Intracranial Pressure Monitoring in Experimental Traumatic Brain Injury: Implications for Clinical Management.

Traumatic brain injury (TBI) is often associated with long-term disability and chronic neurological sequelae. One common contributor to unfavorable outcomes is secondary brain injury, which is potentially treatable and preventable through appropriate management of patients in the neurosurgical intensive care unit. Intracranial pressure (ICP) is currently the predominant neurological-specific physiological parameter used to direct the care of severe TBI (sTBI) patients. However, recent clinical evidence has called into question the association of ICP monitoring with improved clinical outcome. The detailed cellular and molecular derangements associated with intracranial hypertension (IC-HTN) and their relationship to injury phenotype and neurological outcomes are not completely understood. Various animal models of TBI have been developed, but the clinical applicability of ICP monitoring in the pre-clinical setting has not been well-characterized. Linking basic mechanistic studies in translational TBI models with investigation of ICP monitoring that more faithfully replicates the clinical setting will provide clinical investigators with a more informed understanding of the pathophysiology of IC-HTN, thus facilitating development of improved therapies for sTBI patients.

Chronic Upregulation of Cleaved-Caspase-3 Associated with Chronic Myelin Pathology and Microvascular Reorganization in the Thalamus after Traumatic Brain Injury in Rats.

Traumatic brain injury (TBI) is associated with long-term disabilities and devastating chronic neurological complications including problems with cognition, motor function, sensory processing, as well as behavioral deficits and mental health problems such as anxiety, depression, personality change and social unsuitability. Clinical data suggest that disruption of the thalamo-cortical system including anatomical and metabolic changes in the thalamus following TBI might be responsible for some chronic neurological deficits following brain trauma. Detailed mechanisms of these pathological processes are not completely understood. The goal of this study was to evaluate changes in the thalamus following TBI focusing on cleaved-caspase-3, a specific effector of caspase pathway activation and myelin and microvascular pathologies using immuno- and histochemistry at different time points from 24 h to 3 months after controlled cortical impact (CCI) in adult Sprague-Dawley rats. Significant increases in cleaved-caspase-3 immunoreactivity in the thalamus were observed starting one month and persisting for at least three months following experimental TBI. Further, the study demonstrated an association of cleaved-caspase-3 with the demyelination of neuronal processes and tissue degeneration in the gray matter in the thalamus, as reflected in alterations of myelinated fiber integrity (luxol fast blue) and decreases in myelin basic protein (MBP) immunoreactivity. The immunofluorescent counterstaining of cleaved-caspase-3 with endothelial barrier antigen (EBA), a marker of blood-brain barrier, revealed limited direct and indirect associations of cleaved caspase-3 with blood-brain barrier damage. These results demonstrate for the first time a significant chronic upregulation of cleaved-caspase-3 in selected thalamic regions associated with cortical regions directly affected by CCI injury. Further, our study is also the first to report that significant upregulation of cleaved-caspase-3 in selected ipsilateral thalamic regions is associated with microvascular reorganization reflected in the significant increases in the number of microvessels with blood-brain barrier alterations detected by EBA staining. These findings provide new insights into potential mechanisms of TBI cell death involving chronic activation of caspase-3 associated with disrupted cortico-thalamic and thalamo-cortical connectivity. Moreover, this study offers the initial evidence that this upregulation of activated caspase-3, delayed degeneration of myelinated nerve fibers and microvascular reorganization with impaired blood-brain barrier integrity in the thalamus might represent reciprocal pathological processes affecting neuronal networks and brain function at the chronic stages of TBI.

Role of Caspase-3-Mediated Apoptosis in Chronic Caspase-3-Cleaved Tau Accumulation and Blood-Brain Barrier Damage in the Corpus Callosum after Traumatic Brain Injury in Rats.

Traumatic brain injury (TBI) may be a significant risk factor for development of neurodegenerative disorders such as chronic traumatic encephalopathy (CTE), post-traumatic epilepsy (PTE), and Alzheimer's (AD) and Parkinson's (PD) diseases. Chronic TBI is associated with several pathological features that are also characteristic of neurodegenerative diseases, including tau pathologies, caspase-3-mediated apoptosis, neuroinflammation, and microvascular alterations. The goal of this study was to evaluate changes following TBI in cleaved-caspase-3 and caspase-3-cleaved tau truncated at Asp421, and their relationships to cellular markers potentially associated with inflammation and blood-brain (BBB) barrier damage. We studied astrocytes (glial fibrillary acidic protein [GFAP]), microglia (ionized calcium-binding adapter molecule 1 [Iba1]), BBB (endothelial barrier antigen [EBA]), and activated microglia/macrophages (cluster of differentiation 68 [CD68]). We employed immunohistochemistry at different time points from 24 h to 3 months after controlled cortical impact (CCI) injury in rats, with particular interest in white matter. The study demonstrated that CCI caused chronic upregulation of cleaved-caspase-3 in the white matter of the corpus callosum. Increases in cleaved-caspase-3 in the corpus callosum were accompanied by accumulation of caspase-3-cleaved tau, with increasing perivascular aggregation 3 months after CCI. Immunofluorescence experiments further showed cellular co-localization of cleaved-caspase-3 with GFAP and CD68 and its adjacent localization with EBA, suggesting involvement of apoptosis and neuroinflammation in mechanisms of delayed BBB and microvascular damage that could contribute to white matter changes. This study also provides the first evidence that evolving upregulation of cleaved-caspase-3 is associated with accumulation of caspase-3-cleaved tau following experimental TBI, thus providing new insights into potential common mechanisms mediated by caspase-3 and underlying chronic TBI pathologies and neurodegenerative diseases.

Prospective clinical biomarkers of caspase-mediated apoptosis associated with neuronal and neurovascular damage following stroke and other severe brain injuries: Implications for chronic neurodegeneration.

Acute brain injuries, including ischemic and hemorrhagic stroke, as well as traumatic brain injury (TBI), are major worldwide health concerns with very limited options for effective diagnosis and treatment. Stroke and TBI pose an increased risk for the development of chronic neurodegenerative diseases, notably chronic traumatic encephalopathy, Alzheimer's disease, and Parkinson's disease. The existence of premorbid neurodegenerative diseases can exacerbate the severity and prognosis of acute brain injuries. Apoptosis involving caspase-3 is one of the most common mechanisms involved in the etiopathology of both acute and chronic neurological and neurodegenerative diseases, suggesting a relationship between these disorders. Over the past two decades, several clinical biomarkers of apoptosis have been identified in cerebrospinal fluid and peripheral blood following ischemic stroke, intracerebral and subarachnoid hemorrhage, and TBI. These biomarkers include selected caspases, notably caspase-3 and its specific cleavage products such as caspase-cleaved cytokeratin-18, caspase-cleaved tau, and a caspase-specific 120 kDa αII-spectrin breakdown product. The levels of these biomarkers might be a valuable tool for the identification of pathological pathways such as apoptosis and inflammation involved in injury progression, assessment of injury severity, and prediction of clinical outcomes. This review focuses on clinical studies involving biomarkers of caspase-3-mediated pathways, following stroke and TBI. The review further examines their prospective diagnostic utility, as well as clinical utility for improved personalized treatment of stroke and TBI patients and the development of prophylactic treatment chronic neurodegenerative disease.

Animal Models of Posttraumatic Seizures and Epilepsy.

Posttraumatic epilepsy (PTE) is one of the most common and devastating complications of traumatic brain injury (TBI). Currently, the etiopathology and mechanisms of PTE are poorly understood and as a result, there is no effective treatment or means to prevent it. Antiepileptic drugs remain common preventive strategies in the management of TBI to control acute posttraumatic seizures and to prevent the development of PTE, although their efficacy in the latter case is disputed. Different strategies of PTE prophylaxis have been showing promise in preclinical models, but their translation to the clinic still remains elusive due in part to the variability of these models and the fact they do not recapitulate all complex pathologies associated with human TBI. TBI is a multifaceted disorder reflected in several potentially epileptogenic alterations in the brain, including mechanical neuronal and vascular damage, parenchymal and subarachnoid hemorrhage, subsequent toxicity caused by iron-rich hemoglobin breakdown products, and energy disruption resulting in secondary injuries, including excitotoxicity, gliosis, and neuroinflammation, often coexisting to a different degree. Several in vivo models have been developed to reproduce the acute TBI cascade of events, to reflect its anatomical pathologies, and to replicate neurological deficits. Although acute and chronic recurrent posttraumatic seizures are well-recognized phenomena in these models, there is only a limited number of studies focused on PTE. The most used mechanical TBI models with documented electroencephalographic and behavioral seizures with remote epileptogenesis include fluid percussion, controlled cortical impact, and weight-drop. This chapter describes the most popular models of PTE-induced TBI models, focusing on the controlled cortical impact and the fluid percussion injury models, the methods of behavioral and electroencephalogram seizure assessments, and other approaches to detect epileptogenic properties, and discusses their potential application for translational research.

Age-Dependent Effects of Haptoglobin Deletion in Neurobehavioral and Anatomical Outcomes Following Traumatic Brain Injury.

Cerebral hemorrhages are common features of traumatic brain injury (TBI) and their presence is associated with chronic disabilities. Recent clinical and experimental evidence suggests that haptoglobin (Hp), an endogenous hemoglobin-binding protein most abundant in blood plasma, is involved in the intrinsic molecular defensive mechanism, though its role in TBI is poorly understood. The aim of this study was to investigate the effects of Hp deletion on the anatomical and behavioral outcomes in the controlled cortical impact model using wildtype (WT) C57BL/6 mice and genetically modified mice lacking the Hp gene (Hp(-∕-)) in two age cohorts [2-4 mo-old (young adult) and 7-8 mo-old (older adult)]. The data obtained suggest age-dependent significant effects on behavioral and anatomical TBI outcomes and recovery from injury. Moreover, in the adult cohort, neurological deficits in Hp(-∕-) mice at 24 h were significantly improved compared to WT, whereas there were no significant differences in brain pathology between these genotypes. In contrast, in the older adult cohort, Hp(-∕-) mice had significantly larger lesion volumes compared to WT, but neurological deficits were not significantly different. Immunohistochemistry for ionized calcium-binding adapter molecule 1 (Iba1) and glial fibrillary acidic protein (GFAP) revealed significant differences in microglial and astrocytic reactivity between Hp(-∕-) and WT in selected brain regions of the adult but not the older adult-aged cohort. In conclusion, the data obtained in the study provide clarification on the age-dependent aspects of the intrinsic defensive mechanisms involving Hp that might be involved in complex pathways differentially affecting acute brain trauma outcomes.

Biomarkers for acute diagnosis and management of stroke in neurointensive care units.

The effectiveness of current management of critically ill stroke patients depends on rapid assessment of the type of stroke, ischemic or hemorrhagic, and on a patient's general clinical status. Thrombolytic therapy with recombinant tissue plasminogen activator (r-tPA) is the only effective treatment for ischemic stroke approved by the Food and Drug Administration (FDA), whereas no treatment has been shown to be effective for hemorrhagic stroke. Furthermore, a narrow therapeutic window and fear of precipitating intracranial hemorrhage by administering r-tPA cause many clinicians to avoid using this treatment. Thus, rapid and objective assessments of stroke type at admission would increase the number of patients with ischemic stroke receiving r-tPA treatment and thereby, improve outcome for many additional stroke patients. Considerable literature suggests that brain-specific protein biomarkers of glial [i.e. S100 calcium-binding protein B (S100B), glial fibrillary acidic protein (GFAP)] and neuronal cells [e.g., ubiquitin C-terminal hydrolase-L1 (UCH-L1), neuron-specific enolase (NSE), αII-spectrin breakdown products SBDP120, SBDP145, and SBDP150, myelin basic protein (MBP), neurofilament light chain (NF-L), tau protein, visinin-like protein-1 (VLP 1), NR2 peptide] injury that could be detected in the cerebrospinal fluid (CSF) and peripheral blood might provide valuable and timely diagnostic information for stroke necessary to make prompt management and decisions, especially when the time of stroke onset cannot be determined. This information could include injury severity, prognosis of short-term and long-term outcomes, and discrimination of ischemic or hemorrhagic stroke. This chapter reviews the current status of the development of biomarker-based diagnosis of stroke and its potential application to improve stroke care.

Finding effective biomarkers for pediatric traumatic brain injury.

As traumatic brain injury (TBI) continues to affect children and young adults worldwide, research on reliable biomarkers grows as a possible aid in determining the severity of injury. However, many studies have revealed that diverse biomarkers such as S100B and myelin basic protein (MBP) have many limitations, such as their elevated normative concentrations in young children. Therefore, the results of these studies have yet to be translated to clinical applications. However, despite the setbacks of research into S100B and MBP, investigators continue to research viable biomarkers, notably glial fibrillary acidic protein (GFAP) and ubiquitin C-terminal hydrolase L1 (UCH-L1), as possible aids in medical decision making. Studies have revealed that GFAP and UCH-L1 actually are better predictors of injury progression than the before-mentioned biomarkers S100B and MBP. In addition, UCH-L1 has demonstrated an ability to detect injury while CT is negative, suggesting an ability to detect acute intracranial lesions. Here, we evaluate research testing levels of GFAP and UCH-L1 on children diagnosed with TBI and compare our results to those of other tested biomarkers. In a recent study done by Hayes et al., GFAP and UCH-L1 demonstrated the potential to recognize children with the possibility of poor outcome, allowing for more specialized treatments with clinical and laboratory applications. Although studies on GFAP and UCH-L1 have for the most part warranted positive results, further studies will be needed to confirm their role as reliable markers for pediatric TBI.

Role of the prostaglandin E2 EP1 receptor in traumatic brain injury.

Brain injuries promote upregulation of so-called proinflammatory prostaglandins, notably prostaglandin E2 (PGE2), leading to overactivation of a class of its cognate G-protein-coupled receptors, including EP1, which is considered a promising target for treatment of ischemic stroke. However, the role of the EP1 receptor is complex and depends on the type of brain injury. This study is focused on the investigation of the role of the EP1 receptor in a controlled cortical impact (CCI) model, a preclinical model of traumatic brain injury (TBI). The therapeutic effects of post-treatments with a widely studied EP1 receptor antagonist, SC-51089, were examined in wildtype and EP1 receptor knockout C57BL/6 mice. Neurological deficit scores (NDS) were assessed 24 and 48 h following CCI or sham surgery, and brain immunohistochemical pathology was assessed 48 h after surgery. In wildtype mice, CCI resulted in an obvious cortical lesion and localized hippocampal edema with an associated significant increase in NDS compared to sham-operated animals. Post-treatments with the selective EP1 receptor antagonist SC-51089 or genetic knockout of EP1 receptor had no significant effects on cortical lesions and hippocampal swelling or on the NDS 24 and 48 h after CCI. Immunohistochemistry studies revealed CCI-induced gliosis and microglial activation in selected ipsilateral brain regions that were not affected by SC-51089 or in the EP1 receptor-deleted mice. This study provides further clarification on the respective contribution of the EP1 receptor in TBI and suggests that, under this experimental paradigm, the EP1 receptor would have limited effects in modulating acute neurological and anatomical pathologies following contusive brain trauma. Findings from this protocol, in combination with previous studies demonstrating differential roles of EP1 receptor in ischemic, neurotoxic, and hemorrhagic conditions, provide scientific background and further clarification of potential therapeutic application of prospective prostaglandin G-protein-coupled receptor drugs in the clinic for treatment of TBI and other acute brain injuries.

Prostaglandin F2α FP receptor antagonist improves outcomes after experimental traumatic brain injury.

BACKGROUND: Injuries to the brain promote upregulation of prostaglandins, notably the proinflammatory PGF2α, and overactivation of their cognate G-protein-coupled FP receptor, which could exacerbate neuronal damage. Our study is focused on investigation of the FP receptor as a target for novel neuroprotective drugs in a preclinical animal traumatic brain injury (TBI) model. METHODS: Accordingly, the effects of acute intraperitoneal post-treatment with selective FP antagonist AL-8810 were studied in wildtype (WT) and FP receptor knockout (FP-/-) mice after controlled cortical impact (CCI). Neurological impairments were evaluated using neurological deficit scores (NDS) and the grip strength test. Cortical lesions and overall brain pathology were assessed using immunohistochemistry. RESULTS: Morphological analyses of cerebral vasculature and anastomoses revealed no differences between WT and FP-/- mice. CCI produced cortical lesions characterized by cavitation, neuronal loss, and hematoma with a volume of 20.0 ± 1.0 mm(3) and significant hippocampal swelling (146.5 ± 7.4% of contralateral) compared with sham (P < 0.05). Post-treatment with AL-8810 (1 to 10 mg/kg) had no significant effect on cortical lesions, which suggests the irreversible effect of primary CCI injury, but significantly reduced hippocampal swelling to a size not significantly different from the sham group. Post-treatment with AL-8810 at a dose of 10 mg/kg significantly improved NDS at 24 and 48 hours after CCI (P < 0.001 and P < 0.01, respectively). In the AL-8810 group, CCI-induced decrease in grip strength was three-fold (2.93 ± 1.71) less and significantly different than in the saline-treated group. The FP-/- mice had significantly less hippocampal swelling, but not NDS, compared with WT mice. In addition, immunohistochemistry showed that pharmacologic blockade and genetic deletion of FP receptor led to attenuation of CCI-induced gliosis and microglial activation in selected brain regions. CONCLUSION: This study provides, for the first time, demonstration of the unique role of the FP receptor as a potential target for disease-modifying CNS drugs for treatment of acute traumatic injury.

Contribution of PGE2 EP1 receptor in hemin-induced neurotoxicity.

Although hemin-mediated neurotoxicity has been linked to the production of free radicals and glutamate excitotoxicity, the role of the prostaglandin E2 (PGE2)-EP1 receptor remains unclear. Activation of the EP1 receptor in neurons results in increased intracellular calcium levels; therefore, we hypothesize that the blockade of the EP1 receptor reduces hemin neurotoxicity. Using postnatal primary cortical neurons cultured from wild-type (WT) and EP1(-/-) mice, we investigated the EP1 receptor role in hemin neurotoxicity measured by lactate dehydrogenase (LDH) cell survival assay. Hemin (75 µM) induced greater release of LDH in WT (34.7 ± 4.5%) than in EP1(-/-) (27.6 ± 3.3%) neurons. In the presence of the EP1 receptor antagonist SC-51089, the hemin-induced release of LDH decreased. To further investigate potential mechanisms of action, we measured changes in the intracellular calcium level [Ca(2+)]i following treatment with 17-phenyl trinor PGE2 (17-pt-PGE2) a selective EP1 agonist. In the WT neurons, 17-pt-PGE2 dose-dependently increased [Ca(2+)]i. However, in EP1(-/-) neurons, [Ca(2+)]i was significantly attenuated. We also revealed that hemin dose-dependently increased [Ca(2+)]i in WT neurons, with a significant decrease in EP1(-/-) neurons. Both 17-pt-PGE2 and hemin-induced [Ca(2+)]i were abolished by N-methyl-D-aspartic (NMDA) acid receptor and ryanodine receptor blockers. These results suggest that blockade of the EP1 receptor may be protective against hemin neurotoxicity in vitro. We speculate that the mechanism of hemin neuronal death involves [Ca(2+)]i mediated by NMDA acid receptor-mediated extracellular Ca(2+) influx and EP1 receptor-mediated intracellular release from ryanodine receptor-operated Ca(2+) stores. Therefore, blockade of the EP1 receptor could be used to minimize neuronal damage following exposure to supraphysiological levels of hemin.

Putative role of prostaglandin receptor in intracerebral hemorrhage.

Each year, approximately 795,000 people experience a new or recurrent stroke. Of all strokes, 84% are ischemic, 13% are intracerebral hemorrhage (ICH) strokes, and 3% are subarachnoid hemorrhage strokes. Despite the decreased incidence of ischemic stroke, there has been no change in the incidence of hemorrhagic stroke in the last decade. ICH is a devastating disease 37-38% of patients between the ages of 45 and 64 die within 30 days. In an effort to prevent ischemic and hemorrhagic strokes we and others have been studying the role of prostaglandins and their receptors. Prostaglandins are bioactive lipids derived from the metabolism of arachidonic acid. They sustain homeostatic functions and mediate pathogenic mechanisms, including the inflammatory response. Most prostaglandins are produced from specific enzymes and act upon cells via distinct G-protein coupled receptors. The presence of multiple prostaglandin receptors cross-reactivity and coupling to different signal transduction pathways allow differentiated cells to respond to prostaglandins in a unique manner. Due to the number of prostaglandin receptors, prostaglandin-dependent signaling can function either to promote neuronal survival or injury following acute excitotoxicity, hypoxia, and stress induced by ICH. To better understand the mechanisms of neuronal survival and neurotoxicity mediated by prostaglandin receptors, it is essential to understand downstream signaling. Several groups including ours have discovered unique roles for prostaglandin receptors in rodent models of ischemic stroke, excitotoxicity, and Alzheimer disease, highlighting the emerging role of prostaglandin receptor signaling in hemorrhagic stroke with a focus on cyclic-adenosine monophosphate and calcium (Ca(2+)) signaling. We review current ICH data and discuss future directions notably on prostaglandin receptors, which may lead to the development of unique therapeutic targets against hemorrhagic stroke and brain injuries alike.

Halogenated derivatives of aromatic amino acids exhibit balanced antiglutamatergic actions: potential applications for the treatment of neurological and neuropsychiatric disorders.

Glutamate, the major excitatory neurotransmitter, is critical for normal brain development and function. Both extremes of glutamate receptor activity are detrimental for the brain. Glutamate's role in excitotoxicity has driven the search for receptor antagonists as neuroprotective agents, most of which have failed to achieve clinical, i.e. efficacious and safe, neuroprotection. High selectivity and potency provide potential explanations for this failure. For example, targeting individual glutamate receptor subtypes leaves other pathways of glutamatergic excitotoxicity intact. Furthermore, potent depression of glutamate receptor activity causes clinical side effects, such as the symptoms of schizophrenia produced by NMDA receptor antagonists. To produce efficacious neuroprotection devoid of significant side effects, it may be necessary to normalize the function of all components of the glutamatergic system, instead of blocking a single type of glutamate receptors. Halogenated derivatives of aromatic amino acids modulate glutamatergic activity via multiple pre- and postsynaptic actions with moderate efficacy. In addition, these compounds may trap hydroxyl radicals and facilitate hydroxyl radical-impaired glutamate uptake. Their balanced polyvalent action may overcome the limitations of previously tested glutamatergic agents and provide a basis for their use in the treatment of neurological and neuropsychiatric disorders. The properties of this class of compounds and relevant patents are reviewed in this article.

Neuroprotective action of halogenated derivatives of L-phenylalanine.

BACKGROUND AND PURPOSE: The aromatic amino acid L-Phenylalanine (L-Phe) significantly and reversibly depresses excitatory glutamatergic synaptic transmission (GST) via a unique set of presynaptic and postsynaptic mechanisms. Therefore, we hypothesized that endogenous derivatives of L-Phe, which display potent antiglutamatergic activity, may safely and efficaciously protect the brain during conditions characterized by overactivation of glutamate receptors. METHODS: We tested this hypothesis in vitro with a combination of patch-clamp and lactate dehydrogenase (LDH) analyses in rat cultured neurons exposed to simulated ischemia, and in vivo using a rat model of experimental stroke caused by transient middle cerebral artery occlusion (MCAO). RESULTS: 3,5-diiodo-L-tyrosine (DIT) and 3,5-dibromo-L-tyrosine (DBrT), endogenous halogenated derivatives of L-Phe, attenuated GST by similar mechanisms as L-Phe, but with greater potency. For example, the IC50s for DIT and DBrT to depress the frequency of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptor-mediated mEPSCs were 104.6+/-14.1 micromol/L and 127.5+/-13.3 micromol/L, respectively. Depression of GST by DIT and DBrT persisted during energy deprivation. Furthermore, DBrT significantly reduced LDH release in neuronal cultures exposed to oxygen glucose deprivation. In rats subjected to transient MCAO, DBrT decreased the brain infarct volume and neurological deficit score to 52.7+/-14.1% and 57.1+/-12.0% of control values, respectively. DBrT neither altered atrioventricular nodal and intraventricular conduction in isolated heart, nor heart rate and blood pressure in vivo. CONCLUSIONS: DBrT, an endogenous halogenated derivative of L-Phe, shows promise as a representative of a novel class of neuroprotective agents by exerting significant neuroprotection in both in vitro and in vivo models of brain ischemia.

Distribution of neuronal nicotinic acetylcholine receptors containing different alpha-subunits in the submucosal plexus of the guinea-pig.

The subunit composition and localisation of nicotinic acetylcholine receptors (nAChRs) in the submucosal plexus of the guinea-pig ileum were studied using both affinity-purified monoclonal and polyclonal antibodies against alpha3, alpha4, alpha5 and alpha7 nAChR subunits and specific alpha7-containing nAChRs blocker methyllycaconitine (MLA). By means of immunohistochemistry performed in non-dissociated preparations, it was found that only 4% of submucosal ganglia expressed nAChRs. Specific staining, associated with cell membranes, was found with alpha3-, alpha5- and alpha7-, but not alpha4-specific antibodies. Double staining using alpha5- and alpha7-specific antibodies demonstrated that about one-half of the nAChR-positive ganglia contained neurons immunoreactive to both antibodies, while the others possessed either alpha5- or alpha7-immunoreactivity. Nanomolar concentrations of MLA prevented alpha7-specific antibody binding and did not influence the alpha5-specific antibody binding even when applied in micromolar concentrations. In electrophysiological experiments performed using a patch-clamp 'whole-cell' recording method, the neurons were identified by their sensitivity to MLA. In conclusion, submucosal neurons of the guinea-pig ileum express nAChRs containing alpha3-, alpha5- and alpha7-subunits. The co localisation of alpha5- and alpha7-subunits found in immunohistochemical experiments as well as kinetic characteristics of MLA-blocked receptors found by electrophysiological experiments allow us to suggest the presence of heteromeric alpha7-containing nAChRs in the submucosal plexus of the guinea-pig ileum.