After injury, dorsal root (DR) axons regenerate in the peripheral nervous system (PNS), but turn around or stop at the dorsal root entry zone (DREZ), the entrance into the central nervous system (CNS). Examination of the dynamic axon regeneration that occurs following injury to the DR provides the opportunity to advance our understanding of what happens to sensory axons as they approach and arrive at the DREZ and expands our knowledge of sensory axon regeneration failure at the entrance to the spinal cord. Additionally, findings from these studies may offer potential avenues to provide insight into regeneration failure elsewhere in the central nervous system. Nevertheless, our understanding of the cellular and molecular processes underlying the failure of DR axons to regenerate through the DREZ is incomplete. The goal of my thesis work was to determine whether application of the time lapse-in vivo imaging technique is feasible and useful in studying dorsal root regeneration. I have also applied recently developed post-mortem analyses to the axons monitored in vivo, which provided additional insights into the mechanisms that prevent axon regeneration at the DREZ. Results in Chapters 2 and 3 demonstrate that wide-field microscopy is indeed feasible and useful for monitoring regenerating sensory axons immediately before, during, and in the days to weeks after lumbar (L5) DR crush. I was surprised to find that most axons were immobilized abruptly and chronically at the CNS portion of the DREZ, with their axon tips and shafts exhibiting features of differentiated nerve terminals. This observation raises the possibility, which has not been appreciated previously, that DR axons stop at the DREZ because their regeneration is terminated prematurely by forming synaptic contacts with unidentified postsynaptic cells. To confirm the immobilization of DR axons at the DREZ, I applied two-photon microscopy to examine the axon behavior at the DREZ at high resolution. Results described in Chapter 4 confirm those obtained with the time-lapse imaging performed with wide-field microscopy: axons arrested soon after their arrival at the DREZ did not exhibit even subtle movements. Light microscopic analyses of the failed axon tips monitored in vivo demonstrated that almost all axons stopped at the CNS territory of the DREZ, and that axon tips and adjacent shafts intensely immunolabeled with synapse markers. Ultrastructural analyses revealed that numerous axonal profiles had the characteristic features of pre- but not postsynaptic endings. Findings from these studies lead us to speculate that most, if not all, dorsal root axons become arrested as they enter the CNS territory of the DREZ by forming presynaptic terminals on non-neuronal cellular elements that differ from the dystrophic-like endings formed by a few axons. In the chapter 5, I discuss what I have found to be the key factors for successful monitoring of regenerating dorsal root axons in living animals; the feasibility, usefulness and limitations of the available techniques and future directions for studying spinal root injury and regeneration. My thesis work represents the first to employ in vivo imaging to study DR regeneration directly in living animals. This approach was more challenging to develop than we had anticipated but provided unexpected insights into the mechanisms preventing sensory nerve regeneration. Continuous application of the powerful in vivo imaging technique in combination with conventional analyses will elucidate critically important issues that previous static analyses could not decipher. / Cell Biology

A dor neuropática é uma condição debilitante causada por danos no sistema nervoso somatossensorial, como lesões dos nervos periféricos. As células do sistema imune, em particular os monócitos/macrófagos, desempenham um papel fundamental no desenvolvimento deste processo. Embora diversos estudos sugiram o envolvimento dessas células na medula espinal e gânglio da raiz dorsal (GRD) após a indução da neuropatia, a caracterização funcional e fenotípica, bem como a origem dessas células nesses órgãos, ainda não está esclarecida. Na medula espinal, estudos recentes têm demonstrado que apesar da massiva ativação e proliferação da micróglia residente, não há recrutamento de células mielóides para esse tecido após a indução da neuropatia, divergindo dos dados anteriormente descritos na literatura. Diante desses estudos controversos, iniciamos nosso trabalho demonstrando que possivelmente as células mielóides não são capazes de ultrapassar a barreira hematoencefálica e infiltrar na medula espinal após a indução da neuropatia periférica pelo modelo de SNI e assim, a ativação microglial ocorre de maneira independente do infiltrado dessas células neste tecido. No que se refere aos GRDs, trabalhos anteriores demonstram que há um aumento dos marcadores de ativação de macrófagos nesse tecido após a lesão periférica. Com isso, nós caracterizamos as subpopulações de monócitos/macrófagos presentes no GRD e identificamos, células CX3CR1+ e células CCR2+. De maneira interessante, ao isolarmos as células CX3CR1+ observamos que esse subtipo celular possa ser as principais células responsáveis pela produção dos mediadores inflamatórios no GRD após indução de SNI, enquanto as células CCR2+ parecem contribuir apenas de maneira parcial para a produção de IL-1? e TNF-? neste tecido, uma vez que a expressão desses mediadores não foi totalmente suprimida na ausência desse subtipo celular. Por fim, investigamos a origem desses subtipos de monócitos presentes no GRD. Por meio da parabiose entre animais wild type e GFP+, observamos que embora haja um pequeno aumento de células GFP+ no GRD de animais lesionados, essas células não são macrófagos. Corroborando com esses dados, ao realizarmos a parabiose de animais wild type com animais CX3CR1GFP/+CCR2RFP/+ não observamos presença de células CX3CR1 ou CCR2 no GRD após SNI. Em conjunto, nossos dados demonstram que existem duas subpopulações de monócitos no GRD, sendo uma delas residente e contribuindo de maneira efetiva para a produção dos mediadores inflamatórios locais e outra população de células CCR2+ que podem ter um papel mais relevante no sítio da lesão e assim, a exacerbação da inflamação local pode interferir indiretamente, na ativação das células presentes nos GRDs, bem como na produção dos mediadores inflamatórios no tecido, que vão contribuir para o desenvolvimento da dor neuropática. / Neuropathic pain is a debilitating disease due to severe damage to the nervous system, induced by peripheral nerve injury. The cells of the immune system, especially monocytes/macrophages, played a critical role in these process. Several projects have been suggested the role of these cells in the spinal cord and dorsal root ganglia (DRG) after neuropathic pain induction, but the functional and phenotypic characterization, as well as the source of cells, is still unclear. In the spinal cord, recent studies have shown that although massive activation and proliferation of the microglial occurred, there is no recruitment of myeloid cells to this tissue after the neuropathic pain induction, but this is contrary to previous findings in the literature. Based on this controversial studies, we first showed that myeloid cells are not able to overcome the blood-brain barrier and infiltrate in the spinal cord after the peripheral nerve injury by SNI model and thus, the microglial activation occurs independent of the infiltration of these cells in this tissue. With regard to DRGs, previous work has shown that there is an increase in the activation markers of macrophages after peripheral nerve injury.Thus, we characterized the subpopulations of monocytes/macrophages in the DRG and we identified CX3CR1+ and CCR2+ cells. Interestingly, when isolating the CX3CR1+ cells, we observed that this cell subtype may be the main cells responsible for the production of inflammatory mediators in the DRG after SNI induction, whereas CCR2+ cells appear to contribute only partially to the production of IL-1? and TNF-? in this tissue, since the expression of these mediators was not completely suppressed in the absence of this cellular subtype. Finally, we investigated the origin of these monocyte subtypes present in the DRG. Through parabiosis between wild type and GFP+ animals, we observed that although there is a small increase of GFP+ cells in the DRG of injured animals, these cells are not macrophages. Corroborating with these data, when performing the wild type parabiosis with CX3CR1GFP /+ CCR2RFP/+ animals, we did not observe the presence of CX3CR1 or CCR2 cells in the GRD after SNI. Finally, our data demonstrate that there are two subpopulations of monocytes in the DRG, one of them residing and contributing effectively to the production of local inflammatory mediators and another population of CCR2 cells that may have a more relevant role at the lesion site and thus, the exacerbation of local inflammation may indirectly interfere, in the activation of the cells present in the DRGs, as well as in the production of inflammatory mediators in the tissue, which will contribute to the development of neuropathic pain.

Following microsurgical reconstruction of injured peripheral nerves, severed axons are able to undergo spontaneous regeneration. However, the functional result is always unsatisfactory with poor sensory recovery and reduced motor function. One contributing factor is the retrograde neuronal death, which occurs in the dorsal root ganglia (DRG) and in the spinal cord. An additional clinical problem is the loss of nerve tissue that often occurs in the trauma zone and which requires “bridges” to reconnect separated nerve ends. The present thesis investigates the extent of retrograde degeneration in spinal motoneurons and cutaneous and muscular afferent DRG neurons after permanent axotomy and following treatment with N-acetyl-cysteine (NAC). In addition, it examines the survival and growth-promoting effects of nerve reconstructions performed by primary repair and peripheral nerve grafting in combination with NAC treatment. In adult rats, cutaneous sural and muscular medial gastrocnemius DRG neurons and spinal motoneurons were retrogradely labeled with fluorescent tracers from the homonymous transected nerves. Survival of labeled neurons was assessed at different time points after nerve transection, ventral root avulsion and ventral rhizotomy. Axonal regeneration was evaluated using fluorescent tracers after sciatic axotomy and immediate nerve repair. Intraperitoneal or intrathecal treatment with NAC was initiated immediately after nerve injury or was delayed for 1-2 weeks. Counts of labeled gastrocnemius DRG neurons did not reveal any significant retrograde cell death after nerve transection. Sural axotomy induced a delayed loss of DRG cells, which amounted to 43- 48% at 8-24 weeks postoperatively. Proximal transection of the sciatic nerve at 1 week after initial axonal injury did not further increase retrograde DRG degeneration, nor did it affect survival of corresponding motoneurons. In contrast, rhizotomy and ventral root avulsion induced marked 26- 53% cell loss among spinal motoneurons. Primary repair or peripheral nerve grafting supported regeneration of 53-60% of the motoneurons and 47-49% of the muscular gastrocnemius DRG neurons at 13 weeks postoperatively. For the cutaneous sural DRG neurons, primary repair or peripheral nerve grafting increased survival by 19-30% and promoted regeneration of 46-66% of the cells. Regenerating sural and medial gastrocnemius DRG neurons upregulate transcription of peripherin and activating transcription factor 3. The gene expression of the structural neurofilament proteins of high molecular weight was significantly downregulated following injury in both regenerating and non-regenerating sensory neurons. Treatment with NAC was neuroprotective for spinal motoneurons after ventral rhizotomy and avulsion, and sural DRG neurons after sciatic nerve injury. However, combined treatment with nerve graft and NAC had significant additive effect on neuronal survival and also increased the number of sensory neurons regenerating across the graft. In contrast, NAC treatment neither affected the number of regenerating motoneurons nor the number of myelinated axons in the nerve graft and in the distal nerve stump. In summary, the present results demonstrate that cutaneous sural sensory neurons are more sensitive to peripheral nerve injury than muscular gastrocnemius DRG cells. Moreover, the retrograde loss of cutaneous DRG cells taking place despite immediate nerve repair would still limit recovery of cutaneous sensory functions. Experimental data also show that NAC provides a highly significant degree of neuroprotection in animal models of adult nerve injury and could be combined with nerve grafting to further attenuate retrograde neuronal death and to promote functional regeneration.

Successful axon regeneration appears to depend on the development of an injury response. Dorsal root ganglion neurons exemplify the necessity of this injury response in a unique way. Peripheral nerve transection leads to development of an injury response and successful regeneration whereas central root transection does neither. The injury response may involve extracellular and intracellular pathways. To investigate the extraneuronal influences, we performed nerve transection of either the central or peripheral axon branches and studied the expression of GAP-43, a key growth associated protein, and the transcription factors ATF3, c-Jun, and STAT3. Our results show that the responses to peripheral versus central nerve transection are fundamentally different. Peripheral but not central nerve transection increases GAP-43, ATF3, and c-Jun expression. STAT3, however, is upregulated as a result of central but not peripheral nerve transection. To investigate potential intracellular signalling pathways, we applied FGF-2, an extracellular mitogen, or an analog of cAMP, an intracellular second messenger to the cut end of the peripheral axon. Our results indicate that FGF-2 and cAMP act as activators of GAP-43 expression. On the other hand, FGF-2 and cAMP act to downregulate the expression of ATF3. FGF-2 upregulates c-Jun and the activated form of STAT3. Paradoxically, the regulation of GAP-43 expression by cAMP or by FGF-2 in vivo shows opposing results from the previously reported in vitro studies. Our present results suggest that the peripheral nerve injury response may be governed by at least three different signalling pathways.

Successful axon regeneration appears to depend on the development of an injury response. Dorsal root ganglion neurons exemplify the necessity of this injury response in a unique way. Peripheral nerve transection leads to development of an injury response and successful regeneration whereas central root transection does neither. The injury response may involve extracellular and intracellular pathways. To investigate the extraneuronal influences, we performed nerve transection of either the central or peripheral axon branches and studied the expression of GAP-43, a key growth associated protein, and the transcription factors ATF3, c-Jun, and STAT3. Our results show that the responses to peripheral versus central nerve transection are fundamentally different. Peripheral but not central nerve transection increases GAP-43, ATF3, and c-Jun expression. STAT3, however, is upregulated as a result of central but not peripheral nerve transection. To investigate potential intracellular signalling pathways, we applied FGF-2, an extracellular mitogen, or an analog of cAMP, an intracellular second messenger to the cut end of the peripheral axon. Our results indicate that FGF-2 and cAMP act as activators of GAP-43 expression. On the other hand, FGF-2 and cAMP act to downregulate the expression of ATF3. FGF-2 upregulates c-Jun and the activated form of STAT3. Paradoxically, the regulation of GAP-43 expression by cAMP or by FGF-2 in vivo shows opposing results from the previously reported in vitro studies. Our present results suggest that the peripheral nerve injury response may be governed by at least three different signalling pathways.

A dor neuropática é uma condição debilitante causada por danos no sistema nervoso somatossensorial, como lesões dos nervos periféricos. As células do sistema imune, em particular os monócitos/macrófagos, desempenham um papel fundamental no desenvolvimento deste processo. Embora diversos estudos sugiram o envolvimento dessas células na medula espinal e gânglio da raiz dorsal (GRD) após a indução da neuropatia, a caracterização funcional e fenotípica, bem como a origem dessas células nesses órgãos, ainda não está esclarecida. Na medula espinal, estudos recentes têm demonstrado que apesar da massiva ativação e proliferação da micróglia residente, não há recrutamento de células mielóides para esse tecido após a indução da neuropatia, divergindo dos dados anteriormente descritos na literatura. Diante desses estudos controversos, iniciamos nosso trabalho demonstrando que possivelmente as células mielóides não são capazes de ultrapassar a barreira hematoencefálica e infiltrar na medula espinal após a indução da neuropatia periférica pelo modelo de SNI e assim, a ativação microglial ocorre de maneira independente do infiltrado dessas células neste tecido. No que se refere aos GRDs, trabalhos anteriores demonstram que há um aumento dos marcadores de ativação de macrófagos nesse tecido após a lesão periférica. Com isso, nós caracterizamos as subpopulações de monócitos/macrófagos presentes no GRD e identificamos, células CX3CR1+ e células CCR2+. De maneira interessante, ao isolarmos as células CX3CR1+ observamos que esse subtipo celular possa ser as principais células responsáveis pela produção dos mediadores inflamatórios no GRD após indução de SNI, enquanto as células CCR2+ parecem contribuir apenas de maneira parcial para a produção de IL-1? e TNF-? neste tecido, uma vez que a expressão desses mediadores não foi totalmente suprimida na ausência desse subtipo celular. Por fim, investigamos a origem desses subtipos de monócitos presentes no GRD. Por meio da parabiose entre animais wild type e GFP+, observamos que embora haja um pequeno aumento de células GFP+ no GRD de animais lesionados, essas células não são macrófagos. Corroborando com esses dados, ao realizarmos a parabiose de animais wild type com animais CX3CR1GFP/+CCR2RFP/+ não observamos presença de células CX3CR1 ou CCR2 no GRD após SNI. Em conjunto, nossos dados demonstram que existem duas subpopulações de monócitos no GRD, sendo uma delas residente e contribuindo de maneira efetiva para a produção dos mediadores inflamatórios locais e outra população de células CCR2+ que podem ter um papel mais relevante no sítio da lesão e assim, a exacerbação da inflamação local pode interferir indiretamente, na ativação das células presentes nos GRDs, bem como na produção dos mediadores inflamatórios no tecido, que vão contribuir para o desenvolvimento da dor neuropática. / Neuropathic pain is a debilitating disease due to severe damage to the nervous system, induced by peripheral nerve injury. The cells of the immune system, especially monocytes/macrophages, played a critical role in these process. Several projects have been suggested the role of these cells in the spinal cord and dorsal root ganglia (DRG) after neuropathic pain induction, but the functional and phenotypic characterization, as well as the source of cells, is still unclear. In the spinal cord, recent studies have shown that although massive activation and proliferation of the microglial occurred, there is no recruitment of myeloid cells to this tissue after the neuropathic pain induction, but this is contrary to previous findings in the literature. Based on this controversial studies, we first showed that myeloid cells are not able to overcome the blood-brain barrier and infiltrate in the spinal cord after the peripheral nerve injury by SNI model and thus, the microglial activation occurs independent of the infiltration of these cells in this tissue. With regard to DRGs, previous work has shown that there is an increase in the activation markers of macrophages after peripheral nerve injury.Thus, we characterized the subpopulations of monocytes/macrophages in the DRG and we identified CX3CR1+ and CCR2+ cells. Interestingly, when isolating the CX3CR1+ cells, we observed that this cell subtype may be the main cells responsible for the production of inflammatory mediators in the DRG after SNI induction, whereas CCR2+ cells appear to contribute only partially to the production of IL-1? and TNF-? in this tissue, since the expression of these mediators was not completely suppressed in the absence of this cellular subtype. Finally, we investigated the origin of these monocyte subtypes present in the DRG. Through parabiosis between wild type and GFP+ animals, we observed that although there is a small increase of GFP+ cells in the DRG of injured animals, these cells are not macrophages. Corroborating with these data, when performing the wild type parabiosis with CX3CR1GFP /+ CCR2RFP/+ animals, we did not observe the presence of CX3CR1 or CCR2 cells in the GRD after SNI. Finally, our data demonstrate that there are two subpopulations of monocytes in the DRG, one of them residing and contributing effectively to the production of local inflammatory mediators and another population of CCR2 cells that may have a more relevant role at the lesion site and thus, the exacerbation of local inflammation may indirectly interfere, in the activation of the cells present in the DRGs, as well as in the production of inflammatory mediators in the tissue, which will contribute to the development of neuropathic pain.

Les antidépresseurs ont un effet antiallodynique qui dépend de la stimulation des récepteurs β2-adrénergiques. Ceux-ci stimulent la production d’adénosine monophosphate cyclique (AMPc) régulé par les phosphodiestérases de type 4 (PDE4). Nous avons ici étudié l’effet d’inhibiteurs de PDE (iPDE) sur la douleur neuropathique, grâce à des approches de pharmacologie comportementale chez la souris complétées par de l’imagerie calcium et des approches moléculaires. Nos résultats montrent un effet antiallodynique des iPDE4 et des iPDE5. L’action des iPDE4 est liée à une diminution d’expression du TNFα dans le ganglion rachidien et au recrutement des récepteurs delta des opioïdes. Celle des iPDE5 nécessite à la fois les récepteurs mu et delta. Nous montrons aussi que l’action d’un iPDE4 dépend de la dose, l’activation de cellules gliales semblant corrélée à l’effet antiallodynique à faible dose, alors que celle des neurones à forte dose a un effet pronociceptif via les récepteurs TRPV1. / Antidepressants have an antiallodynic action that is dependent on β2-adrenoceptor stimulation. These receptors stimulate the cAMP production, which is regulated by type 4 phosphodiesterases (PDE4). Here, we studied that action of PDE inhibitors (iPDE) on neuropathic pain, using behavioral pharmacology approaches in mice, completed by calcium imaging and molecular approaches. Our results show the iPDE4s and iPDE5s have an antiallodynic action. The iPDE4s act through a decreased expression of TNFα in dorsal root ganglia and the recruitment of the delta opioid receptors. The action of iPDE5 requires both mu and delta opioid receptors. We also show that the action of an iPDE4 depends on the dose, the activation of glial cells at low dose being correlated with an antiallodynic action, while the recruitment of neurons at higher doses has a pronociceptive action via TRPV1 receptors.

While much is known about the influence of ventricular afferent neurons on cardiovascular function in the dog, identification of the neurochemicals transmitting cardiac afferent signals to central neurons is lacking. Accordingly, we identified ventricular afferent neurons in canine dorsal root ganglia (DRG) and nodose ganglia by retrograde labeling after injecting horseradish peroxidase (HRP) into the anterior right and left ventricles. Primary antibodies from three host species were used in immunohistochemical experiments to simultaneously evaluate afferent somata for the presence of HRP and markers for two neurotransmitters. Only a small percentage (2%) of afferent somata were labeled with HRP. About half of the HRP-identified ventricular afferent neurons in T3 DRG also stained for substance P (SP), calcitonin gene-related peptide (CGRP), or neuronal nitric oxide synthase (nNOS), either alone or with two markers colocalized. Ventricular afferent neurons and the general population of T3 DRG neurons showed the same labeling profiles; CGRP (alone or colocalized with SP) being the most common (30-40% of ventricular afferent somata in T3 DRG). About 30% of the ventricular afferent neurons in T2 DRG displayed CGRP immunoreactivity and binding of the putative nociceptive marker IB4. Ventricular afferent neurons of the nodose ganglia were distinct from those in the DRG by having smaller size and lacking immunoreactivity for SP, CGRP, and nNOS. These findings suggest that ventricular sensory information is transferred to the central nervous system by relatively small populations of vagal and spinal afferent neurons and that spinal afferents use a variety of neurotransmitters.

Indiana University-Purdue University Indianapolis (IUPUI) / Increased electrical activity in peripheral sensory neurons contributes to pain. A unique type of sodium current, fast resurgent current, is proposed to increase nerve activity and has been associated with pain pathologies. While sodium channel isoform Nav1.6 has been identified as the main carrier of fast resurgent currents, our understanding of how resurgent currents are modulated in sensory neurons is fairly limited. Thus the goal of this dissertation was to identify resurgent current modulators. In particular, we focused on sodium channel beta subunits (Navβs) and fibroblast growth factor homologous factors (FHFs) in dorsal root ganglion (DRG) neurons. We hypothesized that Navβ4 and FHF2B act as positive regulators by mediating resurgent currents and modulating Nav1.6 inactivation, respectively. In contrast, we hypothesized FHF2A negatively regulates resurgent current by increasing the probability of channels in inactivated states. Thus, the aims of this dissertation were to 1) determine if Navβ4 regulates fast resurgent currents in DRG neurons, 2) examine the effects of Navβ4 knockdown on resurgent currents, firing frequency and pain associated behavior in an inflammatory pain model and 3) determine if FHF2A and FHF2B functionally regulate Nav1.6 currents, including resurgent currents in DRG neurons. To examine the aims, we used biochemical, electrophysiological and behavioral assays. Our results suggest that Navβ4 is a positive regulator of resurgent currents: in particular, the C-terminus likely mediates these currents. Localized knockdown of Navβ4 decreased inflammation-induced enhancement of resurgent currents and neuronal excitability, and prevented the development of persistent pain associated behavior in an inflammatory pain model. FHF2B increased resurgent currents and delayed inactivation. In contrast, FHF2A limited resurgent currents; an effect that is mainly contributed by FHF2A's N-terminus activity that increased accumulation of channels in inactivated states. Interestingly, in an inflammatory pain model FHF2B was upregulated and FHFA isoforms were downregulated. Together these results suggest that FHF2A/B modulation might contribute to enhanced resurgent currents and increased neuronal excitability observed in the inflammatory pain model. Overall, our work has identified three resurgent current modulators FHF2A, FHF2B and Navβ4. Manipulation of these proteins or their activity might result in novel strategies for the study and treatment of pain.

1. The effects of external H-7, a potent protein kinase inhibitor, on the responses mediated by γ-aminobutyric acid A type (GAGA(A))-, nicotinic acetylcholine (nicotinic ACh)-, ionotropic 5-hydroxytryptamine (5-HT3)-, adenosine 5'-triphosphate (ATP)-, N-methyl-D-aspartate (NMDA)- and kainate (KA)-receptors were studied in freshly dissociated rat dorsal root ganglion neurone by use of whole cell patch-clamp technique. 2. External H-7 (1-1000 μM) produced a reversible, dose-dependent inhibition of whole cell currents activated by GABA, ACh and 5-HT. 3. Whole-cell currents evoked by ATP, 2-methylthio-ATP, NMDA and KA were sensitive to external H-7. 4. External H-7 shifted the dose-response curve of GABA-activated currents downward without changing the EC50 significantly (from 15.0 ± 4.0 μM to 18.0 ± 5.0 μM). The maximum response to GABA was depressed by 34.0 ± 5.3%. This inhibitory action of H-7 was voltage-independent. 5. Intracellular application of H-7 (20 μM), cyclic AMP (1 mM) and BAPTA (10 mM) could not reverse the H-7 inhibition of GABA-activated currents. 6. The results suggest that external H-7 selectively and allosterically modulates the functions of GABA(A)-, nicotine ACh- and 5-HT3 receptors via a common conserved site in the external domain of these receptors.