Peripheral nerve injury reduces the excitation-inhibition balance of basolateral amygdala inputs to prelimbic pyramidal neurons projecting to the periaqueductal gray.

Cellular and synaptic mechanisms underlying how chronic pain induces maladaptive alterations to local circuits in the medial prefrontal cortex (mPFC), while emerging, remain unresolved. Consistent evidence shows that chronic pain attenuates activity in the prelimbic (PL) cortex, a mPFC subregion. This reduced activity is thought to be driven by increased inhibitory tone within PL circuits. Enhanced input from the basolateral amygdala (BLA) to inhibitory neurons in PL cortex is one well-received mechanism for this circuit change. In mice, we used retrograde labeling, brain slice recordings, and optogenetics to selectively stimulate and record ascending BLA inputs onto PL neurons that send projections to the periaqueductal gray (PAG), which is a midbrain structure that plays a significant role in endogenous analgesia. Activating BLA projections evoked both excitatory and inhibitory currents in cortico-PAG (CP) neurons, as we have shown previously. We measured changes to the ratio of BLA-evoked excitatory to inhibitory currents in the spared nerve injury (SNI) model of neuropathic pain. Our analysis reveals a reduced excitation-inhibition (E/I) ratio of BLA inputs to PL-CP neurons 7 days after SNI. The E/I ratio of BLA inputs to CP neurons in neighboring infralimbic (IL) cortex was unchanged in SNI animals. Collectively, this study reveals that the overall E/I balance of BLA inputs to PL neurons projecting to the PAG is reduced in a robust neuropathic pain model. Overall, our findings provide new mechanistic insight into how nerve injury produces dysfunction in PL circuits connected to structures involved in pain modulation.

Altered Excitability and Local Connectivity of mPFC-PAG Neurons in a Mouse Model of Neuropathic Pain.

The medial prefrontal cortex (mPFC) plays a major role in both sensory and affective aspects of pain. There is extensive evidence that chronic pain produces functional changes within the mPFC. However, our understanding of local circuit changes to defined subpopulations of mPFC neurons in chronic pain models remains unclear. A major subpopulation of mPFC neurons project to the periaqueductal gray (PAG), which is a key midbrain structure involved in endogenous pain suppression and facilitation. Here, we used laser scanning photostimulation of caged glutamate to map cortical circuits of retrogradely labeled cortico-PAG (CP) neurons in layer 5 (L5) of mPFC in brain slices prepared from male mice having undergone chronic constriction injury (CCI) of the sciatic nerve. Whole-cell recordings revealed a significant reduction in excitability for L5 CP neurons contralateral to CCI in the prelimbic (PL), but not infralimbic (IL), region of mPFC. Circuit mapping showed that excitatory inputs to L5 CP neurons in both PL and IL arose primarily from layer 2/3 (L2/3) and were significantly reduced in CCI mice. Glutamate stimulation of L2/3 and L5 elicited inhibitory inputs to CP neurons in both PL and IL, but only L2/3 input was significantly reduced in CP neurons of CCI mice. We also observed significant reduction in excitability and L2/3 inhibitory input to CP neurons ipsilateral to CCI. These results demonstrating region and laminar specific changes to mPFC-PAG neurons suggest that a unilateral CCI bilaterally alters cortical circuits upstream of the endogenous analgesic network, which may contribute to persistence of chronic pain.SIGNIFICANCE STATEMENT Chronic pain is a significant unresolved medical problem that is refractory to traditional analgesics and can negatively affect emotional health. The role of central circuits in mediating the persistent nature of chronic pain remains unclear. Local circuits within the medial prefrontal cortex (mPFC) process ascending pain inputs and can modulate endogenous analgesia via direct projections to the periaqueductal gray (PAG). However, the mechanisms by which chronic pain alters intracortical circuitry of mPFC-PAG neurons are unknown. Here, we report specific changes to local circuits of mPFC-PAG neurons in mice displaying chronic pain behavior after nerve injury. These findings provide evidence for a neural mechanism by which chronic pain disrupts the descending analgesic system via functional changes to cortical circuits.

Specific Targeting of the Basolateral Amygdala to Projectionally Defined Pyramidal Neurons in Prelimbic and Infralimbic Cortex.

Adjacent prelimbic (PL) and infralimbic (IL) regions in the medial prefrontal cortex have distinct roles in emotional learning. A complete mechanistic understanding underlying this dichotomy remains unclear. Here we explored targeting of specific PL and IL neurons by the basolateral amygdala (BLA), a limbic structure pivotal in pain and fear processing. In mice, we used retrograde labeling, brain-slice recordings, and adenoviral optogenetics to dissect connectivity of ascending BLA input onto PL and IL neurons projecting to the periaqueductal gray (PAG) or the amygdala. We found differential targeting of BLA projections to PL and IL cortex. Activating BLA projections evoked excitatory and inhibitory responses in cortico-PAG (CP) neurons in layer 5 (L5) of both PL and IL cortex. However, all inhibitory responses were polysynaptic and monosynaptic BLA input was stronger to CP neurons in IL cortex. Conversely, the BLA preferentially targeted corticoamygdalar (CA) neurons in layer 2 (L2) of PL over IL cortex. We also reveal that BLA input is projection specific by showing preferential targeting of L5 CP neurons over neighboring L3/5 CA neurons in IL cortex. We conclude by showing that BLA input is laminar-specific by producing stronger excitatory responses CA neurons in L3/5 compared with L2 in IL cortex. Collectively, this study reveals differential targeting of the BLA to PL and IL cortex, which depends both on laminar location and projection target of cortical neurons. Overall, our findings should have important implications for understanding the processing of pain and fear input by the PL and IL cortex.

Pharmacology of triheteromeric N-Methyl-D-Aspartate Receptors.

The N-Methyl-D-Aspartate Receptors (NMDARs) are heteromeric cation channels involved in learning, memory, and synaptic plasticity, and their dysregulation leads to various neurodegenerative disorders. Recent evidence has shown that apart from the GluN1/GluN2A and GluN1/GluN2B diheteromeric ion channels, the NMDAR also exists as a GluN1/GluN2A/GluN2B triheteromeric channel that occupies the majority of the synaptic space. These GluN1/GluN2A/GluN2B triheteromers exhibit pharmacological and electrophysiological properties that are distinct from the GluN1/GluN2A and GluN1/GluN2B diheteromeric subtypes. However, these receptors have not been characterized with regards to their inhibition by conantokins, as well as their allosteric modulation by polyamines and extracellular protons. Here, we show that the GluN1/GluN2A/GluN2B triheteromeric channels showed less sensitivity to GluN2B-specific conantokin (con)-G and con-RlB, and subunit non-specific con-T, compared to the GluN2A-specific inhibitor TCN-201. Also, spermine modulation of GluN1/GluN2A/GluN2B triheteromers switched its nature from potentiation to inhibition in a pH dependent manner, and was 2.5-fold slower compared to the GluN1/GluN2B diheteromeric channels. Unraveling the distinctive functional attributes of the GluN1/GluN2A/GluN2B triheteromers is physiologically relevant since they form an integral part of the synapse, which will aid in understanding spermine/pH-dependent potentiation of these receptors in pathological settings.

Heteromerization of ligand binding domains of N-methyl-D-aspartate receptor requires both coagonists, L-glutamate and glycine.

NMDA receptors (NMDAR) are voltage- and glutamate-gated heteromeric ion channels found at excitatory neuronal synapses, the functions of which are to mediate the mechanisms of brain plasticity and, thereby, its higher order functions. In addition to Glu, the activation of these heteromeric receptors requires Gly or d-Ser as a coagonist. However, it is not fully known as to why coagonism is required for the opening of NMDAR ion channels. We show herein that the ligand binding domains (LBD) of the GluN1 and GluN2A subunits of the NMDAR heterodimerize only when both coagonists, Glu and Gly/d-Ser, bind to their respective sites on GluN2 and GluN1. In the agonist-free state, these domains form homomeric interactions, which are disrupted by binding of their respective agonists. Also, in a heteromer formed by the LBDs, GluN2A is more sensitized to bind Glu, while the affinity of Gly for GluN1 remains unchanged. We thus provide direct evidence to show that coagonism is necessary for heteromeric pairing of LBDs, which is an essential step in forming functional ion channels in NMDARs.

Antagonist properties of Conus parius peptides on N-methyl-D-aspartate receptors and their effects on CREB signaling.

Three members of a family of small neurotoxic peptides from the venom of Conus parius, conantokins (Con) Pr1, Pr2, and Pr3, function as antagonists of N-methyl-D-aspartate receptors (NMDAR). We report structural characterizations of these synthetic peptides, and also demonstrate their antagonistic properties toward ion flow through NMDAR ion channels in primary neurons. ConPr1 and ConPr2 displayed moderate increases in α-helicity after addition of Mg(2+). Native apo-ConPr3 possessed an α-helical conformation, and the helicity increased only slightly on addition of Mg(2+). Additionally, these peptides diminished NMDA/Gly-mediated currents and intracellular Ca(2+) (iCa(2+)) influx in mature rat primary hippocampal neurons. Electrophysiological data showed that these peptides displayed slower antagonistic properties toward the NMDAR than conantokins from other species of cone snails, e.g., ConT and ConG. Furthermore, to demonstrate selectivity of the C. parius-derived conantokins towards specific NMDAR subunits, cortical neurons from GluN2A(-/-) and GluN2B(-/-) mice were utilized. Robust inhibition of NMDAR-mediated stimulation in GluN2A(-/-)-derived mouse neurons, as compared to those isolated from GluN2B(-/-)-mouse brains, was observed, suggesting a greater selectivity of these antagonists towards the GluN2B subunit. These C. parius conantokins mildly inhibited NMDAR-induced phosphorylation of CREB at Ser(133), suggesting that the peptides modulated iCa(2+) entry and, thereby, activation of CREB, a transcription factor that is required for maintaining long-term synaptic activity. Our data mechanistically show that while these peptides effectively antagonize NMDAR-directed current and iCa(2+) influx, receptor-coupled CREB signaling is maintained. The consequence of sustained CREB signaling is improved neuronal plasticity and survival during neuropathologies.

Effect of multimeric structure of CaMKII in the GluN2B-mediated modulation of kinetic parameters of ATP.

Interaction of GluN2B subunit of N-methyl-D-aspartate receptor with calcium/calmodulin dependent protein kinase II (CaMKII) is critical for the induction of long term potentiation at hippocampal CA3-CA1 synapses. We have previously reported that CaMKII binding to GluN2B increases its affinity but abolishes the cooperativity for ATP. In the present study, we demonstrate that the reduction in S(0.5) for ATP of an individual CaMKII subunit seems to be directly induced by the binding of GluN2B to the same subunit, while any GluN2B induced effects on the cooperativity and maximal velocity would additionally require the CaMKII holoenzyme structure. We measured the apparent kinetic parameters for ATP using an association domain truncated monomeric CaMKII and a heteromultimeric CaMKII (having subunits that are either GluN2B binding defective or ATP binding defective), in the presence of GluN2A or GluN2B substrates. The S(0.5) value for ATP of monomeric CaMKII is reduced ∼ 3 fold by the presence of GluN2B suggesting that the induced change in affinity for ATP is independent of the holoenzyme structure. The heteromultimeric mutant of CaMKII, did not exhibit cooperativity of ATP binding probably because of the interspersing of ATP binding defective subunits in the holoenzyme. In contrast to the wild type holoenzyme, presence of GluN2B increased the V(max) of monomeric CaMKII which resulted in an approximately 4.0 fold increase in the apparent catalytic constant (V(max)/S(0.5)) as compared to GluN2A. The kinetic parameter values of the heteromultimeric CaMKII for ATP, on the other hand, did not show any significant difference between the phosphorylation of GluN2B and GluN2A suggesting that modulation requires binding of GluN2B to the same subunit. Overall, our present study provides insights into the role of multimeric structure of CaMKII in GluN2B-mediated regulation.

Calcium/calmodulin dependent protein kinase II bound to NMDA receptor 2B subunit exhibits increased ATP affinity and attenuated dephosphorylation.

Calcium/calmodulin dependent protein kinase II (CaMKII) is implicated to play a key role in learning and memory. NR2B subunit of N-methyl-D-aspartate receptor (NMDAR) is a high affinity binding partner of CaMKII at the postsynaptic membrane. NR2B binds to the T-site of CaMKII and modulates its catalysis. By direct measurement using isothermal titration calorimetry (ITC), we show that NR2B binding causes about 11 fold increase in the affinity of CaMKII for ATPγS, an analogue of ATP. ITC data is also consistent with an ordered binding mechanism for CaMKII with ATP binding the catalytic site first followed by peptide substrate. We also show that dephosphorylation of phospho-Thr(286)-α-CaMKII is attenuated when NR2B is bound to CaMKII. This favors the persistence of Thr(286) autophosphorylated state of CaMKII in a CaMKII/phosphatase conjugate system in vitro. Overall our data indicate that the NR2B- bound state of CaMKII attains unique biochemical properties which could help in the efficient functioning of the proposed molecular switch supporting synaptic memory.

Phosphorylation status of the NR2B subunit of NMDA receptor regulates its interaction with calcium/calmodulin-dependent protein kinase II.

Ca(2+) influx through NMDA-type glutamate receptor at excitatory synapses causes activation of post-synaptic Ca(2+)/calmodulin-dependent protein kinase type II (CaMKII) and its translocation to the NR2B subunit of NMDA receptor. The major binding site for CaMKII on NR2B undergoes phosphorylation at Ser1303, in vivo. Even though some regulatory effects of this phosphorylation are known, the mode of dephosphorylation of NR2B-Ser1303 is still unclear. We show that phosphorylation status at Ser1303 enables NR2B to distinguish between the Ca(2+)/calmodulin activated form and the autonomously active Thr286-autophosphorylated form of CaMKII. Green fluorescent protein-alpha-CaMKII co-expressed with NR2B sequence in human embryonic kidney 293 cells was used to study intracellular binding between the two proteins. Binding in vitro was studied by glutathione-S-transferase pull-down assay. Thr286-autophosphorylated alpha-CaMKII or the autophosphorylation mimicking mutant, T286D-alpha-CaMKII, binds NR2B sequence independent of Ca(2+)/calmodulin unlike native wild-type alpha-CaMKII. We show enhancement of this binding by Ca(2+)/calmodulin. Phosphorylation or a phosphorylation mimicking mutation on NR2B (NR2B-S1303D) abolishes the Ca(2+)/calmodulin-independent binding whereas it allows the Ca(2+)/calmodulin-dependent binding of alpha-CaMKII in vitro. Similarly, the autonomously active mutants, T286D-alpha-CaMKII and F293E/N294D-alpha-CaMKII, exhibited Ca(2+)-independent binding to non-phosphorylatable mutant of NR2B under intracellular conditions. We also show for the first time that phosphatases in the brain such as protein phosphatase 1 and protein phosphatase 2A dephosphorylate phospho-Ser1303 on NR2B.

Regulation of Ca2+/calmodulin-dependent protein kinase II catalysis by N-methyl-D-aspartate receptor subunit 2B.

Binding of CaMKII (Ca(2+)/calmodulin-dependent protein kinase II) to the NR2B subunit of the NMDAR (N-methyl-D-aspartate-type glutamate receptor) in the PSD (postsynaptic density) is essential for the induction of long-term potentiation. In this study, we show that binding of NR2B to the T-site (Thr(286)-autophosphorylation site binding pocket) of CaMKII regulates its catalysis as reflected in the kinetic parameters. The apparent S(0.5) (substrate concentration at half maximal velocity) and V(max) values for ATP were lower for phosphorylation of a GST (glutathione transferase)-fusion of NR2B((1271-1311)) (with the phosphorylation site Ser(1303)) when compared with phosphorylation of the analogous sequence motif from NR2A. The co-operative behaviour exhibited by the CaMKII holoenzyme towards ATP for phosphorylation of GST-NR2A was significantly altered by the interaction with GST-NR2B. Disrupting the T-site-mediated binding by mutagenesis of either NR2B or CaMKII abolished the modulation of CaMKII activity by NR2B. The active site residue of alpha-CaMKII, Glu(96), participates in effecting the modulation. The CaMKII-binding motif of the Drosophila voltage-gated potassium channel Eag interacted with the T-site of CaMKII with lower affinity and caused catalytic modulation to a lesser extent. The kinetic parameters of ATP for the Thr(286)-autophosphorylation reaction of CaMKII were also altered by NR2B in a similar manner. Interestingly, the NR2B sequence motif caused increased sensitivity of CaMKII activity to ATP, and saturation by lower concentrations of ATP, which, in effect, resulted in a constant level of activity of CaMKII over a broad range of ATP concentrations. Our findings indicate that CaMKII at the PSD may be regulated by bound NR2B in a manner that supports synaptic memories.

Influence of a mutation in the ATP-binding region of Ca2+/calmodulin-dependent protein kinase II on its interaction with peptide substrates.

CaMKII (Ca2+/calmodulin-dependent protein kinase II) is expressed in high concentrations in the brain and is found enriched in the postsynaptic densities. The enzyme is activated by the binding of calmodulin to the autoregulatory domain in the presence of high levels of intracellular Ca2+, which causes removal of auto-inhibition from the N-terminal catalytic domain. Knowledge of the 3D (three-dimensional) structure of this enzyme at atomic resolution is restricted to the association domain, a region at the extreme C-terminus. The catalytic domain of CaMKII shares high sequence similarity with CaMKI. The 3D structure of the catalytic core of CaMKI comprises ATP- and substrate-binding regions in a cleft between two distinct lobes, similar to the structures of all protein kinases solved to date. Mutation of Glu-60, a residue in the ATP-binding region of CaMKII, to glycine exerts different effects on phosphorylation of two peptide substrates, syntide and NR2B ( N -methyl-D-aspartate receptor subunit 2B) 17-mer. Although the mutation caused increases in the Km values for phosphorylation for both the peptide substrates, the effect on the kcat values for each was different. The kcat value decreased in the case of syntide, whereas it increased in the case of the NR2B peptide as a result of the mutation. This resulted in a significant decrease in the apparent kcat/Km value for syntide, but the change was minimal for the NR2B peptide. These results indicate that different catalytic mechanisms are employed by the kinase for the two peptides. Molecular modelling suggests structural changes are likely to occur at the peptide-binding pocket in the active state of the enzyme as a consequence of the Glu-60-->Gly mutation.