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PKA and neural facilitation (Short and long term potentiation - a model for learning): Studies in mammals (part 2/2)

Activation of adenylyl cyclase and the consequent production of cAMP is a process that has been shown to be central to invertebrate model systems of information storage. In the vertebrate brain, it has been suggested that a presynaptic cascade involving Ca influx, cAMP production, and subsequent activation of cAMP-dependent protein kinase is necessary for induction of long-term potentiation (LTP) at the cerebellar parallel fiber-Purkinje cell synapse. It has been suggested that use-dependent modification of the strength of the parallel fiber-Purkinje cell synapse in the cerebellar cortex is necessary for certain forms of motor learning, including adaptation of the vestibulo-ocular reflex and associative eyeblink conditioning. One cellular model system that has been proposed as a mechanism for such information storage is cerebellar long-term depression (LTD), in which coactivation of climbing fiber and parallel fiber inputs to a Purkinje cell induces a persistent, input-specific depression of the parallel fiber-Purkinje cell synapse. The converse phenomenon, cerebellar LTP, has also been described. In this protocol, the parallel fiber-Purkinje cell synapse is strengthened by repetitive parallel fiber stimulation at low (2-8 Hz) frequencies, thus endowing this synapse with the important capacity of use-dependent bidirectional modification. Mutant mice in which the major Ca-sensitive adenylyl cyclase isoform of cerebellar cortex (type I) is deleted have been used to show that activation of cAMP-dependent protein kinase results in an 65% reduction in cerebellar Ca-sensitive cyclase activity and a nearly complete blockade of cerebellar LTP, assessed by using granule cell-Purkinje cell pairs in culture. This blockade is not accompanied by alterations in a number of basal electrophysiological parameters and may be bypassed by application of an exogenous cAMP analog, suggesting that it results specifically from deletion of the type I adenylyl cyclase (Storm, 1998).

The electrophysiological findings reported here are consistent with a previously proposed model for hippocampal mossy fiber-CA3 and cerebellar LTP induction. In this model, Ca influx through presynaptic voltage-gated channels activates a Ca/CaM-sensitive adenylyl cyclase, resulting in production of cAMP, activation of PKA, and the consequent phosphorylation of proteins that control synaptic strength. PKA could conceivably exert its effect either locally, in the presynaptic terminal, or at other sites through activation of cascades linked to a diffusable messenger. However, several findings have been proposed in support of the hypothesis that the locus of cerebellar LTP expression is presynaptic. Cerebellar LTP expression is associated with a decrease in the rate of synaptic failures when measured in granule cell-Purkinje cell pairs. In addition, it is associated with a decrease in paired-pulse facilitation. Perhaps the most convincing evidence for a presynaptic locus of expression comes from work in cell culture that takes advantage of the fact that the rapid inward current produced in glial cells by activation of adjacent granule cells is 90% mediated by activation of AMPA/kainate receptors and 10% by electrogenic glutamate re-uptake. Stimulation of the granule neuron can give rise to the LTP of either the total glial synaptic current or the pharamacologically isolated glial re-uptake current. LTP recorded using either of these detector currents has properties indistinguishable from that of granule neuron-Purkinje neuron LTP, as seen in the current study. Therefore, the site of the adenylyl cyclase and PKA action that produces LTP is almost certainly presynaptic (Storm, 1998).

The mechanism(s) responsible for PKA enhancement of neurotransmitter release have not been elucidated but may involve direct phosphorylation of proteins of the secretory machinery. Treatment of cultured cerebellar granule cells with forskolin or cAMP analogs results in an increase in presynaptic vesicular cycling as detected using an immunocytochemical technique. Forskolin or cAMP analogs do not affect resting Ca levels in cultured hippocampal neurons, and these drugs can potentiate transmitter release evoked directly by ruthenium red. More recently, Forskolin-induced potentiation of the parallel fiber-Purkinje cells EPSC is not associated with alterations in intraterminal resting Ca, evoked Ca influx, or changes in the presynaptic fiber volley, but is associated with an increase in mEPSC frequency. Taken together, these studies suggest that the effect of increasing intraterminal cAMP on the probability of release can be completely accounted for by changes in the secretory machinery. The hypothesis that PKA activation exerts its effects on release through alteration of secretory machinery is particularly appealing because the synaptic vesicle protein Rab3A, which is an effector for the PKA substrate rabphilin 3, is essential for hippocampal mossy fiber LTP (Storm, 1998 and references).

Long-term potentiation (LTP) is a form of synaptic plasticity that has been extensively studied as a putative mechanism underlying learning and memory. A late phase of LTP occurring 3-5 hours after stimulation and depending on transcription, protein synthesis and cyclic-AMP-dependent protein kinase (protein kinase A, or PKA) has been described, but it is not known whether transcription of presynaptic and/or postsynaptic genes is required to support late-phase LTP. Late-phase LTP can be obtained in rat hippocampal CA1 mini-slices in which the cell bodies of presynaptic Schaffer collateral/commissural fibers are removed. Thus, transcription of presynaptic genes is not necessary to support maintenance of late-phase LTP. The AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate) receptor is the predominant mediator of the ionotropic response to synaptically released glutamate in the hippocampus and it has been implicated in LTP maintenance. Synthesis of AMPA receptor subunits is increased three hours after LTP induction: this effect on the synthesis of the AMPA receptor is blocked by inhibitors of PKA and of transcription. These results support the idea of a postsynaptic mechanism maintaining late-phase LTP, in which AMPA receptor synthesis is increased as a result of PKA-dependent gene transcription (Nayak. 1998).

In 1937, Papez proposed that the neuroanatomical substratum of emotion consists of the limbic lobe, a ring of primitive cortex that surrounds the brain stem. One of the major advances in the modern neurobiological study of emotion has been the realization that of the various structures of the limbic lobes, only the amygdala is critical for the expression of emotion. Lesions of the amygdala in humans interfere with the perception of pain, and electrical stimulation of the amygdala produces feelings of fear and apprehension. Moreover, functional magnetic resonance imaging (fMRI) studies have revealed that stimuli that elicit fear affect blood flow to the amygdala in humans. In experimental animals, the amygdala similarly is essential for both instinctive and learned expressions of fear. Damage to the amygdala produces tameness and affects a variety of autonomic responses associated with emotional behavior. Recent physiological experiments on the role of the amygdala in emotion have focused on the lateral nucleus because it is important for conditioned fear produced by pairing a neutral tone with a fear-inducing electric shock. Auditory information critical for conditioning of fear comes into the lateral nucleus of the amygdala via two routes: directly from the medial geniculate nucleus and indirectly from the auditory cortex. The synapses of both of these projections undergo long-term potentiation (LTP) thought to be essential for memory storage related to fear (Y, Y, Huang, 1998 and references).

Whereas much is now known about the behavioral importance of the lateral nucleus of the amygdala for the storage of implicit memories of fear, little is known in molecular terms about the signal transduction pathways required for long-term potentiation (LTP) in this nucleus. Using brain slices containing the amygdala, LTP was studied in the pathway from external capsule to the lateral nucleus, a pathway that mediates information from the auditory cortex important for fear conditioning. The induction of LTP is postsynaptic; it is dependent on postsynaptic depolarization, on the influx of Ca2+ into the postsynaptic cell and, at least in part, on the activation of N-methyl-D-aspartate (NMDA) receptors. To determine whether the induction of amygdala LTP is pre- or postsynaptic, an examination was carried out to see if Ca2+ influx into the postsynaptic neuron is required. To address this question, the postsynaptic cell was loaded with the Ca2+ chelator BAPTA. In cells loaded with BAPTA, one train of tetanus (100 Hz, 1 S), which normally induces LTP, produces only a transient increase of the EPSP that decays to the baseline by 5 min after tetanus. The LTP is reduced from 145% in control cells to 98% in the BAPTA-loaded cells by 20 min after the tetanus. The effect of the Ca2+ chelator on LTP supports the involvement of a postsynaptic mechanism (Y. Y. Huang, 1998).

These results indicated that induction of LTP in the lateral amygdala is dependent on postsynaptic depolarization and on Ca2+ influx into the postsynaptic cell. Where is the site of expression for this form of LTP? As a first step in localizing the site of expression, the effects of LTP were examined on PPF, a well-defined presynaptic process in which the residual Ca2+ influx following the first of two presynaptic action potentials leads to enhancement of the transmitter release in response to the second action potential. The LTP is associated with a decrease of paired-pulse facilitation (PPF) and is blocked by bath application but not blocked by postsynaptic injection of inhibitors of the cyclic adenosine monophosphate-dependent (cAMP-dependent) protein kinase (PKA). Consistent with the possibility that expression of LTP might involve PKA presynaptically, the adenylyl cyclase activator forskolin induces synaptic potentiation of this pathway that also is associated with a decrease of PPF, and this potentiation occludes the tetanus-induced LTP (Y. Y. Huang, 1998).

Thus the LTP produced in lateral amygdala by stimulation of the external capsule mediating information from the auditory cortex is dependent on postsynaptic depolarization, on Ca2+ influx into the postsynaptic cell, and at least under certain conditions, on activation of NMDA receptors. These steps are similar to those utilized in Schaffer collateral LTP and are different from mossy fiber LTP. However, LTP in the amygdala is associated with a profound depression of PPF. In this feature, LTP of the amygdala is more reminiscent of LTP evident in the hippocampal mossy fiber and cerebellar parallel fiber pathways, two forms of LTP involving a PKA-mediated presynaptic mechanism. Consistent with a possible involvement of PKA in amygdala LTP, it was found that the LTP is blocked by Rp-cAMPs (an inhibitor of cyclic AMP) when that inhibitor is put in the bathing solution but not when it is injected into the postsynaptic cell. Moreover, forskolin, the adenylyl cyclase activator, induces a potentiation that occludes LTP induced by tetanus, and like the tetanus, forskolin also leads to a depression of paired-pulse facilitation. A PKA-dependent mechanism for presynaptic facilitation has earlier been found in Aplysia and crayfish. The finding that PKA may be important for LTP in the amygdala, and the previous finding of its importance for LTP in both the mossy fiber and parallel fiber pathways, raise the possibility that PKA may be commonly involved in the early phase of LTP when the expression of that phase has a presynaptic component. Moreover, comparison of the properties of LTP in the amygdala, and in the Schaffer collateral, mossy fiber, and parallel fiber pathways, suggests that a variety of subtly different cellular physiological forms of LTP are likely to exist in the brain, each with its own distinctive properties. It seems attractive to suggest that these different forms of LTP may, however, operate by recruiting different combinations of common and conserved signal transduction modules made up of a relatively restricted repertoire of kinase and phosphatase pathways (Y. Y. Huang, 1998 and references).

There are at least two temporally distinct phases of memory storage: a short-term memory lasting minutes and a long-term memory lasting days or longer. These two phases differ not only in their time courses, but also in their molecular mechanisms: long-term memory, but not the short-term form, requires the synthesis of new proteins. Recent studies in Aplysia and mice have revealed that these distinct stages in behavioral memory are reflected in distinct phases of synaptic plasticity. In Aplysia, these stages have been particularly well studied in the context of sensitization, a form of learning in which an animal learns to strengthen its reflex responses to previously neutral stimuli following the presentation of an aversive stimulus. The short- and long-term memory for sensitization are mirrored by the short- and long-term facilitation of the synaptic connections between the sensory and motor neurons that mediate this reflex. This monosynaptic component can be examined not only in the intact animal, but also in a single sensory neuron cultured with its target postsynaptic motor neuron. At this cultured synapse, one pulse of 5-HT, a neurotransmitter released in vivo by interneurons activated by the sensitizing tail stimuli, produces a PKA- and PKC-mediated short-term facilitation lasting only minutes. By contrast, five spaced pulses of 5-HT elicit a long-term facilitation lasting more than 24 hr. With five pulses of 5-HT, PKA recruits MAP kinase and both translocate to the nucleus. Here, they activate CREB1 and derepress CREB2, leading to the induction of a set of immediate-early genes. Long-term facilitation is further associated with the growth of new synaptic connections (Casadio, 1999 and references therein).

A single bifurcated sensory neuron is plated in contact with two spatially separated postsynaptic motor neurons; a single synapse or group of synapses can be modified independently in a protein synthesis-dependent manner. This spatially restricted synapse specific facilitation requires the activity of CREB1 in the nucleus as well as local protein synthesis in the 5-HT-treated processes of the sensory cell. In addition to synapse-specific facilitation, a second phenomenon referred to as 'synaptic capture' has been characterized. Once synapse-specific long-term facilitation has been initiated by microperfusing five pulses of 5-HT onto one branch of the sensory neuron, a single pulse of 5-HT, which per se induces only transient facilitation, is able to recruit long-term facilitation when applied to a second branch. This synaptic capture does not require local protein synthesis (Casadio, 1999 and references therein).

As is generally the case with neurons, the sensory neuron is a highly polarized cell with several functionally distinct and spatially separate compartments, each of which is selectively innervated. In fact, in the intact animal, the endings of different serotonergic interneurons contact the sensory neurons at different sites, including its pre- and post-synaptic terminals and processes and its cell body. What then are the functional consequences of applying 5-HT selectively to the cell body? Five pulses of 5-HT applied selectively to the soma of the sensory neuron produce a cell-wide long-term facilitation (a third phenomenon) that requires CREB1-mediated transcription, but that does not persist and is not associated with growth. A similar cell-wide facilitation is obtained by injecting recombinant phospho-CREB1 protein into the sensory neuron. Cell-wide facilitation can be sustained and growth can be captured by a single pulse of 5-HT. Thus, a single pulse of 5-HT serves two functions: (1) it allows for 24 hr facilitation and growth by means of a PKA-mediated covalent modification; (2) it stabilizes growth and facilitation in a protein synthesis-dependent way to produce facilitation that persists at least 72 hr. By systematically comparing cell-wide facilitation to synapse-specific facilitation and synaptic capture, it has been found that each represents a distinct form of CREB-dependent, long-lasting synaptic plasticity. Thus, a single neuron has multiple long-term mechanisms for temporally and spatially integrating stimuli to produce transcription-dependent long-lasting changes in synaptic strength (Casadio, 1999).

These studies demonstrate that CREB-mediated long-term plasticity is not a unitary phenomenon. In fact, the same neuron can undergo four different CREB-mediated forms of long-term synaptic plasticity, depending on the site and the number of repetitions of modulatory input: synapse-specific long-term facilitation, capture of the synapse-specific form, cell-wide long-term facilitation, and capture of the cell-wide form (a fourth phenomenon). The experimental paradigm for the fourth phenomenon, capture of the cell-wide form, involves 5 pulses of 5-HT to the cell body rather than to a single branch as is carried out in the synapse specific long term paradigm. These five pulses create cell wide facilitation. In the case of this cell-wide capture, a single pulse to a branch, constituting the capture event, results in new varicosities. Although all four processes share at least one common nuclear mechanism (CREB-mediated transcription), each has distinct properties. Synapse-specific long-term facilitation initiated by five repeated pulses of 5-HT stimuli at a synapse is accompanied by growth, persists for at least 72 hr, and requires local protein synthesis for both its retrograde signal and for the stabilization of growth. Capture of the synapse-specific long-term facilitation by a marked synapse is smaller in amplitude than synapse-specific facilitation but is also accompanied by growth, is persistent, and requires local protein synthesis only for stabilization. Cell-wide long-term facilitation, generated by repeated modulatory stimuli at the cell body, is transient. This cell-wide long-term facilitation, however, can be captured by a marking signal that stimulates growth and converts the facilitation to a persistent form (referring to the fourth phenomenon of capture of the cell-wide form) (Casadio, 1999).

The finding that cell-wide, CREB-dependent cell-wide facilitation can occur in the absence of any synaptic growth was unexpected, since long-lasting increases in synaptic strength and long-term facilitation have been thought to result, at least in part, from the growth of new synaptic connections. How, then, might an increase in synaptic strength that requires transcription and translation persist for 24 hr in the absence of synaptic growth? One possibility is the recruitment of 'silent synapses'. In the intact animal, approximately 60% of the presynaptic varicosities of the sensory neurons do not have active release sites. When long-term sensitization is produced in the animal, it leads to both an increase in the number of varicosities and to an increase in the percentage of varicosities that have active zones. Whereas the increase in varicosity number persists as long as the memory, changes in the size of active zones only last a few days. Since only 50% of the varicosities in cultured sensory neurons have active zones, cell body application of 5-HT might change synaptic strength at 24 hr by increasing the incidence of active zones (Casadio, 1999).

CREB-mediated transcription appears to be necessary for the initial establishment of all four forms of synaptic plasticity, but it is not sufficient for the self-maintained stabilization of the plastic changes. To obtain persistent facilitation and the growth of new synaptic connections, one also needs a single pulse of 5-HT applied to the synapse. This single pulse by itself is only able to induce short-term facilitation. But in a cell where CREB-mediated transcription is induced by repeated stimuli, the single pulse also marks a synapse so as to allow long-term facilitation to become persistent and to be accompanied by growth of new terminals. Thus, one of the surprising functions of the short-term process is to convert a transient long-term process into a persistent one. These results illustrate again, as did earlier studies, that the short-term process serves two functions: (1) a single pulse of 5-HT alone produces a selective synapse-specific enhancement of synaptic strength that contributes to short-term memory lasting minutes; (2) in conjunction with the activation of CREB by repeated stimuli given to any other synapse or to the cell body, a single pulse of 5-HT will also act to ''mark'' that synapse for persistent synaptic facilitation and growth (Casadio, 1999).

Studies of the molecular nature of the mark necessary for synapse-specific facilitation and for capture suggest that there are two components to the marking signals: a covalent mark for the initiation of synapse-specific plasticity, mediated by PKA, and a mark for its stabilization, mediated by local protein synthesis. The initiation of synapse-specific facilitation is reflected by the facilitation (both functional and morphological) expressed at 24 hr, while the stabilization is reflected by the facilitation (both functional and morphological) expressed at 72 hr (Casadio, 1999).

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