Chronic pain hypersensitivity depends on N-type voltage-gated calcium channels (CaV2.2). However, the use of CaV2.2 blockers in pain therapeutics is limited by side effects that result from inhibited physiological functions of these channels. Here we report suppression of both inflammatory and neuropathic hypersensitivity by inhibiting the binding of the axonal collapsin response mediator protein 2 (CRMP-2) to CaV2.2, thus reducing channel function. A 15-amino acid peptide of CRMP-2 fused to the transduction domain of HIV TAT protein (TAT-CBD3) decreases neurotransmitter release from nociceptive dorsal root ganglion neurons, reduces meningeal blood flow, reduces nocifensive behavior induced by subcutaneous formalin injection or following corneal capsaicin application, and reverses neuropathic hypersensitivity produced by the antiretroviral drug 2’,3’-dideoxycytidine. TAT-CBD3 was mildly anxiolytic but innocuous on sensorimotor and cognitive functions and despair. By preventing CRMP-2-mediated enhancement of CaV2.2 function, TAT-CBD3 alleviates inflammatory and neuropathic hypersensitivity, an approach that may prove useful in managing clinical pain.

Excessive CNS synapses are eliminated during development to establish mature patterns of neuronal connectivity. A complement cascade protein, C1q, is involved in this process. Mice deficient in C1q fail to refine retinogeniculate connections resulting in excessive retinal innervation of lateral geniculate neurons. We hypothesized that C1q knockout (KO) mice would exhibit defects in neocortical synapse elimination resulting in enhanced excitatory synaptic connectivity and epileptiform activity. We recorded spontaneous and evoked field potential activity in neocortical slices and obtained video-EEG recordings from implanted C1q KO and wild-type (WT) mice. We also used laser scanning photostimulation of caged glutamate and whole cell recordings to map excitatory and inhibitory synaptic connectivity. Spontaneous and evoked epileptiform field potentials occurred at multiple sites in neocortical slices from C1q KO, but not WT mice. Laser mapping experiments in C1q KO slices showed that the proportion of glutamate uncaging sites from which excitatory postsynaptic currents (EPSCs) could be evoked ("hotspot ratio") increased significantly in layer IV and layer V, although EPSC amplitudes were unaltered. Density of axonal boutons was significantly increased in layer V pyramidal neurons of C1q KO mice. Implanted KO mice had frequent behavioral seizures consisting of behavioral arrest associated with bihemispheric spikes and slow wave activity lasting from 5 to 30 s. Results indicate that epileptogenesis in C1q KO mice is related to a genetically determined failure to prune excessive excitatory synapses during development. complement cascade | seizure | synaptic pruning | development | axonal boutons E arly brain development is marked by an exuberant outgrowth of axons that innervate multiple targets. These projections are later refined through axonal pruning, a developmental regulatory process by which excessive connectivity is eliminated and mature patterns of neuronal innervation become established (1). Cortical synaptic pruning events, such as selective branch elimination for segregating layer V cortical neuronal projections to subcortical targets (1), and refinement of interhemispheric (2) and intracortical (3) are important for the development of precisely wired connections and normal information processing. The activation of a number of genes and proteins is crucial for the process of synaptic pruning in the mammalian brain (4). Recently, molecules ordinarily associated with immune and inflammatory processes have been shown to play nonimmune roles in synaptic pruning (5). C1q and C3 genes of the classical complement cascade are necessary for synapse elimination in the retino-geniculate pathway during development (6). Activation of C1q protein by astrocytes leads to triggering of a protease cascade, deposition of C3 and opsonization or "tagging" of synapses that are likely eliminated by microglia. Lateral geniculate relay neurons in C1q knockout (KO) mice deficient in these proteins retain multiple synaptic contacts f...

Formation of new recurrent excitatory circuits after brain injuries has been hypothesized as a major factor contributing to epileptogenesis. Increases in total axonal length and the density of synaptic boutons are present in layer V pyramidal neurons of chronic partial isolations of rat neocortex, a model of posttraumatic epileptogenesis. To explore the functional consequences of these changes, we used laser-scanning photostimulation combined with whole-cell patch-clamp recording from neurons in layer V of somatosensory cortex to map changes in excitatory synaptic connectivity after injury. Coronal slices were submerged in artificial CSF (23°C) containing 100 M caged glutamate, APV (2-amino-5-phosphonovaleric acid), and high divalent cation concentration to block polysynaptic responses. Focal uncaging of glutamate, accomplished by switching a pulsed UV laser to give a 200 -400 s light stimulus, evoked single-or multiple-component composite EPSCs. In neurons of the partially isolated cortex, there were significant increases in the fraction of uncaging sites from which EPSCs could be evoked ("hot spots") and a decrease in the mean amplitude of individual elements in the composite EPSC. When plotted along the cortical depth, the changes in EPSCs took place mainly between 150 and 200 m above and below the somata, suggesting a specific enhancement of recurrent excitatory connectivity among layer V pyramidal neurons of the undercut neocortex. These changes may shift the balance within cortical circuits toward increased synaptic excitation and contribute to epileptogenesis.

In the mature brain, the K(+)/Cl- cotransporter KCC2 is important in maintaining low [Cl-]i, resulting in hyperpolarizing GABA responses. Decreases in KCC2 after neuronal injuries result in increases in [Cl-]i and enhanced neuronal excitability due to depolarizing GABA responses. We used the gramicidin perforated-patch technique to measure E(Cl) ( approximately E(GABA)) in layer V pyramidal neurons in slices of partially isolated sensorimotor cortex of adult rats to explore the potential functional consequence of KCC2 downregulation in chronically injured cortex. E(GABA) was measured by recording currents evoked with brief GABA puffs at various membrane potentials. There was no significant difference in E(Cl) between neurons in control and undercut animals (-71.2 +/- 2.6 and -71.8 +/- 2.8 mV, respectively). However, when loaded with Cl- by applying muscimol puffs at 0.2 Hz for 60 s, neurons in the undercut cortex had a significantly shorter time constant for the positive shift in E(Cl) during the Cl- loading phase (4.3 +/- 0.5 s for control and 2.2 +/- 0.4 s for undercut, P < 0.01). The positive shift in E(Cl) 3 s after the beginning of Cl- loading was also significantly larger in the undercut group than in the control, indicating that neurons in undercut cortex were less effective in maintaining low [Cl-]i during repetitive activation of GABA(A) receptors. Application of furosemide eliminated the difference between the control and undercut groups for both of these measures of [Cl-]i regulation. The results suggest an impairment in Cl- extrusion resulting from decreased KCC2 expression that may reduce the strength of GABAergic inhibition and contribute to epileptogenesis.

Brain-derived neurotrophic factor (BDNF) promotes postnatal maturation of GABAergic inhibition in the cerebral and cerebellar cortices, and its expression and release are enhanced by neuronal activity, suggesting that it acts in a feedback manner to maintain a balance between excitation and inhibition during development. BDNF promotes differentiation of cerebellar, hippocampal, and neostriatal inhibitory neurons, but its effects on the dendritic development of neocortical inhibitory interneurons remain unknown. Here, we show that BDNF mediates depolarization-induced dendritic growth and branching in neocortical interneurons. To visualize inhibitory interneurons, we biolistically transfected organotypic cortical slice cultures from neonatal mice with green fluorescent protein (GFP) driven by the glutamic acid decarboxylase (GAD)67 promoter. Nearly all GAD67-GFP-expressing neurons were nonpyramidal, many contained GABA, and some expressed markers of neurochemically defined GABAergic subtypes, indicating that GAD67-GFP-expressing neurons were GABAergic. We traced dendritic trees from confocal images of the same GAD67-GFP-expressing neurons before and after a 5 d growth period, and quantified the change in total dendritic length (TDL) and total dendritic branch points (TDBPs) for each neuron. GAD67-GFP-expressing neurons growing in control medium exhibited a 20% increase in TDL, but in 200 ng/ml BDNF or 10 mm KCl, this increase nearly doubled and was accompanied by a significant increase in TDBPs. Blocking action potentials with TTX did not prevent the BDNF-induced growth, but antibodies against BDNF blocked the growth-promoting effect of KCl. We conclude that BDNF, released by neocortical pyramidal neurons in response to depolarization, enhances dendritic growth and branching in nearby inhibitory interneurons.