Alistair K. Dixon

Tissue distribution of adenosine receptor mRNAs in the rat

1 A degree of ambiguity and uncertainty exists concerning the distribution of mRNAs encoding the four cloned adenosine receptors. In order to consolidate and extend current understanding in this area, the expression of the adenosine receptors has been examined in the rat by use of in situ hybridisation and the reverse transcription‐polymerase chain reaction (RT‐PCR). 2 In accordance with earlier studies, in situ hybridisation revealed that the adenosine A1 receptor was widely expressed in the brain, whereas A2A receptor mRNA was restricted to the striatum, nucleus accumbens and olfactory tubercle. In addition, A1 receptor mRNA was detected in large striatal cholinergic interneurones, 26% of these neurones were also found to express the A2A receptor gene. Central levels of mRNAs encoding adenosine A2B and A3 receptors were, however, below the detection limits of in situ hybridisation. 3 The more sensitive technique of RT‐PCR was then employed to investigate the distribution of adenosine receptor mRNAs in the central nervous system (CNS) and a wide range of peripheral tissues. As a result, many novel sites of adenosine receptor gene expression were identified. A1 receptor expression has now been found in the heart, aorta, liver, kidney, eye and bladder. These observations are largely consistent with previous functional data. A2A receptor mRNA was detected in all brain regions tested, demonstrating that expression of this receptor is not restricted to the basal ganglia. In the periphery A2A receptor mRNA was also found to be more widely distributed than generally recognised. The ubiquitous distribution of the A2B receptor is shown for the first time, A2B mRNA was detected at various levels in all rat tissues studied. Expression of the gene encoding the adenosine A3 receptor was also found to be widespread in the rat, message detected throughout the CNS and in many peripheral tissues. This pattern of expression is similar to that observed in man and sheep, which had previously been perceived to possess distinct patterns of A3 receptor gene expression in comparison to the rat. 4 In summary, this work has comprehensively studied the expression of all the cloned adenosine receptors in the rat, and in so doing, resolves some of the uncertainty over where these receptors might act to control physiological processes mediated by adenosine.

Glucose‐receptive neurones in the rat ventromedial hypothalamus express KATP channels composed of Kir6.1 and SUR1 subunits

1 Patch‐clamp recordings were made from rat ventromedial hypothalamic neurones in slices of brain tissue in vitro. In cell‐attached recordings, removal of extracellular glucose or metabolic inhibition with sodium azide reduced the firing rate of a subpopulation of cells through the activation of a 65 pS channel that was blocked by the sulphonylureas tolbutamide and glibenclamide. 2 In whole‐cell patch‐clamp recordings, in the absence of ATP in the electrode solution, glucose‐receptive neurones gradually hyperpolarized due to the induction of an outward current at ‐60 mV. This outward current and the resultant hyperpolarization were blocked by the sulphonylureas tolbutamide and glibenclamide. 3 In recordings where the electrode solution contained 4 mM ATP, this outward current was not observed. Under these conditions, 500 μM diazoxide was found to induce an outward current that was blocked by tolbutamide. 4 In cell‐attached recordings diazoxide and the active fragment of leptin (leptin 22–56) reduced the firing rate of glucose‐receptive neurones by the activation of a channel with similar properties to that induced by removal of extracellular glucose. 5 Reverse transcription followed by the polymerase chain reaction using cytoplasm from single glucose‐receptive neurones demonstrated the expression of the ATP‐sensitive potassium (KATP) channel subunits Kir6.1 and SUR1 but not Kir6.2 or SUR2. 6 It is concluded that glucose‐receptive neurones within the rat ventromedial hypothalamus exhibit a KATP channel current with pharmacological and molecular properties similar to those reported in other tissues.

Serotonin receptor mRNA expression in rat dorsal root ganglion neurons.

In the present study, we have used in situ hybridization to examine the distribution of serotonin (5-HT) receptors in rat dorsal root ganglion (DRG) neurons. Within DRG neurons, mRNAs for 5-HT1B, 5-HT1D, 5-HT2A, 5-HT2B, 5-HT3B and 5-HT4 receptors were readily detected in small (<25 microm), medium (25-45 microm) and large (>45 microm) diameter neurons. In contrast mRNAs for 5-HT1A, 5-HT1E, 5-HT2C, 5-HT5A, 5-HT5B, 5-HT6 and 5-HT7 receptors were undetectable in these neurons. The present study provides an insight into the molecular profile of 5-HT receptor subtypes in neurons responsible for modulating sensory information.

Desensitisation of the Adenosine A1 Receptor by the A2A Receptor in the Rat Striatum

Abstract: The influence of the adenosine A2A receptor on the A1 receptor was examined in rat striatal nerve terminals, a model for other cells in which these receptors are coexpressed. Incubation of striatal synaptosomes with the A2A receptor agonist 2‐p‐(2‐carboxyethyl)phenethylamino‐5′‐N‐ethylcarboxamidoadenosine (CGS 21680) caused the appearance of a low‐affinity binding site for the A1 receptor agonist 2‐chloro‐N6‐cyclopentyladenosine (CCPA). This effect was blocked by the A2A receptor antagonist ZM241385 and by the protein kinase C inhibitor chelerythrine, but not by the protein kinase A inhibitor N‐(2‐guanidinoethyl)‐5‐isoquinolinesulfonamide (HA 1004). The effect was not seen with striatal membranes or with hypotonically lysed synaptosomes. These results demonstrate a protein kinase C‐mediated heterologous desensitisation of the A1 receptor by the A2A receptor.

Identification of MEK1 as a novel target for the treatment of neuropathic pain

In the present study we have attempted to identify changes in gene expression which are associated with neuropathic pain using subtractive suppression hybridization analysis of the lumbar spinal cord of animals suffering streptozocin induced diabetic neuropathy. Using this approach, we found a significant up‐regulation of several key components of the extracellular signal‐regulated kinase (ERK) cascade. These findings were confirmed by Western blot analysis, which demonstrated that the levels of active ERK1 and 2 correlated with the onset of streptozocin‐induced hyperalgesia. Intrathecal administration of the selective MAPK/ERK‐kinase (MEK) inhibitor PD 198306 dose‐dependently (1–30 μg) blocked static allodynia in both the streptozocin and the chronic constriction injury (CCI) models of neuropathic pain. The antihyperalgesic effects of PD 198306, in both the streptozocin and CCI models of neuropathic pain, correlated with a reduction in the elevated levels of active ERK1 and 2 in lumbar spinal cord. Intraplantar administration of PD 198306 had no effect in either model of hyperalgesia, indicating that changes in the activation of ERKs and the effect of MEK inhibition are localized to the central nervous system. In summary, we have demonstrated for the first time that the development of neuropathic pain is associated with an increase in the activity of the MAPK/ERK‐kinase cascade within the spinal cord and that enzymes in this pathway represent potential targets for the treatment of this condition.

β3, a novel auxiliary subunit for the voltage-gated sodium channel, is expressed preferentially in sensory neurons and is upregulated in the chronic constriction injury model of neuropathic pain

Adult dorsal root ganglia (DRG) have been shown to express a wide range of voltage‐gated sodium channel α‐subunits. However, of the auxiliary subunits, β1 is expressed preferentially in only large‐ and medium‐diameter neurons of the DRG while β2 is absent in all DRG cells. In view of this, we have compared the distribution of β1 in rat DRG and spinal cord with a novel, recently cloned β1‐like subunit, β3. In situ hybridization studies demonstrated high levels of β3 mRNA in small‐diameter c‐fibres, while β1 mRNA was virtually absent in these cell types but was expressed in 100% of large‐diameter neurons. In the spinal cord, β3 transcript was present specifically in layers I/II (substantia gelatinosa) and layer X, while β1 mRNA was expressed in all laminae throughout the grey matter. Since the pattern of β3 expression in DRG appears to correlate with the TTX‐resistant voltage‐gated sodium channel subunit PN3, we co‐expressed the two subunits in Xenopus oocytes. In this system, β3 caused a 5‐mV hyperpolarizing shift in the threshold of activation of PN3, and a threefold increase in the peak current amplitude when compared with PN3 expressed alone. On the basis of these results, we examined the expression of β‐subunits in the chronic constriction injury model of neuropathic pain. Results revealed a significant increase in β3 mRNA expression in small‐diameter sensory neurons of the ipsilateral DRG. These results show that β3 is the dominant auxiliary sodium channel subunit in small‐diameter neurons of the rat DRG and that it is significantly upregulated in a model of neuropathic pain.

Developmental expression of the novel voltage-gated sodium channel auxiliary subunit β3, in rat CNS

1 We have compared the mRNA distribution of sodium channel alpha subunits known to be expressed during development with the known auxiliary subunits Naβ1.1 and Naβ2.1 and the novel, recently cloned subunit, β3. 2 In situ hybridisation studies demonstrated high levels of Nav1.2, Nav1.3, Nav1.6 and β3 mRNA at embryonic stages whilst Naβ1.1 and Naβ2.1 mRNA was absent throughout this period. 3 Naβ1.1 and Naβ2.1 expression occurred after postnatal day 3 (P3), increasing steadily in most brain regions until adulthood. β3 expression differentially decreased after P3 in certain areas but remained high in the hippocampus and striatum. 4 Emulsion‐dipped slides showed co‐localisation of β3 with Nav1.3 mRNA in areas of the CNS suggesting that these subunits may be capable of functional interaction. 5 Co‐expression in Xenopus oocytes revealed that β3 could modify the properties of Nav1.3; β3 changed the equilibrium of Nav1.3 between the fast and slow gating modes and caused a negative shift in the voltage dependence of activation and inactivation. 6 In conclusion, β3 is shown to be the predominant β subunit expressed during development and is capable of modulating the kinetic properties of the embryonic Nav1.3 subunit. These findings provide new information regarding the nature and properties of voltage‐gated sodium channels during development.

Gene-expression analysis at the single-cell level

The manner in which a cell responds to and influences its environment is ultimately determined by the genes that it expresses. To fully understand and manipulate cellular function, identification of these expressed genes is essential. Techniques such as RT-PCR enable examination of gene expression at the tissue level. However, the study of complex heterogeneous tissue, such as the CNS or immune system, requires gene analysis to be performed at much higher resolution. In this article, the various methods that have been developed to enable RT-PCR to be performed at the level of the single cell are reviewed. In addition, how, when carried out in combination with techniques such as patch-clamp recording, single-cell gene-expression studies extend our understanding of biological systems is discussed.

Identification of an ATP-sensitive potassium channel current in rat striatal cholinergic interneurones

1 Whole‐cell patch‐clamp recordings were made from rat striatal cholinergic interneurones in slices of brain tissue in vitro. In the absence of ATP in the electrode solution, these neurones were found to gradually hyperpolarize through the induction of an outward current at −60 mV. This outward current and the resultant hyperpolarization were blocked by the sulphonylureas tolbutamide and glibenclamide and by the photorelease of caged ATP within neurones. 2 This ATP‐sensitive outward current was not observed when 2 mM ATP was present in the electrode solution. Under these conditions, 500 μM diazoxide was found to induce an outward current that was blocked by tolbutamide. 3 Using permeabilized patch recordings, neurones were shown to hyperpolarize in response to glucose deprivation or metabolic poisoning with sodium azide (NaN3). The resultant hyperpolarization was blocked by tolbutamide. 4 In cell‐attached recordings, metabolic inhibition with 1 mM NaN3 revealed the presence of a tolbutamide‐sensitive channel exhibiting a unitary conductance of 44.1 pS. 5 Reverse transcription followed by the polymerase chain reaction using cytoplasm from single cholinergic interneurones demonstrated the expression of the ATP‐sensitive potassium (KATP) channel subunits Kir6.1 and SUR1 but not Kir6.2 or SUR2. 6 It is concluded that cholinergic interneurones within the rat striatum exhibit a KATP channel current and that this channel is formed from Kir6.1 and SUR1 subunits.

Expression of voltage-gated calcium channel subunits in rat dorsal root ganglion neurons

In the present study, we have used in situ hybridisation to examine the distribution of calcium channel subunits in rat dorsal root ganglion (DRG) neurons. Within DRG neurons, the calcium channel alpha subunit mRNAs alpha(1A), alpha(1B), alpha(1C), alpha(1D), alpha(1E), alpha(1I) and alpha(1S) were readily detected in small (<25 microm), medium (25-45 microm) and large (>45 microm) diameter neurons. alpha(1F) was present at very low levels in these neurons whilst alpha(1G) was virtually undetectable. The calcium channel auxiliary subunits alpha(2)delta(1) and alpha(2)delta(2) showed a complementary pattern of distribution to that of alpha(2)delta(3) in DRG neurons. alpha(2)delta(1) and alpha(2)delta(2) transcripts were expressed predominantly in small c-type sensory neurons and were present at lower levels in large Abeta-type sensory neurons. In contrast, alpha(2)delta(3) mRNA was present in high quantities in the large-diameter cells but was expressed at lower levels in small-diameter neurons of the DRG. The present study provides an insight into the molecular profile of calcium channel alpha(1) and alpha(2)delta subunits in the neurons responsible for transmitting sensory information.