled scattered cells in corresponding fields of TH mRNA-containing neurons in both the ventral tegmental area and the medial substantia nigra pars compacta but, in contrast to NT-3 cRNA, labeled fewer cells in these areas. Somata containing BDNF mRNA were also present in surrounding regions, including the interfascicular and interpeduncular nuclei, the supramammillary region, the periaqueductal grey matter, and fields dorsal to the lateral substantia nigra. Hybridization of NGF cRNA was not observed in the ventral mesencephalon. These results demonstrate that mRNAs for NT-3 and BDNF are expressed by neurons in both the substantia nigra and ventral tegmental area of adult rat and suggest that trophic support for the dopaminergic neurons in these areas may arise from local synthesis. Moreover, these results raise the possibility that perturbations in local neurotrophin synthesis might contribute to dopamine-related disorders including Parkinsons disease and schizophrenia.

Proliferative zones of postnatal rat brain express epidermal growth factor receptor mRNA

Two ligands for the epidermal growth factor receptor (EGF-R), EGF and transforming growth factor-alpha (TGF alpha), have recently been shown to influence the proliferation, differentiation or survival of diverse populations of fetal and neonatal neuronal and glial cells in culture. These findings suggest that EGF, TGF alpha, or another EGF-R ligand play a role in the regulation of similar cellular developmental events in vivo. In the present study, in situ hybridization with an 35S-labeled cRNA probe was used to determine if mRNA for EGF-R is expressed in two principal germinal zones of the postnatal rat brain, the forebrain ventricular/subventricular zone and the cerebellar external granule layer. Cells labeled with the EGF-R cRNA were distributed throughout the subventricular zone, particularly in the dorsolateral aspect, from birth to adulthood, although the numbers of labeled cells as well as the density of hybridization diminished during development. In the developing cerebellum, virtually all cells in the external granule layer were densely labeled with the EGF-R cRNA, as were numerous perikarya throughout the molecular layer. EGF-R mRNA was also transiently expressed at lower levels by neurons of the internal granule layer and deep cerebellar nuclei. By adulthood, cerebellar expression of EGF-R mRNA was not detected. These results demonstrate prominent expression of EGF-R mRNA within germinal zones of the developing brain and indicate a role for EGF, TGF alpha, or another member of the EGF-related family in regulating the activities of neuronal and glial progenitor cells in vivo.

Co-localization of enkephalin and cholecystokinin in discrete areas of rat brain

A double-label immunofluorescence technique was used to demonstrate that immunoreactivities for the functionally antagonistic neuropeptides enkephalin and cholecystokinin octapeptide (CCK) are co-localized within individual neurons and processes in discrete areas of rat midbrain and forebrain. Coexistence was most prominent within varicose pericellular axons extending from the periaqueductal gray matter to a field overlying the medial lemniscus, axons and terminal-like puncta in the central medial, paracentral, interanterodorsal and ventral anterior thalamic nuclei, and perikarya and proximal axonal fragments in layers II and III of neo- and allocortex, and in the anterior olfactory nucleus. The former two systems of axons lie in areas of spinothalamic tract termination. These data suggest that some of the antagonism of opioid analgesia by CCK occurs at the synaptic level in nociceptive areas of brain-stem and thalamus where CCK and enkephalin are co-localized and presumably co-released.

Multiple trophic actions of heparin‐binding epidermal growth factor (HB‐EGF) in the central nervous system

The epidermal growth factor (EGF) family of ligands interacts with the epidermal growth factor receptor (EGF‐R) to produce numerous direct and indirect actions on central nervous system cells. They induce the proliferation of astrocytes and multipotent progenitors (‘stem’ cells) and promote the survival and differentiation of postmitotic neurons. Heparin‐binding epidermal growth factor (HB‐EGF) interacts with both EGF‐R and a related receptor, ErbB4, whereas transforming growth factor alpha (TGFα) interacts only with EGF‐R. Because of the unique characteristics of HB‐EGF and the potential utility of EGF family members in brain repair, we examine the effects of HB‐EGF on rat and mouse CNS cells in vitro and compare them to those of TGFα. We find that, like TGFα, HB‐EGF stimulates the proliferation of CNS astrocytes and multipotent progenitors. These proliferative effects require the expression of EGF‐R, as no such effects are observed in cells derived from EGF‐R–/– mice. Both HB‐EGF and TGFα enhanced the survival of neurons derived from the neocortex and the striatum. Within these neuron‐enriched cultures, nestin‐positive cells but not neurons express EGF‐R mRNA, indicating that the neurotrophic actions of EGF‐R ligands are a result of indirect stimulation mediated by non‐neuronal cells. The neurotrophic actions of HB‐EGF and TGFα are accompanied by an elevation in immunoreactive dual phosphorylated mitogen‐activated protein kinase (MAP kinase) in neurons, providing evidence that the MAP kinase cascade mediates these actions. In situ hybridization studies demonstrate that HB‐EGF mRNA is present within the brainstem as early as E14 and subsequently is found in the developing cortical plate, hippocampus, cerebellar Purkinje cells and ventrobasal thalamus, among other brain areas. These findings indicate that HB‐EGF may be an important trophic factor in the developing CNS and is a useful candidate molecule for brain repair strategies.

Mild Experimental Brain Injury Differentially Alters the Expression of Neurotrophin and Neurotrophin Receptor mRNAs in the Hippocampus

The molecular events responsible for impairments in cognition following mild traumatic brain injury are poorly understood. Neurotrophins, such as brain-derived neurotrophic factor (BDNF), have been identified as having a role in learning and memory. We have previously demonstrated that following experimental brain trauma of moderate severity (2.0-2.1 atm), mRNA levels of BDNF and its high-affinity receptor, trkB, are increased bilaterally in the hippocampus for several hours, whereas NT-3 mRNA expression is decreased. In the present study, we used in situ hybridization to compare BDNF, trkB, NT-3, and trkC mRNA expression in rat hippocampus at 3 or 6 h after a lateral fluid percussion brain injury (FPI) of mild severity (1.0 atm) to sham-injured controls at equivalent time points. Mild FPI induced significant increases in hybridization levels for BDNF and trkB mRNAs, and a decrease in NT-3 mRNA in the hippocampus. However, in contrast to the bilateral effects of moderate experimental brain injury, the present changes with mild injury were restricted to the injured side. These findings demonstrate that even a mild traumatic brain injury differentially alters neurotrophin and neurotrophin receptor levels in the hippocampus. Such alterations may have important implications for neural plasticity and recovery of function in people who sustain a mild head injury.

Distribution of VIP- and NPY-like immunoreactivities in rat main olfactory bulb

The distribution of vasoactive intestinal peptide (VIP)- and neuropeptide Y (NPY)-like immunoreactivities in the Sprague-Dawley rat main olfactory bulb was analyzed using the peroxidase-antiperoxidase light microscopic immunocytochemical technique. VIP-like immunoreactivity was most prominently localized within a large number of intermediate-sized neurons whose perikarya and extensively branched varicose processes remained confined to the external plexiform layer (EPL). A few small short-axon type neurons in the mitral cell layer and granule cell layer (GRL) and even fewer large neurons in the glomerular layer (GL)/EPL border region contained immunoreactivity for VIP as well. Neuropeptide Y-like immunoreactivity (NPY-I) was principally localized within sparsely distributed large multipolar neurons of the deep GRL and within axons distributed with diminishing density from deep to superficial GRL. In addition, dense NPY-I was localized within very few large superficial short-axon type neurons of the GL/EPL border region. The restricted laminar and cellular distribution of NPY-I and VIP-I suggests that both peptides may act to modulate granule cell activity, and therefore, indirectly, olfactory bulb output.