Spaced training improves learning in Ts65Dn and Ube3a mouse models of intellectual disabilities.

Benefits of distributed learning strategies have been extensively described in the human literature, but minimally investigated in intellectual disability syndromes. We tested the hypothesis that training trials spaced apart in time could improve learning in two distinct genetic mouse models of neurodevelopmental disorders characterized by intellectual impairments. As compared to training with massed trials, spaced training significantly improved learning in both the Ts65Dn trisomy mouse model of Down syndrome and the maternally inherited Ube3a mutant mouse model of Angelman syndrome. Spacing the training trials at 1 h intervals accelerated acquisition of three cognitive tasks by Ts65Dn mice: (1) object location memory, (2) novel object recognition, (3) water maze spatial learning. Further, (4) spaced training improved water maze spatial learning by Ube3a mice. In contrast, (5) cerebellar-mediated rotarod motor learning was not improved by spaced training. Corroborations in three assays, conducted in two model systems, replicated within and across two laboratories, confirm the strength of the findings. Our results indicate strong translational relevance of a behavioral intervention strategy for improving the standard of care in treating the learning difficulties that are characteristic and clinically intractable features of many neurodevelopmental disorders.

Ampakines cause sustained increases in brain-derived neurotrophic factor signaling at excitatory synapses without changes in AMPA receptor subunit expression.

Recent demonstrations that positive modulators of AMPA-type glutamate receptors (ampakines) increase neuronal brain-derived neurotrophic factor (BDNF) expression have suggested a novel strategy for treating neurodegenerative diseases. However, reports that AMPA and BDNF receptors are down-regulated by prolonged activation raise concerns about the extent to which activity-induced increases in BDNF levels can be sustained without compromising glutamate receptor function. The present study constitutes an initial test of whether ampakines can cause enduring increases in BDNF content and signaling without affecting AMPA receptor (AMPAR) expression. Prolonged (12-24 h) treatment with the ampakine CX614 reduced AMPAR subunit (glutamate receptor subunit (GluR) 1-3) mRNA and protein levels in cultured rat hippocampal slices whereas treatment with AMPAR antagonists had the opposite effects. The cholinergic agonist carbachol also depressed GluR1-3 mRNA levels, suggesting that AMPAR down-regulation is a global response to extended periods of elevated neuronal activity. Analyses of time courses and thresholds indicated that BDNF expression is influenced by lower doses of, and shorter treatments with, the ampakine than is AMPAR expression. Accordingly, daily 3 h infusions of CX614 chronically elevated BDNF content with no effect on GluR1-3 concentrations. Restorative deconvolution microscopy provided the first evidence that chronic up-regulation of BDNF is accompanied by increased activation of the neurotrophin's TrkB-Fc receptor at spine synapses. These results show that changes in BDNF and AMPAR expression are dissociable and that up-regulation of the former leads to enhanced trophic signaling at excitatory synapses. These findings are encouraging with regard to the feasibility of using ampakines to tonically enhance BDNF-dependent functions in adult brain.

Integrins regulate neuronal neurotrophin gene expression through effects on voltage-sensitive calcium channels.

Integrin adhesion receptors regulate gene expression during growth and differentiation in various cell types. Recent work, implicating integrins in functional synaptic plasticity, suggest they may have similar activities in adult brain. The present study tested if integrins binding the arginine-glycine-aspartate (RGD) matrix sequence regulate neurotrophin and neurotrophin receptor gene expression in cultured hippocampal slices. The soluble RGD-containing peptide glycine-arginine-glycine-aspartate-serine-proline (GRGDSP) increased neurotrophin mRNA levels in transcript- and subfield-specific fashions. Integrin ligand effects were greatest for brain-derived neurotrophic factor transcripts I and II and barely detectable for transcript III. In accordance with increased nerve growth factor mRNA levels, GRGDSP increased c-fos expression as well. In contrast, growth-associated protein-43, amyloid precursor protein and fibroblast growth factor-1 mRNAs were not elevated. Ligand effects on brain-derived neurotrophic factor transcript II and c-fos mRNA did not depend on the integrity of the actin cytoskeleton, neuronal activity, or various signaling pathways but were blocked by L-type voltage-sensitive calcium-channel blockers. These results indicate that in mature hippocampal neurons integrin engagement regulates expression of a subset of growth-related genes at least in part through effects on calcium influx. Accordingly, these synaptic adhesion receptors may play the same role in maintaining an adult, differentiated state in brain as they do in other tissues and changes in integrin activation and/or engagement may contribute to dynamic changes in neurotrophin expression and to neuronal calcium signaling.

Arrival of afferents and the differentiation of target neurons: studies of developing cholinergic projections to the dentate gyrus.

This study examined the relationship between the development of cholinergic axons originating from the septum and a group of their target cells, the granule cells of the dentate gyrus of the rat. Acetylcholinesterase histochemistry was used to identify septal cholinergic afferents to the dentate gyrus; parallel studies used anterograde movement of a carbocyanine dye to label the septal projections. Septal cholinergic axons are present in the molecular layer of the internal blade of the dentate gyrus shortly after birth, but these axons do not reach the external blade until several days later. Results demonstrate that acetylcholinesterase positive septal axons grow into the external blade of the dentate gyrus only after the recently generated granule cells have coalesced to form a clearly defined layer. Results from studies using in situ hybridization techniques demonstrate that dentate gyrus granule cells express messenger RNAs for brain derived neurotrophic factor and for neurotrophic factor 3 shortly after formation of the granule cell layer. Ingrowth of septal cholinergic axons follows two days after the formation of the external blade of the dentate gyrus and the expression of neurotrophin messenger RNAs by the dentate granule cells. These data support the hypothesis that target cell development is a prerequisite for attracting the ingrowth of septal afferent axons.

Enhanced CREB phosphorylation in immature dentate gyrus granule cells precedes neurotrophin expression and indicates a specific role of CREB in granule cell differentiation.

Differentiation and maturation of dentate gyrus granule cells requires coordinated interactions of numerous processes. These must be regulated by protein factors capable of integrating signals mediated through diverse signalling pathways. Such integrators of inter and intracellular physiological stimuli include the cAMP-response element binding protein (CREB), a leucine-zipper class transcription factor that is activated through phosphorylation. Neuronal activity and neurotrophic factors, known to be involved in granule cell differentiation, are major physiologic regulators of CREB function. To examine whether CREB may play a role in governing coordinated gene transcription during granule cell differentiation, we determined the spatial and temporal profiles of phosphorylated (activated) CREB throughout postnatal development in immature rat hippocampus. We demonstrate that CREB activation is confined to discrete, early stages of granule cell differentiation. In addition, CREB phosphorylation occurs prior to expression of the neurotrophins BDNF and NT-3. These data indicate that in a signal transduction cascade connecting CREB and neurotrophins in the process of granule cell maturation, CREB is located upstream of neurotrophins. Importantly, CREB may be a critical component of the machinery regulating the coordinated transcription of genes contributing to the differentiation of granule cells and their integration into the dentate gyrus network.

Positive modulation of AMPA receptors increases neurotrophin expression by hippocampal and cortical neurons.

This study investigated whether positive modulators of AMPA-type glutamate receptors influence neurotrophin expression by forebrain neurons. Treatments with the ampakine CX614 markedly and reversibly increased brain-derived neurotrophic factor (BDNF) mRNA and protein levels in cultured rat entorhinal/hippocampal slices. Acute effects of CX614 were dose dependent over the range in which the drug increased synchronous neuronal discharges; threshold concentrations for acute responses had large effects on mRNA content when applied for 3 d. Comparable results were obtained with a second, structurally distinct ampakine CX546. Ampakine-induced upregulation was broadly suppressed by AMPA, but not NMDA, receptor antagonists and by reducing transmitter release. Antagonism of L-type voltage-sensitive calcium channels blocked induction in entorhinal cortex but not hippocampus. Prolonged infusions of suprathreshold ampakine concentrations produced peak BDNF mRNA levels at 12 hr and a return to baseline levels by 48 hr. In contrast, BDNF protein remained elevated throughout a 48 hr incubation with the drug. Nerve growth factor mRNA levels also were increased by ampakines but with a much more rapid return to control levels during chronic administration. Finally, intraperitoneal injections of CX546 increased hippocampal BDNF mRNA levels in aged rats and middle-aged mice. The present results provide evidence of regional differences in mechanisms via which activity regulates neurotrophin expression. Moreover, these data establish that changes in synaptic potency produce sufficient network level physiological effects for inducing neurotrophin genes, indicate that the response becomes refractory during prolonged ampakine exposure, and raise the possibility of using positive AMPA modulators to regulate neurotrophin levels in aged brain.

Subpopulations of striatal interneurons can be distinguished on the basis of neurotrophic factor expression.

Substantial evidence supports a role for trophic activities in the function and survival of fully mature striatal neurons, but little is known regarding trophic factor expression in adult striatum. In situ hybridization was used to identify the distribution and the neurotransmitter phenotypes (i.e., cholinergic and gamma-aminobutyric acid [GABA]-ergic) of cells expressing acidic fibroblast growth factor (aFGF), glial cell line-derived neurotrophic factor (GDNF), or nerve growth factor (NGF) mRNA in adult rat striatum. Each trophic factor mRNA was localized to large, sparsely scattered striatal cells that corresponded to interneurons. Double-labeling studies demonstrated that NGF mRNA was expressed by GABAergic and never by cholinergic cells, whereas aFGF and GDNF mRNAs were expressed by both cell types. Approximately 75% of aFGF+ and GDNF+ cells in dorsal striatum and 46% of aFGF+ and 61% of GDNF+ cells in ventral striatum were cholinergic. Conversely, about 32% of aFGF+ and 24% of GDNF+ cells in dorsal striatum and 55% of aFGF+ and 27% of GDNF+ cells in ventral striatum were GABAergic. A portion of aFGF+ and NGF+ cells was of the parvalbumin GABAergic subtype. The colocalization of trophic factor expression was also examined. Of aFGF+ cells, 20% and 41% were NGF+ and 67% and 83% were GDNF+ in dorsal and ventral striata, respectively. These findings demonstrate that aFGF, GDNF, and NGF are synthesized by discrete but overlapping populations of striatal interneurons. The expression of these survival factors may contribute to the resistance of striatal interneurons to various insults including excitotoxicity.

Anterograde transport of neurotrophin proteins in the CNS--a reassessment of the neurotrophic hypothesis.

The basic tenets of the neurotrophic hypothesis are that i) limiting quantities of a given factor are produced in specific target tissues; ii) responsive neurons projecting to these targets compete for the limiting amounts of the factor; iii) the factor is bound within the target by selective receptors on afferent terminals, internalized, and retrogradely transported to the neuronal cell body where it provides signals affecting neuronal survival and differentiation. Although originally formulated on the basis of evidence for NGF's actions on peripheral sensory and sympathetic neurons, the neurotrophic hypothesis appeared to be upheld for CNS neuronal systems as well, where NGF was found to function primarily as a target-derived trophic factor for basal forebrain cholinergic neurons. With the discovery of additional neurotrophins sharing considerable structural homology with NGF, the question arose of whether the neurotrophic hypothesis held true for all members of this protein family. Recent investigations into the localization and function of neurotrophins other than NGF, particularly BDNF and NT-3, have provided evidence indicating that these molecules may not act in a manner consistent with the neurotrophic hypothesis, as originally postulated. Numerous studies in the peripheral and central nervous systems have now demonstrated that BDNF (and NT-3) may be preferentially trafficked anterogradely along axonal processes and stored within pre-synaptic terminals. Other studies have suggested that these factors may be released in an activity-dependent, rather than constitutive, manner and can act in autocrine or paracrine fashions to subserve an assortment of biological functions including anterograde effects on cell survival and differentiation, as well as more novel roles in synaptic transmission. These recent findings strongly suggest that, while the various neurotrophin proteins may be grouped into a single family based upon their structural homology, they should be considered as a heterogeneous group of trophic factors based upon function and mode of action.

Transcript-specific effects of adrenalectomy on seizure-induced BDNF expression in rat hippocampus.

Activity-induced brain-derived neurotrophic factor (BDNF) expression is negatively modulated by circulating adrenal steroids. The rat BDNF gene gives rise to four major transcript forms that each contain a unique 5' exon (I-IV) and a common 3' exon (V) that codes for BDNF protein. Exon-specific in situ hybridization was used to determine if adrenalectomy has differential effects on basal and activity-induced BDNF transcript expression in hippocampus. Adrenalectomy alone had only modest effects on BDNF mRNA levels with slight increases in exon III-containing mRNA with 7-10-day survival and in exon II-containing mRNA with 30-days survival. In the dentate gyrus granule cells, adrenalectomy markedly potentiated increases in exon I and II cRNA labeling, but not increases in exon III and IV cRNA labeling, elicited by one hippocampal afterdischarge. Similarly, for the granule cells and CA1 pyramidal cells, hilus lesion (HL)-induced recurrent limbic seizures elicited greater increases in exon I and II cRNA hybridization in adrenalectomized (ADX) as compared to adrenal-intact rats. In this paradigm, adrenalectomy modestly potentiated the increase in exon III-containing mRNA in CA1 but had no effect on exon IV-containing mRNA content. These results demonstrate that the negative effects of adrenal hormones on activity-induced BDNF expression are by far the greatest for transcripts containing exons I and II. Together with evidence for region-specific transcript expression, these results suggest that the effects of stress on adaptive changes in BDNF signalling will be greatest for neurons that predominantly express transcripts I and II.

Neonatal treatment with 192 IgG-saporin produces long-term forebrain cholinergic deficits and reduces dendritic branching and spine density of neocortical pyramidal neurons.

The role of basal forebrain-derived cholinergic afferents in the development of neocortex was studied in postnatal rats. Newborn rat pups received intraventricular injections of 192 IgG-saporin. Following survival periods ranging from 2 days to 6 months, the brains were processed to document the cholinergic lesion and to examine morphological consequences. Immunocytochemistry for choline acetyltransferase (ChAT) and in situ hybridization for ChAT mRNA demonstrate a loss of approximately 75% of the cholinergic neurons in the medial septum and nucleus of the diagonal band of Broca in the basal forebrain. In situ hybridization for glutamic acid decarboxylase mRNA reveals no loss of basal forebrain GABAergic neurons. Acetylcholinesterase histochemistry demonstrates a marked reduction of the cholinergic axons in neocortex. Cholinergic axons are reduced throughout the cortical layers; this reduction is more marked in medial than in lateral cortical areas. The thickness of neocortex is reduced by approximately 10%. Retrograde labeling of layer V cortico-collicular pyramidal cells reveals a reduction in cell body size and also a reduction in numbers of branches of apical dendrites. Spine densities on apical dendrites are reduced by approximately 20-25% in 192 IgG-saporin-treated cases; no change was detected in number of spines on basal dendrites. These results indicate a developmental or maintenance role for cholinergic afferents to cerebral cortical neurons.

Lysosomal dysfunction reduces brain-derived neurotrophic factor expression.

Brain-derived neurotrophic factor (BDNF) expression in hippocampus and cortex is considerably reduced in Alzheimer's disease. The present study tested if lysosomal disturbances, a concomitant of brain aging, impair basal and/or induced expression of BDNF. Cultured hippocampal slices were incubated with N- CBZ-L-phenylalanyl-L-alanine-diazomethylketone (ZPAD), an inhibitor of cathepsins B and L, for 6 days and processed for in situ hybridization using radiolabeled cRNA probes against BDNF mRNA. Multiple densitometric readings were collected from each of the three principal hippocampal subdivisions. Within-slice averages were substantially lower in the ZPAD-treated group compared to controls. Treatment with the inhibitor did not change average neuron diameter or packing density. Intense stimulation of glutamate receptors with kainate for 30 min (followed by a 90-min recovery period) caused a nearly threefold increase in BDNF mRNA concentrations in the dentate gyrus while having only marginal effects in the other subdivisions. Slice averages of ZPAD-exposed cultures treated with kainate were lower than those of controls exposed to the excitotoxin; however, on a percentage basis, the kainate-induced increase in the dentate gyrus was comparable for the two groups (175 +/- 31 vs 179 +/- 39%). Kainate for 1 h (with a 5-h recovery) affected BDNF mRNA in a manner similar to that found with shorter infusions, i.e., induction in stratum granulosum but not elsewhere, lower overall slice averages with ZPAD treatment, and no evidence that ZPAD blocked the percentage increase in the dentate gyrus. These results provide evidence that lysosomal dysfunction occurring during brain aging could disrupt ongoing BDNF production without substantially impairing the neurotrophin response to intense physiological activity. The first observation suggests a plausible aging sequence leading to pathology while the second may be of interest with regard to possible therapeutics.

Distribution of brain-derived neurotrophic factor (BDNF) protein and mRNA in the normal adult rat CNS: evidence for anterograde axonal transport.

A sensitive immunohistochemical technique was used, along with highly specific affinity-purified antibodies to brain-derived neurotrophic factor (BDNF), to generate a detailed mapping of BDNF immunoreactivity (BDNF-ir) throughout the adult rat CNS. A parallel analysis of sites of BDNF synthesis was performed with in situ hybridization techniques using a cRNA probe to the exon encoding mature rat BDNF protein. These combined data revealed (1) groups of cell bodies containing diffuse BDNF-ir throughout the CNS that were strongly correlated with fields of cells containing BDNF mRNA; (2) varying degrees of BDNF-ir outside of cell bodies, in what appeared to be fibers and/or terminals; and (3) many regions containing extremely heavy BDNF-immunoreactive fiber/terminal labeling that lacked BDNF mRNA (e.g., medial habenula, central nucleus of the amygdala, bed nucleus of stria terminalis, lateral septum, and spinal cord). The latter observation suggested that in these regions BDNF was derived from anterograde axonal transport by afferent systems. In the two cases in which this hypothesis was tested by the elimination of select afferents, BDNF immunostaining was completely eliminated. These data, along with the observation that BDNF-ir was rarely found within dendrites or fibers en passage, suggest that BDNF protein produced in adult CNS neurons is polarized primarily along axonal processes and is preferentially stored in terminals within the innervation target.

Differential effects of protein synthesis inhibition on the activity-dependent expression of BDNF transcripts: evidence for immediate-early gene responses from specific promoters.

In the adult rat forebrain, brain-derived neurotrophic factor (BDNF) expression is very rapidly induced by neuronal activity, suggesting that this might occur without intervening protein synthesis. The rat BDNF gene has four differentially regulated promoter regions; each gives rise to an mRNA containing a unique 5' exon (I-IV) and a common 3' exon (V) that codes for mature BDNF protein. The present study used exon-specific in situ hybridization and both in vivo and in vitro preparations to determine whether activity induces BDNF as an "immediate-early gene" (IEG) from specific promoter regions and to compare the regulation of BDNF and nerve growth factor (NGF). In cultured hippocampal slices, kainic acid markedly increased pan-BDNF (exon V) and NGF mRNA content; cycloheximide attenuated the effect of kainic acid on both. In vivo stimulation of a paroxysmal afterdischarge increased both pan-BDNF and NGF mRNA levels in the dentate gyrus granule cells; pretreatment with anisomycin modestly attenuated the paroxysmal afterdischarge-induced increase of both transcripts. To determine whether partial drug effects on BDNF expression reflect the differential regulation of transcript species, levels of mRNAs containing exons I-IV were evaluated. A single afterdischarge increased exon I-IV-containing mRNA levels; anisomycin significantly attenuated the increase in exon I- and II-containing mRNAs but had no effect on the increase in exon III- and IV-containing mRNAs. These data show that for mature forebrain neurons, activity induces the expression of BDNF exon III- and IV-containing transcripts as IEG responses.

Acidic fibroblast growth factor mRNA is expressed by basal forebrain and striatal cholinergic neurons.

Evidence for the importance of the basal forebrain cholinergic system in the maintenance of cognitive function has stimulated efforts to identify trophic mechanisms that protect this cell population from atrophy and dysfunction associated with aging and disease. Acidic fibroblast growth factor (aFGF) has been reported to support cholinergic neuronal survival and has been localized in basal forebrain with the use of immunohistochemical techniques. Although these data indicate that aFGF is present in regions containing cholinergic cell bodies, the actual site of synthesis of this factor has yet to be determined. In the present study, in situ hybridization techniques were used to evaluate the distribution and possible colocalization of mRNAs for aFGF and the cholinergic neuron marker choline acetyltransferase (ChAT) in basal forebrain and striatum. In single-labeling preparations, aFGF mRNA-containing neurons were found to be codistributed with ChAT mRNA+ cells throughout all fields of basal forebrain, including the medial septum/diagonal band complex and striatum. By using a double-labeling (colormetric and isotopic) technique, high levels of colocalization (over 85%) of aFGF and ChAT mRNAs were observed in the medial septum, the diagonal bands of Broca, the magnocellular preoptic area, and the nucleus basalis of Meynert. The degree of colocalization was lower in the striatum, with 64% of the cholinergic cells in the caudate and 33% in the ventral striatum and olfactory tubercle labeled by the aFGF cRNA. These data demonstrate substantial regionally specific patterns of colocalization and support the hypothesis that, via an autocrine mechanism, aFGF provides local trophic support for cholinergic neurons in the basal forebrain and the striatum.

A subpopulation of striatal gabaergic neurons expresses the epidermal growth factor receptor.

Epidermal growth factor and transforming growth factor alpha are mitogenic polypeptides that act at the epidermal growth factor receptor, a protein tyrosine kinase.10,16,18,24 Studies have shown that epidermal growth factor and transforming growth factor alpha support the survival and promote the differentiation of central nervous system neurons in vitro.13,21,33 Messenger RNAs for both transforming growth factor alpha and the epidermal growth factor receptor have been identified in the adult and developing mammalian central nervous system, particularly within the neostriatum of young animals.11,15,27,28,30 However, the cell types that synthesize these messenger RNAs in striatum are not well understood. The present study investigates the hypothesis that epidermal growth factor receptor and transforming growth factor alpha are synthesized by striatal GABAergic neurons using double-labeling in situ hybridization in the rat. Most neurons within the neostriatum that intensely expressed messenger RNA for the 67,000 mol. wt isoform of glutamate decarboxylase also expressed messenger RNA for the epidermal growth factor receptor. Scattered striatal cells with neuronal morphology were immunoreactive for epidermal growth factor receptor protein, indicating that epidermal growth factor receptor messenger RNA expressed by striatal neurons is translated. Striatal neurons that expressed high levels of the 67,000 mol. wt isoform of glutamate decarboxylate messenger RNA did not appear to express transforming growth factor alpha messenger RNA. The present study indicates that epidermal growth factor receptor is synthesized by a subpopulation of GABAergic striatal neurons, supporting the hypothesis that transforming growth factor alpha and epidermal growth factor act directly upon neurons to produce their neurotrophic effects. These neurons may be GABAergic interneurons, which have been shown to be relatively resistant to degeneration in Huntington's disease and excitotoxic models of this disease.6,1,9

Expression of agrin mRNA is altered following seizures in adult rat brain.

Agrin mRNA is broadly distributed throughout the adult rat brain, consistent with its proposed role in synaptogenesis and the organization of synaptic proteins in the central nervous system. The present study examined the effect of neuronal activity on agrin mRNA expression in adult rat forebrain using the hilus lesion paradigm for seizure induction and in situ hybridization and polymerase chain reaction techniques for quantification and characterization of agrin mRNA content. Seizures induced rapid, prolonged, and region-specific changes in agrin mRNA expression with the most prominent alterations occurring in hippocampal and cortical neurons. However, there were no detectable perturbations in the relative abundance of alternatively spliced agrin transcripts in affected brain regions. Activity-dependent changes in agrin expression suggest a role for this protein in modifications of synaptic structure associated with functional synaptic plasticity.

NGF mRNA is expressed by GABAergic but not cholinergic neurons in rat basal forebrain.

Nerve growth factor (NGF) supports the survival and biosynthetic activities of basal forebrain cholinergic neurons and is expressed by neurons within lateral aspects of this system including the horizontal limb of the diagonal bands and magnocellular preoptic areas. In the present study, colormetric and isotopic in situ hybridization techniques were combined to identify the neurotransmitter phenotype of the NGF-producing cells in these two areas. Adult rat forebrain tissue was processed for the colocalization of mRNA for NGF with mRNA for either choline acetyltransferase, a cholinergic cell marker, or glutamic acid decarboxylase, a GABAergic cell marker. In both regions, many neurons were single-labeled for choline acetyltransferase mRNA, but cells containing both choline acetyltransferase and NGF mRNA were not detected. In these fields, virtually all NGF mRNA-positive neurons contained glutamic acid decarboxylase mRNA. The double-labeled cells comprised a subpopulation of GABAergic neurons; numerous cells labeled with glutamic acid decarboxylase cRNA alone were codistributed with the double-labeled neurons. These data demonstrate that in basal forebrain GABAergic neurons are the principal source of locally produced NGF.

Localization and alternative splicing of agrin mRNA in adult rat brain: transcripts encoding isoforms that aggregate acetylcholine receptors are not restricted to cholinergic regions.

Agrin is a protein implicated in the formation and maintenance of the neuromuscular junction. In addition to motor neurons, agrin mRNA has been detected in the brains of embryonic rat and chick and adult marine ray, suggesting that this molecule may also be involved in the formation of synapses between neurons. As a step toward understanding agrin's role in the CNS, we utilized Northern blot and in situ hybridization techniques to analyze the regional distribution and cellular localization of agrin mRNA in the spinal cord and brain of adult rats. The results of these studies indicate that the agrin mRNA is expressed predominantly by neurons broadly distributed throughout the adult CNS. Moreover, expression of agrin mRNA is not restricted to cholinergic structures or regions of the brain receiving cholinergic input. Recently, RNA isolated from rat embryonic spinal cord was shown to contain four alternatively spliced agrin mRNAs, referred to as agrin0, agrin8, agrin11, and agrin19, each of which encodes agrin proteins that are active in acetylcholine receptor aggregating assays (Ferns et al., 1992). Using the polymerase chain reaction we demonstrate that all four of these agrin transcripts are expressed within the adult CNS. Agrin0, agrin8, and agrin19 were present in all regions analyzed. In contrast, agrin11 was detected only in forebrain. Results of these studies indicate that both the level of expression and pattern of alternative splicing of agrin mRNA are differentially regulated in the brain. The broad and predominantly neuronal distribution of agrin mRNA in the adult brain suggests that, in addition to its role at the neuromuscular junction, agrin may play a role in formation and maintenance of synapses between neurons in the CNS.

Cellular localization of NGF and NT-3 mRNAs in postnatal rat forebrain.

The presence of transiently elevated levels of mRNA for nerve growth factor (NGF) and neurotrophin-3 (NT-3) in postnatal development of several brain areas suggests that these factors may be expressed by a greater number of cell types in the immature than in the adult brain. To evaluate this possibility, in situ hybridization was used to determine the cellular localization of NGF mRNA and NT-3 mRNA in hippocampus, cingulate cortex, posterolateral neocortex, thalamus, and cerebellum of postnatal rat. In areas expressing both neurotrophins (i.e., hippocampus, cingulate cortex, and anteroventral thalamus), NT-3 mRNA was detected at earlier ages than NGF mRNA. Patterns of hybridization in hippocampus and cerebellum indicate that NT-3 is expressed by neurons soon after leaving the mitotic cycle whereas NGF expression is a feature of more mature neurons. The exception to this pattern was NGF expression in the lateral geniculate nuclei which was present by Postnatal Day 1 and retained in the adult. Both neurotrophins were transiently expressed in several brain areas. The loss of expression with age was most striking in thalamus with transient expression of NT-3 mRNA by the majority of dorsal thalamic relay nuclei and of NGF mRNA by fewer nuclei including the posterior, anteroventral, ventrolateral, and ventromedial nuclei. NT-3 expression also was transient in caudal cingulate/retrosplenial cortex, hippocampal CA3 stratum pyramidale, and the granule cells of archicerebellum. In early postnatal cingulate and retrosplenial cortices there were reciprocal rostrocaudal gradients of NGF and NT-3 expression. These results suggest both distinct and overlapping functions for NT-3 and NGF in early developmental processes including involvement of NT-3 in cerebellar development and of NGF in the development and maintenance of visual afferents to thalamus. Patterns of neurotrophin expression in medial limbic cortex may establish trophic gradients which influence the topography of thalamic innervation.