Introduction

Psychiatric and neurological illnesses are among the most common and serious health problems. The clinical treatment of neurological and psychiatric diseases requires advances in neuroscience for their elucidation, prevention, and treatment. In the past 20 years or so, we see successful applications of molecular genetics and cell biology to the central nervous system. With the complete mapping of both human and mouse genomes, new disease-related genes and proteins are being identified at a rapid speed. Discovery of diseases-related genes and proteins opens the door for the future understanding of the pathogenesis and lays the ground for their potential clinical treatment of patients with brain illnesses

'Department of Physiology, Faculty of Medicine, University of Toronto Center for the Study of Pain, University of Toronto, Ontario, Canada M5S 1A8; [email protected]

such as schizophrenia, autism, mental retardation, chronic pain, depression, and Alzheimer and Parkinson diseases.

Knowing the sequence of a new gene or the structure of a new protein is not enough for understanding its role in brain functions. Neurons communicate with each other through a structure called the synapse. One major principle explaining how synapses work is that synaptic strength between two neurons should increase when the neurons exhibit coincident activity as proposed by Donald Hebb in his now-famous book, The Organization of Behavior. Subsequent findings of long-term potentiation (LTP) in the hippocampus provide a key synaptic model for studying brain functions. Recent advances in the ability to genetically modify mice made it possible to relate specific genes to both synaptic transmission/plasticity and intact animal behaviors, including memory, drug addiction, and persistent

While we enjoy the success of connecting genes in synaptic function to behavioral phenotypic changes, we are confronted with a more difficult question: how changes in synaptic plasticity such as LTP may affect brain functions at systemic and behavioral levels? Recent progresses in neurobiology and genetics have significantly helped us to address it. First is the introduction of various genetically modified mice. This approach can overcome the lack of drugs for the interested proteins as well as their potential side effects. Due to the introduction of this approach in neuroscience of learning and memory by Susumu and Kandel, there are increasing numbers of publications relating molecules to behavioral phenotypes, learning, memory, pain, drug addiction, and various genetical diseases. Second is the cross marriage of chemistry and biology. Based on the understanding of the structures of proteins, many chemical inhibitors have been generated. In the near future, combination of gene-knockout mice with the use of pharmacological inhibitors will provide better studies of the mechanisms of brain functions. One major task for neuroscientists is to integrate recent findings in molecular and cellular research to the neuronal network and physiological functions of intact animals.

Here, I review recent progress in sensory-related central synapses, and their roles in sensory transmission, modulation, as well as long-term storage of fear-related information. I propose that these synaptic mechanisms provide the basis for future neuronal network studies as well as for design of better medicines to treat chronic pain and fear.

3. SPINAL DORSAL HORN: THE FIRST SENSORY SYNAPSE 3.1. Sensory Transmission

Neurons in the spinal cord dorsal horn and related areas receive sensory inputs, including noxious information, and convey them to supraspinal structures. Studies using pharmacological and behavioral approaches show that glutamate and neuropeptides, including substance P (SP), are excitatory transmitters for pain. Electrophysiological investigation of sensory synaptic responses between primary afferent fibers and dorsal horn neurons provided evidence that glutamate is the principal fast excitatory transmitter, and synaptic responses are mediated by postsynaptic glutamate receptors. While a-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors mediate the largest component of postsynaptic currents, kainate (KA) receptors preferentially contribute to synaptic responses induced by higher (noxious) stimulation intensities. Consistent with this, antagonism of both KA and AMPA receptor yields greater analgesic effects in adult animals than AMPA receptor antagonism alone. These findings suggest that sensory modality may be coded in part by different postsynaptic neurotransmitter receptors5.

In addition to glutamate, several neuropeptides, including SP, are thought to act as sensory transmitters. For many years, there has been a lack of electrophysiological evidence that SP can mediate monosynaptic responses, since SP-mediated responses had a very slow onset. Recent studies using whole-cell patch-clamp recordings reveal relatively fast SP- and neurokinin A-mediated synaptic currents in synapses between primary afferent fibers and dorsal horn neurons. Excitatory postsynaptic currents (EPSCs) in response to the burst activity of the primary afferent fibers may affect the excitability of spinal dorsal horn neurons. Together with glutamate-mediated synaptic responses, these neuropeptide-mediated EPSCs may cause dorsal horn neurons to fire action potentials at a high frequency for a long period of time. Therefore, the combination of glutamate- and neuropeptide-mediated EPSCs allow nociceptive information to be conveyed from the periphery to the central nervous system5.

3.2. Serotonin (5-HT)-Induced Potentiation

Spinal dorsal neurons receive innervations from descending serotonin systems from the brainstem6. Application of 5-HT or 5-HT2 subtype receptor agonist induced long-term facilitation of synaptic response7. One mechanism for the facilitation is the recruitment of silent synapses through interaction of glutamate AMPA receptors with proteins containing postsynaptic density-95/discs large/zona occludens-1 (PDZ) domains. To examine the functional significance of GluR2/3-PDZ interactions in sensory synaptic transmission, we made a synthetic peptide corresponding to the last 10 amino acids of GluR2 ("GluR2-SVKI": NVYGIESVKI) that disrupts binding of GluR2 to GRIP7. As expected, GluR2-SVKI peptide blocked the facilitatory effect of 5-HT. The effect of GluR2-SVKI on synaptic facilitation is rather selective because baseline EPSCs and currents evoked by glutamate application did not change over time in these neurons7. Furthermore, synaptic facilitation induced by phorbol 12,13-dibutyrate (PDBu) was also blocked by GluR2-SVKI, suggesting that synaptic facilitation mediated by protein kinase C activation is similar to that produced by 5-HT in its dependence on GluR2/3 C-terminal interactions7 (Figure 25.1).

Activation of several receptors for sensory transmitters such as glutamate and calcitonin gene related peptide (CGRP) has been reported to raise cAMP levels. In a recent study, application of forskolin did not significantly affect synaptic responses induced by dorsal root stimulation in slices of adult mice. However, co-application of 5-HT and forskolin produced long-lasting facilitation of synaptic responses. Possible contributors to the increase in the cAMP levels are calcium-sensitive adenylyl cyclases (AC). We found that the facilitatory effect induced by 5-HT and forskolin was completely blocked in mice lacking AC1, indicating that calcium-sensitive AC1 is important8. The interaction between cAMP and 5-HT may provide an associative heterosynaptic form of central plasticity in the spinal dorsal horn to allow sensory inputs from the periphery to act synergistically with central modulatory influences descending from the brainstem rostroventral medulla (RVM).

KAR(GluR5)

KAR(GluR5)

cAMP

KAR(GluR6)

cAMP

Ca2+

KAR(GluR6)

Figure 25.1. Signaling Pathways Contribute to Synaptic Potentiation in Spinal Sensory Synapses. Glutamate (Glu) is the major fast excitatory transmitter in the spinal cord. Both AMPA and KA GluR6 receptors contribute to synaptic responses in normal conditions. Activation of glutamate NMDA receptors leads to an increase in postsynaptic Ca2+ in dendritic spines. Ca2+ serves as an important intracellular signal for triggering a series of biochemical events that contribute to the potentiation of synaptic transmission. Ca2+ binds to CaM and leads to activation of calcium-stimulated ACs, including AC1 and AC8 and Ca2+/CaM dependent protein kinases (PKC, CaMKII, and CaMKIV) (not shown). The Ca2+/CaM dependent protein kinases phosphorylate glutamate AMPA receptors, increase their sensitivity to glutamate and increase the number of functional synapses. In addition to the homosynaptic LTP, recruitment of postsynaptic AMPA receptors contributes to serotonin-induced facilitation. Neurons in the rostroventral medulla (RVM) project to the spinal dorsal horn and modulate sensory synaptic transmission in the spinal cord. Serotonin (5-HT) is the transmitter that mediates this facilitatory effect. The facilitation induced by 5-HT likely requires the activation of specific subtypes of serotonin receptors and coactivation of cAMP signaling pathways to induce facilitation in adult spinal dorsal horn neurons. 5-HT activates postsynaptic PKC through G protein receptors. PKC activation and subsequent AMPA receptor and GRIP interactions cause the recruitment of AMPA receptors to the synapse. Due to enhanced synaptic efficacy between primary afferent fibers and dorsal horn neurons, spike (action potential) responses to the stimulation of afferent fibers and behavioral nociceptive responses are enhanced (e.g., decrease in response latencies). Glutamatergic transmission is also under presynaptic regulation through the activation of auto-KA GluR5 receptors.

3.3. Homosynaptic LTP

Studies of LTP in spinal dorsal horn neurons draw much attention because it is believed that potentiation of sensory responses after injury may explain chronic pain9. While it has been demonstrated that spike responses of dorsal horn neurons to peripheral stimulation are enhanced after injury9, it remains to be investigated if enhanced spike responses are simply due to enhanced synaptic transmission between the dorsal root ganglion (DRG) cells and dorsal horn neurons. Unlike synapses in other areas such as hippocampus, synaptic potentiation in the spinal dorsal horn neurons is not induced by strong tetanic stimulation. Recent studies further show that LTP only happens in some spinal projecting cells10 (Figure 25.1). Activation of neurokinin subtype 1 (NK1) receptors or NMD A receptors is required for spinal LTP. Additionally, only spinal cord dorsal horn neurons that express SP receptors undergo potentiation, although in many other areas of the brain, there is no requirement of SP for the induction of NMDA receptor dependent LTP10. It will be important in the future to investigate why LTP cannot be induced in dorsal horn neurons that do not express SP receptors.

Trying to link changes in the spinal cord with behavioral responses has proved to be difficult. Not only do spinal sensory synapses receive inputs from the periphery, but they also receive biphasic modulation from supraspinal structures. In addition, many supraspinal structures also contribute to pain-related behavioral responses. Therefore, it is premature to directly connect what found in the spinal cord with behavioral changes.

4. AMYGDALA: FEAR AND ITS LONG-TERM STORAGE

4.1. Amygdala and Fear

Fear memory requires the involvement of higher brain structures including the hippocampus, the amygdala, and related cortical structures2,11-13. Studies from animals and humans consistently suggest that the amygdala is important for fear memory formation14-16. Two forms of fear memory are commonly studied in animals: auditory and contextual fear memory. For auditory fear memory, evidence from experiments using different approaches suggests that the amygdala plays an important role in the acquisition and retention of the memory2. Neurons in the amygdala, particularly in the lateral amygdala (LA), receive auditory (used as conditioning stimulus, CS, in fear conditioning) and somatosensory inputs (used as unconditioned stimuli, US) from the periphery2. LA neurons receive auditory inputs from both the auditory thalamus and the auditory cortex. Both pathways contribute to auditory fear memory, although they may play differential and complementary roles in different forms of auditory memory. For somatosensory inputs, including noxious inputs (used as US), neurons in the LA receive somatosensory inputs from thalamus. LA neurons are responsive to nociceptive stimulation, and some LA cells respond to both auditory and noxious stimuli17. For contextual fear memory, it is believed to require the involvement of both the amygdala and hippocampus18,19. In addition to the amygdala and hippocampus, cortical areas that process nociceptive stimuli project to the LA and other amygdala nuclei20,21.

4.2. Fear and LTP

How is new information learned and stored in amygdala circuits? One leading hypothesis is that synaptic transmission undergoes long-term plastic changes after training in the amygdala. Studies using multiple approaches, including electrophysiological recordings in brain slices and single units or field synaptic responses in whole animals, consistently indicate that LTP is the most likely synaptic mechanism underlying fear memory in the amygdala. First, electrophysiological studies using in vitro amygdala slices or in vivo recordings showed that auditory afferent pathways, including auditory thalamus-amygdala and auditory cortex-amygdala pathways, undergo synaptic potentiation after LTP-inducing stimulation paradigm22-24. Second, the associative nature of LTP in the amygdala supports the idea that fear conditioning requires the convergence of auditory and nociceptive inputs onto single neurons in the LA24. Third, neural activity in the LA has been shown to be modified during auditory fear conditioning in a manner similar to that observed after artificial LTP induction25,26. Fourth, drugs that inhibit the induction and/or consolidation of LTP in the amygdala also inhibit fear memory27-29 (see ref. 2 for reviews). Finally, a recent study shows that fear conditioning occluded LTP-induced presynaptic enhancement of synaptic transmission in the cortical pathway to the lateral amygdala30.

4.3. Thalamic-Amygdala LTP

Synaptic mechanisms for LTP have been best investigated in the amygdala slices. It has been noted that the contribution of NMDA receptors and/or L-type voltage gated calcium channels (L-VGCCs) to the LTP induction are somewhat dependent on induction protocols31 (Figure 25.2). Activation of NMDA receptors is required for the induction of the thalamic-amygdala LTP. Interestingly, bath application of a selective NR2B receptor antagonist blocked the induction of LTP32,33. The role of NR2A containing NMDA receptors has not yet been investigated. In addition to the NMDA receptors, KA GluR6 receptors are reported to be important for the LTP. Field potential recordings and whole-cell patch-clamp recordings revealed that LTP is blocked in mice lacking GluR6 subunit. In contrast, LTP in the GluR5 subunit knockout mice is normal. The exact synaptic mechanisms for the GluR6 contribution to the LTP remain to be investigated. Additionally, inhibition of metabotropic glutamate receptor (mGluR) subtype 5 has been reported to block LTP32. Thus, it is likely that multiple receptor systems are involved in synaptic potentiation in the thalamic-amygdala pathways.

The involvement of protein kinases in amygdala LTP has also been investigated. cAMP-dependent protein kinase (PKA) and CaMKII are reported to contribute to the induction of LTP. In addition, CaMKIV has been implicated in early LTP in the amygdala34. The expression of LTP in the thalamic-amygdala pathways contains both presynaptic and postsynaptic mechanisms. A recent study suggests that during LTP, paired-pulse facilitation (PPF) was altered in the thalamic-amygdala pathways, raising the possibility for presynaptic contribution to LTP. Furthermore, inhibition of the production of nitric oxide (NO), a retrograde messenger, blocked the LTP35 (see Figure 25.2 for a model), suggesting a possible role of NO as a retrograde messenger in the expression of thalamic-amygdala LTP.

4.4. LTP in the Cortical-Amygdala Pathway

Induction of LTP in the cortical pathway to the LA is postsynaptic, whereas the expression of LTP is presynaptic. The induction of LTP is dependent on postsynaptic depolarization, on the influx of Ca2+ into the postsynaptic cell either through NMDA receptors23,30 or L-VGCCs30. In a well-executed experiment by Tsvekov et al., it was found that LTP was completely blocked when both NMDA receptor and L-VDCCs were blocked. Small conductance Ca2+ activated K+ channels (SK) have also been reported to contribute to the LTP in the cortical-amygdala pathways36. Blockade of SK channels greatly enhanced the LTP. These results suggest that Ca2+ influx through NMDA receptor activates SK channels and shunts the resultant excitatory postsynaptic potential.

The expression of the cortical-amygdala LTP is mostly presynaptic23,30. LTP is associated with a decrease of PPF and is blocked by bath application but not by postsynaptic injection of inhibitors of PKA. Moreover, forskolin could induce LTP in this pathway, which occluded the tetanus-induced LTP. Many other signaling molecules/proteins have been reported to contribute to PI-3 kinase inhibitors blocked tetanus-induced LTP (field recordings) in cortico-amygdala pathway. Tetanus and forskolin-induced activation of MAPK was blocked by PI-3 kinase inhibitors, which also inhibited CREB phosphorylation. These results suggest PI-3 is upstream for MAPK and CREB activation, thereby contributing to synaptic plasticity in the amygdala.

Ca2 Cam Mapk

Figure 25. 2. Presynaptic and Postsynaptic Signaling Pathways that Contribute to LTP in the Amygdala. NMDA receptors are important for the induction of LTP. Ca2+ serves as an important intracellular signal for triggering a series of biochemical events that contribute to the expression of LTP. The Ca2+/CaM dependent protein kinases phosphorylate glutamate AMPA receptors, increase their sensitivity to glutamate and the number of functional AMPA receptors. Activation of CaMKIV, a kinase predominantly expressed in the nuclei, will trigger CaMKIV-dependent CREB that contribute to LTP. The involvement of L-VGCCs has also been implicated, depending on the LTP induction protocol. The production of retrograde messenger nitric oxide (NO) is thought to be important for the expression of LTP.

Figure 25. 2. Presynaptic and Postsynaptic Signaling Pathways that Contribute to LTP in the Amygdala. NMDA receptors are important for the induction of LTP. Ca2+ serves as an important intracellular signal for triggering a series of biochemical events that contribute to the expression of LTP. The Ca2+/CaM dependent protein kinases phosphorylate glutamate AMPA receptors, increase their sensitivity to glutamate and the number of functional AMPA receptors. Activation of CaMKIV, a kinase predominantly expressed in the nuclei, will trigger CaMKIV-dependent CREB that contribute to LTP. The involvement of L-VGCCs has also been implicated, depending on the LTP induction protocol. The production of retrograde messenger nitric oxide (NO) is thought to be important for the expression of LTP.

The late phase of LTP (L-LTP) in the cortico-amgdala is mediated by PKA and MAPK37. The L-LTP is associated with phosphorylation of CREB and protein synthesis. Most molecules studied are involved in the thalamic-amygdala LTP, including GluR138, NR2B33, GluR639, NO35, CaMKIV34, and CaMKII31, but their roles in cortical LTP has not been elucidated yet.

5. ANTERIOR CINGULATE CORTEX (ACC): AN INTEGRATIVE CENTER

5.1. Synaptic Transmission in the ACC

Glutamate is the major fast excitatory transmitter in the ACC7. Different types of glutamate receptors, including AMPA, KA, NMDA, and mGluRs, are found in the ACC. Whole-cell patch-clamp recordings from ACC pyramidal neurons of rats and mice showed that excitatory synaptic responses are mediated by glutamate AMPA/KA receptors7,40. While the majority of fast EPSCs are mediated by postsynaptic AMPA receptors, a recent study reported that some synaptic currents can be mediated by glutamate KA receptors in ACC neurons of adult mice40. Furthermore, genetic studies using a KA subtype receptor knockout mice showed that postsynaptic KA receptor-mediated currents require GluR5 and GluR6 subunits40. In addition to fast synaptic responses, NMDA receptor-mediated slow synaptic responses were also recorded from adult ACC slices at physiological temperatures or in vivo recordings from the ACC of freely moving animals41,42. These findings indicate that under physiological conditions, postsynaptic NMDA receptors can contribute to sensory synaptic transmission in the ACC.

Glutamatergic synapses in the ACC can undergo long-lasting potentiation in response to theta-burst stimulation, a paradigm more closely related to the activity of ACC neurons by using field recording in ACC slices of adult rats or mice. The potentiation lasts for at least 40-120 min34. LTP can be recorded using whole-cell patch-clamp recordings in slices of adult mice. Long-term synaptic potentiation can be induced by using other two different induction protocols43. Activation of postsynaptic NMDA receptors and postsynaptic Ca2+ signaling is critical for the induction of LTP. During LTP, no obvious changes in PPF are detected, suggesting that potentiation is unlikely due to pure presynaptic mechanisms. Activation of NMDA NR2A and NR2B subunits is critical for the induction of LTP43. Blockade of both NR2A and NR2B subunits is required for the complete inhibition of the induction of cingulate LTP. In addition to NMDA receptors, activation of KA GluR6 receptors was also reported to contribute to cingulate LTP39.

Cyclic AMP is a key second messenger in neurons. Recent studies using geneknockout mice and pharmacological activators/inhibitors found that calcium-stimulated AC1 and AC8 are important for the cingulate LTP induced by theta-burst stimulation or pairing training protocol in pyramidal cells. Activation of MAP kinases is also found to be important for cingulate LTP. In addition, CaMKIV is also required for the induction of LTP34. Thus, it is likely that multiple signaling pathways are critical for the induction of cingulate LTP. Future studies are needed to address the possible contribution of AMPA receptor subunits in cingulate LTP (Figure 25.3).

5.3. Behavioral Fear and the ACC

It has been reported that stimulation of ACC generates fear memory, and the neuronal activity in the ACC is required for retrieval of remote fear memory44.

Recent studies using pharmacological and genetic approaches showed that inhibition of NMDA NR2B receptors in the ACC significantly reduced the formation of classic fear memory, indicating that ACC neuronal activity contributes to fear memory formation43. To support the roles of the ACC in fear, Tang and co-workers reported that stimulation of the ACC generates fear memory without classic foot shocks42. This ACC stimulation-induced fear memory requires activity in the amygdala, suggesting that the ACC and amygdala are likely to work together to memorize fearful cues.

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