Sensory and Sympathetic Neuron Development
In a few instances, neurotrophins have been shown to regulate the pathways of differentiation selected by neural precursors and to regulate the differentiation process, helping to determine the levels of expression of proteins essential for the normal physiological functions of differentiated neurons, such as neurotransmitters, ion channels, and receptors. For example, in vitro and in vivo, NGF promotes the differentiation of sympathoadrenal precursors into sympathetic neurons as opposed to adrenal chromaffin cells (Levi-Montalcini 1987
, Anderson 1993
). In contrast, glucocorticoids have been shown to suppress these responses to NGF, inhibiting differentiation into neurons and promoting differentiation into mature adrenal chromaffin cells. As glucocorticoids are present in high concentrations in the adrenal gland, they seem likely to regulate differentiation of sympathoadrenal precursors similarly in vivo. In vitro and in vivo, the actions of NGF and glucocorticoids on these precursors are largely irreversible and dramatic. The sympathetic neurons formed are permanently dependent on NGF for survival, whereas the chromaffin cells are not. The two differentiated cell types differ in their predominant transmitter (norepinephrine vs epinephrine). Morphologically they are distinct, and this clearly reflects differences in many molecular constituents, which are regulated either directly or indirectly by NGF and the glucocorticoids. In PC12 cells, a very brief exposure to NGF has been shown to result in long-term induction of a sodium channel gene (Toledo-Aral et al 1995
). This may serve as a model system for investigating how neurotrophins can cause irreversible fate changes within neurons.
As described above, precursors of murine sensory neurons do not appear to express Trk receptors in vivo or be affected directly by deficiencies in neurotrophins (Fariñas et al 1998
, 2000; Huang et al 1999a
). In mouse DRG, at least, initial generation of TrkB- and TrkC-expressing versus TrkA-expressing neurons appears to occur in two waves, dependent on sequential expression of neurogenins 2 and 1 (Ma et al 1999
). The neurotrophins function to maintain the viability of these neurons. They do not appear to guide their initial determination. There is some evidence indicating that the situation may be more complicated in the chicken embryo. There, evidence has been obtained that suggests a subpopulation of DRG precursors expresses TrkC, and available evidence suggests there is more dynamic regulation of Trk receptor expression after neurogenesis than is observed in mouse sensory ganglia (e.g. Rifkin et al 2000
). It seems unlikely, however, that the role of neurotrophins is very different there than in murine sensory ganglia.
Neurotrophins are important in regulating aspects of later sensory neuron development that, in some instances, control important aspects of neuronal phenotype. For example, NGF-responsive sensory neurons primarily convey nociceptive information and extend either unmyelinated C-fibers or thinly myelinated Aδ
fibers. They express small peptide transmitters, such as CGRP and substance P, specific receptors such as the capsaicin receptor, and distinct Na+
channels isoforms (e.g. Amaya et al 2000
, reviewed by Mendell et al 1999
). Expression levels of most of these proteins are regulated by NGF (e.g. Fjell et al 1999
; reviewed in Lewin & Barde 1996
, Mendell 1999
; Mendell et al 1999
). Although the absence of NGF during embryogenesis results in loss of almost all nociceptive neurons, at later ages withdrawal of NGF no longer kills these neurons. Instead, perturbation of NGF levels results in phenotypic changes. When NGF is sequestered during the early postnatal period, the properties of Aδ
fibers are dramatically changed (Ritter et al 1991
). Normally, many of these fibers are responsive to high threshold mechanical stimulation and are classified as high-threshold mechanoreceptors (HTMRs). Postnatal deficiency in NGF results in almost complete loss of HTMRs with a proportional increase in D-hair fibers, which respond to light touch. As this change is not associated with elevated apoptosis, the results suggest that the neurons of origin for the HTMR fibers are not lost but instead undergo a change in phenotype, becoming D-hair fibers that function as touch, but not pain, receptors. The central projections of D-hair and HTMR fibers have different termination zones in the substantia gelatinosa, and it is interesting that the phenotypic conversion induced by withdrawal of NGF does not result in inappropriate innervation by D-hair fibers of zones normally innervated by HTMR fibers (Lewin & Mendell 1996
). Central projections appear to be regulated to maintain appropriate modality connectivity.
Overexpression of NGF in skin using the keratin-14 promoter results in increased survival of both the C and Aδ
classes of nociceptive neurons and, in addition, affects the functional properties of these neurons (Stucky et al 1999
, Mendell et al 1999
, Stucky & Lewin 1999
). The percentage of Aδ
fibers responsive to nociceptive stimuli increases from 65% to 97%, which may reflect selective survival of these neurons. Even more notably, the percentage of C-fibers responsive to heat increases from 42% to 96%, an effect too large to be accounted for by their selective survival. In addition, the chronic presence of NGF in skin also affects the functional properties of heat-sensitive C-fibers, increasing their thermal responsiveness and lowering their mechanical responsiveness. It seems likely that regulation of VR-1, the capsaicin receptor, may be involved in these phenotypic changes.
NGF is not the only neurotrophic factor to regulate the phenotype of nociceptive neurons. Although all nociceptors are believed initially to express TrkA, a proportion of these begin subsequently to express c-ret together with one or more of the GFR adapter subunits (e.g. Snider & McMahon 1998
). In addition to expressing c-ret, these neurons are distinguished by expression of a binding site for the plant lectin isolectin B4. As development proceeds, their survival becomes dependent on GDNF family members (Molliver et al 1997
). Targeted disruption of the GFR-α
2 gene does not cause loss of cells expressing the isolectin B4 ligand but does result in a three-fold reduction in the percentage of isolectin B4 ligand-expressing neurons sensitive to heat (CL Stucky, J Rossi, MS Airaksinen, GR Lewin, unpublished observations). The results indicate that signaling mediated by a GDNF family member, most likely neurturin, is necessary for these neurons to manifest a nociceptive phenotype.
Continued presence of D-hair afferents has been shown to depend on NT-3 in early postnatal development and on NT-4 at later times (Airaksinen et al 1996
, Stucky et al 1998
). It is not certain whether these phenotypes are caused by neuronal loss or changes in neuronal phenotype.
Control of Target Innervation
Each of the neurotrophins has been shown to promote neurite outgrowth by responsive neurons in vitro. Elegant experiments have demonstrated that local NGF regulates the advance of sympathetic neuron growth cones (Campenot 1977
). The presence of NGF within a compartment was shown to be essential for axons to grow into that compartment, even when neurons received adequate trophic support. When neurons were seeded between two chambers, one with NGF and one with no neurotrophin, axons invaded only the chamber containing NGF. If at a later time NGF was withdrawn from a chamber, the axons stopped growing and slowly retracted. In addition to promoting growth, gradients of neurotrophins are able to steer growth cones in vitro (Gundersen & Barrett 1979
). It is intriguing that in these assays, whether a neurotrophin acts as a chemoattractant or a chemorepellent depends on cyclic nucleotide levels within neurons (Song et al 1997
, Song & Poo 1999
). The chemoattractive activities of NGF and BDNF, acting through TrkA and TrkB, respectively, are converted to chemorepellent activities by inhibitors of the cAMP signaling cascade. Effects of a PI-3 kinase inhibitor and of NGF signaling through a TrkA mutant lacking a putative PI-3 kinase docking site suggest that activation of PI-3 kinase is required for the chemoattractive response (Ming et al 1999
). It is intriguing that although the different Trk receptors are believed to function through similar signal transduction pathways, the chemoattractive activity of NT-3, acting through TrkC, is not affected by agents that affect cAMP-mediated signaling. Instead, inhibitors of cGMP signaling convert this chemotrophic response from attractive to repulsive (Song & Poo 1999
). These observations argue persuasively that there are fundamental differences in the signaling mediated by different Trk receptors.
Since the discovery of NGF, it has been appreciated that systematically applied neurotrophins affect innervation patterns in vivo (e.g. Levi-Montalcini 1987
). NGF was shown to increase innervation of tissues that receive sympathetic or sensory innervation normally and to induce aberrant innervation of tissues that normally are not innervated. In adults, neurotrophins are generally concentrated in targets of sensory and sympathetic targets (e.g. see Reichardt & Fariñas 1997
). Analyses of transgenic animals either lacking or expressing ectopically neurotrophins have provided many examples where disruptions of normal expression patterns of a neurotrophin results in perturbations of innervation, including aberrant routing of axons and interference with innervation of specific targets. For example, elevation of NGF in pancreatic islets using the insulin promoter induces dense sympathetic innervation of cells within the pancreatic islets, which normally are not innervated (Edwards et al 1989
). Elevation of NGF in the epidermis using the keratin-14 promoter induces similarly dense sympathetic innervation of the epidermis (Guidry et al 1998
). In this case also, the pattern of innervation is perturbed. Sympathetic innervation of the footpad vasculature and sweat glands is strongly inhibited. Instead, the sympathetic fibers are found in a plexus together with sensory fibers in the dermis. Sympathetic innervation is also distributed aberrantly in the vicinity of the mystacial pads (Davis et al 1997
). Overexpression of NGF under control of the keratin-14 promoter also increases greatly the density of sensory innervation, selectively promoting innervation by NGF-dependent nociceptors (Stucky et al 1999
). In contrast to sympathetic fibers, aberrant targeting of these endings was not detected. As a final example, in mice that overexpress BDNF under control of the nestin promoter, sensory fibers dependent on this neurotrophin appear to stall at sites of ectopic BDNF expression at the base of the tongue and fail to reach the gustatory papillae (Ringstedt et al 1999
). Fibers that do not traverse these sites of ectopic BDNF expression are able to reach and innervate their targets normally. Taken together, the results of these studies indicate that elevated expression of a neurotrophin in a region usually results in an increased density of innervation by axons from neurons that normally innervate that region. Overexpression in regions that are normally not innervated often, but not always, results in aberrant innervation by neurons responsive to that neurotrophin. This suggests that guidance and targeting clues are either masked or overridden by the presence of high levels of a neurotrophin. Consistent with this concept, recent studies in tissue culture have shown that uniform exposure of TrkA-expressing neurons to NGF results in a desensitization of chemotactic responses to gradients of netrin, BDNF, or myelin-associated glycoprotein, which suggests that these factors share common cytosolic signaling pathways (Ming et al 1999
In some instances, deficits or aberrancies in innervation observed in transgenic animals may reflect competitive phenomena. In analyses of innervation by sensory neuron fibers of mystacial pads, elevated innervation by TrkA-dependent sympathetic fibers was observed in mutants lacking BDNF or TrkB (Rice et al 1998
). Excessive innervation by TrkA-dependent sensory endings was seen in mice lacking BDNF, NT-4, or TrkB. When innervation of different classes of endings that detect mechanosensation was examined in these mutants, each ligand and Trk receptor was shown to support innervation of at least one type of mechanoreceptor (Fundin et al 1997
). Innervation of some endings is dependent on more than one neurotrophin or Trk receptor. For example, NT-3 is important for formation of all types of endings, but it may signal through different Trk receptors as development proceeds. In addition, the results suggested that BDNF signaling through TrkB may suppress Merkel innervation whereas NT-3 signaling through TrkC suppresses Ruffini innervation. There is not a single compelling explanation for these observations. Some sprouting may be attributable to loss of competition for a neurotrophin. Absence of TrkC-dependent endings, for example, may result in less NT-3 being transported out of the region. The elevated level of NT-3 remaining in the region may mediate sprouting of endings otherwise supported by NGF alone. Not all observations appear compatible with this model, however. Instead, the results suggest that neurotrophin-mediated signaling regulates, both positively and negatively, responses to other factors involved in sensory fiber targeting and differentiation.
Sensory Neuron Function
Neurotrophins have multiple interesting effects on the functional properties of sensory neurons extending beyond regulation of their survival. Essentially all modalities of sensory information are modulated in different ways by changes in the levels of these factors.
During early postnatal rodent development, both NT-3 and BDNF have been shown to regulate the development of the synapses formed between Ia afferents and motor neurons (Seebach et al 1999
). Chronic NT-3 results in larger monosynaptic excitatory postsynaptic potentials (EPSPs) and reduced polysynaptic components, whereas BDNF actually reduces the size of the monosynaptic EPSPs and increases the contribution of polysynaptic signaling. Infusion with TrkB-Ig also results in the appearance of larger monosynaptic EPSPs, which suggests that endogenous BDNF is an important modulator of development of these synapses. Infusion with TrkC-Ig had little effect. These data argue that levels of endogenous BDNF within the spinal cord control the comparative efficiencies of monosynaptic and polysynaptic signaling between Ia afferents and motor neurons. The role of endogenous NT-3 is less certain.
During the first postnatal week, but not subsequently, direct application of NT-3 has been shown to acutely potentiate the α
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor-mediated monosynaptic EPSP at the synapses formed by Ia afferents on motor neurons (Arvanov et al 2000
). Potentiation is long lasting and is prevented by an inhibitor of Trk receptor kinase activity. Initiation, but not maintenance, of potentiation requires N-methyl-d
-aspartate (NMDA) receptors and postsynaptic Ca2+
. The dependence on NMDA receptor function and Ca2+
are similar to the requirements for generation of long-term potentiaion (LTP) in the hippocampus (see Malenka & Nicoll 1999
). It is intriguing that EPSPs with similar properties evoked by stimulation of a different innervation pathway are not potentiated by NT-3, so the effects of NT-3 are synapse specific. NT-3 is effective at potentiating the strength of the Ia afferent-motor neuron synapse only during the first week, very likely because NMDA receptors are downregulated at later times.
Effects of peripheral nerve transection on monosynaptic Ia EPSPs, however, suggest that NT-3 from peripheral sources is also important in regulating the efficiency of synaptic function in adult animals. Peripheral transection results in long-term declines in conduction velocity and monosynaptic EPSP amplitude (Mendell et al 1999
). These declines can be prevented by infusion of NT-3 in the vicinity of the cut nerve ending, which suggests that interruption of NT-3 transport is the cause of the synaptic deficiencies observed after transection.
Although overexpression of NGF induces the changes described in the previous section that are likely to involve gene expression, acute NGF also has striking effects on nociceptors. Application of NGF to the undersurface of a patch of skin acutely sensitizes nociceptive C and Aδ
fibers to heat within 10 min (e.g. Shu & Mendell 1999a
). Sensitization is not seen in the skin of animals depleted of mast cells, so a major pathway mediating this response is believed to involve activation of mast cells by NGF, resulting in secretion from these cells of serotonin, histamine, and other agents, including NGF (e.g. see Levi-Montalcini et al 1996
). Acute application of NGF also sensitized the subsequent response of sensory neurons to capsaicin (Shu & Mendell 1999b
). As sensitization is seen in dissociated sensory neuron cultures, NGF must be acting directly on the sensory neurons.
One of the proteins known to be upregulated by NGF in sensory neurons is the neurotrophin BDNF (Michael et al 1997
). There is evidence that BDNF is transported to both peripheral and central terminals of nociceptive sensory neurons. In the periphery, BDNF and NT-4 have been shown to acutely sensitize nociceptive fibers by a pathway that requires the presence of mast cells (Shu et al 1999
, Rueff & Mendell 1996
). Sensory neuron–derived BDNF also appears to act centrally (Mannion et al 1999
, Woolf & Costigan 1999
). Perfusion of the spinal cord with TrkB-IgG has been shown to prevent the progressive hypersensitivity elicited by low-intensity tactile stimulation of inflamed tissues.
Cortical Circuitry and Function
Several neurotrophins are expressed in the neocortex and hippocampus during development, and their expression continues in adult animals, which suggests that they have functions extending beyond initial development. NGF, for example, is widely expressed in both the developing and adult neocortex (e.g. Large et al 1986
). Projections from the cholinergic basal forebrain extend throughout the neocortex and hippocampus (e.g. Mesulam et al 1983
). The fibers of these projections express TrkA (e.g. Sobreviela et al 1994
), and expression in these neurons of proteins associated with cholinergic function, such as choline-o-acetyl transferase, is increased by infusion of NGF (e.g. Hefti et al 1989
). NGF infusion has been shown to attenuate the behavioral deficits associated with cholinergic atrophy (e.g. Fischer et al 1987
). Maintenance of normal function of these neurons in adult animals is sensitive to small perturbations of NGF levels. For example, animals heterozygous for a mutation in the NGF
gene express approximately half the normal level of NGF mRNA and protein and have significant deficits in memory acquisition and retention, which can be corrected by prolonged infusion of NGF (Chen et al 1997
). Mice lacking TrkA have also been shown to have deficits in cholinergic projections from the basal forebrain (Smeyne et al 1994
Both BDNF and TrkB are widely expressed in the developing and adult hippocampus and neocortex (e.g. Cellerino et al 1996
). BDNF mRNA is present in excitatory pyramidal neurons, but not in GABAergic inhibitory interneurons. TrkB is expressed by both classes of neurons, although its expression is higher in inhibitory interneurons. In addition, expression of BDNF is regulated by both sensory input and electrical activity. For example, induction of seizures in the hippocampus strongly induces BDNF expression (e.g. Kornblum et al 1997
). In the visual and somatosensory cortices, expression has been shown to be regulated by sensory inputs, with deprivation reducing expression of this neurotrophin (e.g. Castren et al 1992
, Rocamora et al 1996
, Singh et al 1997
). Several promoters control expression of BDNF mRNA, and one of these is regulated by Ca2+
acting through Ca2+
-calmodulin–dependent protein kinase IV to phosphorylate and activate the transcription factor CREB (Tao et al 1998
, Shieh et al 1998
). BDNF is sorted into a regulated secretory pathway in hippocampal neurons (e.g. Farhadi et al 2000
), so increases in neuronal activity should both activate transcription of the BDNF
gene and increase secretion of the BDNF protein.
Expression of TrkB has also been shown to be modestly increased by activity (e.g. Castren et al 1992
). Equally important, surface expression of TrkB is also regulated by activity (Meyer-Franke et al 1998
). In the absence of activity, this protein appears to be largely sequestered into cytoplasmic vesicles. This result suggests that neurons become more responsive to BDNF as a result of activity. At the subcellular level, regulation of TrkB distribution may provide a mechanism by which active and inactive synapses differ in their responsiveness to BDNF, thereby regulating actin dynamics, glutamate receptor activity, and other functions important for adjusting synaptic function. In the absence of a direct demonstration of activity-regulated TrkB trafficking in vivo, however, this should be considered only an intriguing possibility.
In the neocortex, BDNF signaling through TrkB has been implicated in both development and maintainance of cortical circuitry. BDNF expression in excitatory neurons is promoted by activity, whereas increased release of BDNF can be expected to enhance the effectiveness of inhibitory interneurons. This has raised the possibility that BDNF-to-TrkB signaling modulates an autoregulatory circuit between excitatory pyramidal cells and inhibitory interneurons. In mixed cultures of postnatal rat cortical neurons, activity blockage has been shown to reduce reversibly GABA expression in interneurons and to reduce GABA-mediated inhibition on pyramidal cells (Rutherford et al 1997
). In these cultures, the rates of firing are stabilized by scaling of the amplitude of AMPA receptor-mediated synaptic inputs (Rutherford et al 1998
). These effects appear to be modulated by endogenous BDNF, as effects of activity blockade can be prevented by exogenous BDNF and effects of activity blockade are mimicked by a BDNF scavenger (Desai et al 1999
). It is attractive to imagine that this autoregulatory circuit functions in vivo.
Formation of Ocular Dominance Columns
The density of innervation of layer IV by afferents from the thalamus is increased by exogenous BDNF and reduced by a scavenger of endogenous BDNF, TrkB-IgG (Cabelli et al 1997
). Both agents appear to interfere with sorting of these afferents into ocular dominance columns, raising the possibility that competition for limiting amounts of BDNF by these afferents is involved in some manner in the sorting mechanism. In addition, infusion of NT-4 into the visual cortex during the critical period has been shown to prevent many of the consequences of monocular deprivation (Gillespie et al 2000
). In the presence of NT-4, neurons remain responsive to stimuli from the deprived eye. Even after responses to the deprived eye are lost, infusion of NT-4 is able to restore them. These observations suggest that TrkB activation during the critical period promotes connectivity independent of correlated activity.
The function of inhibitory interneurons is essential for formation of ocular dominance columns (e.g. Hensch et al 1998
), and maturation of these neurons is regulated by BDNF in vitro and in vivo. In initial analysis of the BDNF
mutant, several deficits in interneuron maturation were detected, including expression of Ca2+
-binding proteins and peptide neurotransmitters (Jones et al 1994
). Recently, BDNF has been overexpressed in excitatory pyramidal neurons by use of the Ca2+
-calmodulin–dependent protein kinase II promoter (Huang et al 1999c
). In these animals, maturation of interneurons is accelerated, as assessed by expression and synaptic localization of glutamate decarboxylase, expression of parvalbumin, and the strength of inhibitory postsynaptic potentials. In addition, the critical period of ocular dominance plasticity begins and terminates precociously, and the acuity of vision increases on an accelerated time course (Huang et al 1999c
, Hanover et al 1999
). As visual stimulation also increased expression of BDNF within pyramidal neurons, the results suggest that early sensory stimulation acts to promote maturation of interneurons through BDNF-to-TrkB signaling. Interneurons in turn promote the refinements in synaptic connectivity needed for maturation of the cortex. The results suggest that refinement of cortical circuitry is driven by intracortical mechanisms, which then drive the sorting of thalamic afferents. Recent work supports this model (e.g. Trachtenberg et al 2000
Another mechanism by which neurotrophins may control development and changes in cortical circuitry is through control of dendritic and axonal arbors. Neurotrophins affect neuronal morphologies at many levels in the visual pathway. Local application of BDNF or of a BDNF scavenger, for example, has been shown to decrease and increase, respectively, the complexity of Xenopus retinal ganglion cell dendritic arbors (Lom & Cohen-Cory 1999
). Local application to the optic tectum of these same agents has different effects on axonal branching patterns, with BDNF increasing the complexity of retinal ganglion cell–derived axonal arbors and a BDNF scavenger having the opposite effect. NT-4 has been shown to prevent the atrophy of lateral geniculate neurons seen after monocular visual deprivation (Riddle et al 1995
). Applications of BDNF, NT-3, or NT-4 to slices of neonatal neocortex have been shown to regulate the dendritic morphologies of pyramidal cells over comparatively short time spans (e.g. Horch et al 1999
; reviewed in McAllister et al 1999
). The effects are distinct, cell specific, and layer specific. For example, BDNF was observed to promote dendritic arborization of neurons in layers IV and V, but BDNF actually inhibited arborization by neurons in layer VI (McAllister et al 1997
; see also Castellani & Boltz 1999
). In layer IV, NT-3 was shown to oppose the stimulation of dendritic arborization promoted by BDNF. In layer VI, BDNF inhibited the stimulation of arborization induced by NT-3. Apical and basal dendrites of the same neurons responded differently to the same neurotrophin (e.g. McAllister et al 1995
). Many of the observed effects were prevented by blocking electrical activity (e.g. McAllister et al 1996
). Specific deletion of the TrkB
gene in pyramidal neurons also results in striking changes in these cells during postnatal development, with significant retraction of dendrites observed at 6 weeks and loss of many neurons seen at 10 weeks of age (Xu et al 2000b
). Although the changes in dendritic morphology and subsequent loss of neurons were cell autonomous, requiring deletion of the TrkB
gene within the affected neurons, changes in gene expression were also seen that were not cell autonomous, i.e. they were observed in neurons that continued to express TrkB. It seems likely that circuit perturbation as a result of alterations of dendritic morphologies accounts for these changes.
Taken together, the results described above indicate that neurotrophins are important in regulating establishment and function of cortical circuits. Within the visual system, they regulate development of retinal ganglion cell axonal and dendritic arbors, thalamic afferents, cortical pyramidal cells, and cortical interneurons, with profound effects on cortical function. Although mechanisms by which neurotrophins influence axonal and dendritic morphologies in vivo have not be examined, they almost certainly involve regulation of the Cdc-42/Rac/Rho family of small GTPases. Aberrant expression of constitutively active and dominant negative mutants of these proteins have been shown to have dramatic effects on dendritic branch patterns and spine density (e.g. Li et al 2000
, Nakayama et al 2000
Synaptic Strength and Plasticity
Mechanisms underlying establishment of LTP between afferents from CA3 pyramidal cells and postsynaptic CA1 pyramidal neurons in the hippocampus have been of intense interest, as these mechanisms are believed to provide a paradigm for regulation of synaptic strength and plasticity (reviewed in Malenka & Nicoll 1999
). BDNF is expressed in CA3 and CA1 pyramidal neurons within the hippocampus, and TrkB is expressed by almost all hippocampal neurons, including dentate granule cells, CA3 and CA1 pyramidal cells, and inhibitory interneurons. It is interesting that LTP is greatly reduced in BDNF
mutants, both in homozygotes and in heterozygotes (e.g. Korte et al 1995
, Patterson et al 1996
). Long-lasting, protein synthesis-dependent LTP is not seen in these mutants (Korte et al 1998
). There are also deficits in long-lasting LTP and in memory consolidation in the hippocampus in mutant mice lacking NT-4 (Xie et al 2000
). CA1 LTP is also reduced in TrkB mutant heterozygotes and in a mouse mutant that expresses reduced levels of TrkB (Minichiello et al 1999
, Xu et al 2000a
). Signaling through p75NTR does not appear to be important, because there is very little expression of this receptor within the hippocampus and because functional antibodies to p75NTR do not affect LTP (Xu et al 2000a
). Loss of TrkB from excitatory pyramidal neurons in the hippocampus and forebrain interferes with memory acquisition and consolidation in many learning paradigms (Minichiello et al 1999
In these mutants, the observed reductions in synaptic plasticity probably reflect functional, not developmental, deficits. First, the hippocampi of these animals appear to be morphologically normal. Second, a very similar inhibition of LTP can be seen following acute application of the BDNF/NT-4 scavenger TrkB-IgG to hippocampal slices (e.g. Chen et al 1999
). Finally, the deficits observed in BDNF mutant heterozygotes and homozygotes can be rescued by exposure of hippocampal slices to BDNF (Korte et al 1995
, Patterson et al 1996
). The enhanced efficiency of synaptic transmission observed after induction of LTP is largely mediated by an NMDA receptor-dependent increase in AMPA receptor function (reviewed in Malenka & Nicoll 1999
). Most evidence suggests, however, that BDNF-to-TrkB signaling is not directly involved in the biochemical changes underlying LTP within the postsynaptic cells, but instead modulates the competence of presynaptic nerve terminals to generate the repetitive exocytotic events needed to modify the responses of these postsynaptic neurons. In one set of experiments, LTP was generated normally in a TrkB hypomorph by a low-frequency–paired depolarization protocol that specifically assesses properties of postsynaptic cells and puts minimal demands on presynaptic terminal function (Xu et al 2000a
). In addition, AMPA and NMDA receptor functions appeared to be normal in the postsynaptic neurons. In contrast, the ability of presynaptic nerve terminals to respond to repetitive pulses of stimulation was clearly impaired. Consistent with a presynaptic deficit, BDNF has been shown to enhance synaptic vesicle release in response to tetanic stimulation, possibly by promoting docking of synaptic vesicles to the presynaptic membrane (e.g. Gottschalk et al 1998
, Pozzo-Miller et al 1999
). Also consistent with a presynaptic deficit, LTP is further reduced in a TrkB
mutant heterozygote by elimination of the remaining functional TrkB
gene from excitatory pyramidal cells in both the CA3 and CA1 regions, eliminating TrkB from both presynaptic and postsynaptic neurons (Minichiello et al 1999
). LTP is not further reduced, however, by deletion of the TrkB
gene solely within the postsynaptic CA1 pyramidal neurons in the mouse mutant that expressed reduced TrkB levels (Xu et al 2000a
). All these data argue that BDNF-to-TrkB signaling is crucial in presynaptic nerve terminals in CA1 that are derived from neurons in CA3. In addition, BDNF has been shown to decrease inhibitory postsynaptic currents on CA1 pyramidal cells (Tanaka et al 1997
, Frerking et al 1998
), so it is possible that part of the LTP deficit reflects an increase in inhibitory signaling by GABAergic interneurons in the absence of normal TrkB function within these cells. BDNF has been shown to affect NMDA function in hippocampal neurons in culture by increasing the open probability of their channels (Levine et al 1998
). The experiments described above suggest that TrkB did not modify the function of these channels during tetanic stimulation.
At many developing and mature synapses, application of a neurotrophin acutely stimulates neurotransmitter release. This has been most intensely studied at the CA1 synapse in the hippocampus and in developing Xenopus neuromuscular synapses (e.g. Kang & Schuman 1996
, Wang & Poo 1997
). In the developing neuromuscular cultures, the neurotrophins have both pre- and postsynaptic effects, increasing spontaneous and evoked release of synaptic vesicles and changing the kinetics of opening of the acetylcholine receptor (e.g. Wang & Poo 1997
; Schinder et al 2000
). Low levels of neurotrophin act synergistically with synaptic terminal depolarization (e.g. Boulanger & Poo 1999b
). To be effective, the cell must be depolarized during the period of neurotrophin exposure. A cAMP agonist also synergizes with BDNF to potentiate spontaneous and action potential–evoked neurotransmitter release (Boulanger & Poo 1999a
). The synergistic effect of depolarization appears to be caused by an increase in the level of cAMP. What is surprising is that a very similar presynaptic potentiation induced by NT-3 application is not affected by agents that inhibit cAMP-mediated signaling.
In hippocampal cultures, BDNF also potentiates release from presynaptic nerve terminals, but potentiation depends on inositol tris-phosphate gated Ca2+
stores in presynaptic nerve terminals (Li et al 1998a
). LTP of synaptic transmission at the CA1 synapse in hippocampal slices has been seen by some, but not all, workers (e.g. Kang & Schuman 1995
, Figurov et al 1996
). The difference appears to be caused by differences in slice culture and conditions of application of BDNF. BDNF must be applied rapidly to slices to observe potentiation (Kang et al 1996
). In conditions where it is observed, LTP also depends on inositol tris-phosphate gated Ca2+
stores, but in addition it requires local protein synthesis (Kang & Schuman 1996
). In studies using slices of postnatal rat visual cortex, potentiation of synaptic transmission from layer IV cells to cells in layers II/III was seen only with very high concentrations of BDNF (Akaneya et al 1997
). At lower concentrations, BDNF enhanced the magnitude of LTP without potentiating basal synaptic transmission. In both the hippocampus and postnatal visual cortex, BDNF enhances LTP in conditions where it does not potentiate synaptic transmission. The acute changes in BDNF concentration needed to potentiate synaptic transmission probably occur only rarely in vivo. BDNF also potentiates transmitter release from brain synaptosomes (Jovanovic et al 2000
). In this case, it has been elegantly demonstrated that a MAP kinase phosphorylation of synapsin I mediates this response. The response is not seen in synaptosomes isolated from a synapsin I
mutant and is prevented by inhibitors of the MAP kinase cascade. Phosphorylation of synapsins by MAP kinase has been shown to regulate their interactions with the actin cytoskeleton (Jovanovic et al 1996
), so the MAP kinase cascade may potentiate synaptic transmission by releasing synaptic vesicles from the cytoskeleton, facilitating their entry into a exocytosis-competent pool. It will be interesting to determine whether BDNF-to-TrkB signaling regulates generation of LTP in mice lacking synapsin I.