Spinal lamina I neurons that express neurokinin 1 receptors: morphological analysis

The morphology of neurons in lamina I of the dorsal horn of the lumbar spinal cord which express neurokinin 1 receptors in the rat has been investigated. On the basis of soma and dendritic measurements, these neurons form two populations. One group consists of large neurons that stain intensely for the neurokinin 1 receptor with the immunochemical methods employed. They have a large soma, typically giving rise to between three and five thick principal dendrites. The dendritic tree, however, is relatively sparse, with the principal dendrites giving rise to small numbers of second- and third-order branches. All these dendrites are almost spine free. The dendritic tree spreads extensively in the rostrocaudal (approximately 550microm) and mediolateral (approximately 30microm) orientations, with few ventrally directed branches. These cells give rise to a single axon from their soma or a principal dendrite that generates a few local branches and also ramifies sparsely in deeper laminae (II-IV). The details of axonal morphology were established from intracellularly labelled material. Ultrastructural analysis of the synaptic input to these neurons reveals that they receive synapses with both clear round, flattened and dense-core vesicles; however, they do not form components of glomerular synapses.The second neuron type stains less intensely and typically has a small fusiform soma, giving rise to dendrites at its rostral and caudal poles. The dendritic tree is long in the rostrocaudal orientation (approximately 350microm), but restricted mediolaterally (approximately 40microm). The primary dendrites of these neurons bifurcate and soon give rise to third-order branches that are spiny. No pattern of organization could be detected for the distribution of either neuron type. These observations are discussed in the light of other recent studies indicating a central role for lamina I neurons expressing neurokinin 1 in the perception of severe pain.     

Involvement of the dorsolateral funiculus in the descending spinal projections responsible for diffuse noxious inhibitory controls in the rat

Recordings were made from convergent neurons in the lumbar dorsal horn of the spinal cord of the rat. These neurons were activated by both innocuous and noxious mechanical stimuli applied to their excitatory receptive fields located on the extremity of the hindpaw. Transcutaneous application of suprathreshold 2-ms square-wave electrical stimuli to the center of the excitatory field, resulted in responses to C-fiber activation being observed. This type of response was inhibited by applying a noxious thermal conditioning stimulus on the muzzle. The immersion of the muzzle in a 52 degrees C waterbath resulted in a strong reduction of the response during the application of the noxious conditioning stimulus and this was followed by long lasting poststimulus effects. Such inhibitory processes have been termed diffuse noxious inhibitory controls (DNIC). The effects on these inhibitions of lesions including the dorsolateral funiculus (DLF) were investigated in acute experiments: tests were performed before and at least 30 min after the DLF lesion. A lesion including the DLF ipsilateral to the neuron under study completely abolished the inhibitory processes triggered from the muzzle. Concomitantly, a facilitation of C-fiber responses was observed. Nevertheless, DNIC was still impaired even using a juxtathreshold current to elicit a weak C-fiber response. To ascertain further the main, if not entire, participation of the ipsilateral DLF in the descending projections responsible for the heterotopic inhibitory processes, the effects of a lesion of the contralateral DLF were investigated. Neither the inhibitory processes nor the unconditioned C-fiber responses were altered by this procedure. Again, a second lesion including the ipsilateral DLF induced a blockade of DNIC. It is concluded that the descending projections involved in the triggering of DNIC are mainly, if not entirely, confined to the DLF ipsilateral to the neuron under study. The contralateral DLF did not appear to play a role in these processes.     

Spinal interneurons that receive input from muscle afferents are differentially modulated by dorsolateral descending systems

The possibility that descending systems have differential actions on the spinal interneurons that receive input from muscle afferents was investigated. Prolonged, physiological inputs were generated by stretch of the triceps surae muscles. The resulting firing patterns of 25 lumbosacral interneurons were recorded before and during a reversible cold block of the dorsolateral white matter at the thoracic level in nonparalyzed, decerebrate preparations. The strength of group I muscle afferent input was assessed from the response to sinusoidal tendon vibration, which activated muscle spindle Ia afferents directly and tendon organ Ib afferents via the resulting reflex force. The stretch-evoked responses of interneurons with strong responses to vibration were markedly suppressed by dorsal cold block, whereas the stretch-evoked responses of interneurons with weak vibration input were enhanced. The cells most strongly activated by vibration received their primary input from Ia afferents and all of these cells were inhibited by the cold block. These results suggest that a disruption of the descending system, such as occurs in spinal cord injury, will lead to a suppression of the interneuronal pathways with group Ia input while enhancing excitability within interneuronal pathways transmitting actions from higher threshold afferents. One possible consequence of this suppression would be a decreased activity among the Ia inhibitory interneurons that mediate reciprocal inhibition, resulting in abnormal reciprocal relations between antagonists and promoting anomalous muscle cocontraction.     

NK-1 receptors modulate the excitability of ON cells in the rostral ventromedial medulla

The role of neurokinin-1 (NK-1) receptors in the rostral ventromedial medulla (RVM) was studied using extracellular single-unit recording combined with microiontophoresis. In rats, on- and off-type neurons were identified using noxious heat or mechanical stimuli applied to the tail. Responses evoked by iontophoretic application of N-methyl-d-aspartate (NMDA) were determined before and after intraplantar injection of capsaicin or iontophoretic application of substance P. In off cells, capsaicin produced an extended pause in ongoing activity but did not alter the subsequent spontaneous discharge rate or NMDA-evoked responses. In contrast, spontaneous discharge rates of on cells increased after capsaicin, and their responses to NMDA increased >100% above control values. The increased responses to NMDA after capsaicin were attenuated by iontophoretic application of the selective NK-1 receptor antagonist L-733,060. Similarly to capsaicin, iontophoretic application of the selective NK-1 receptor agonist, [Sar(9),Met(O(2))(11)]-substance P (SM-SP), increased the spontaneous discharge rate and NMDA-evoked responses of on cells by >100% of control values. These effects were antagonized by L-733,060. Immunohistochemical studies showed that a subset of neurons in the RVM labeled NK-1 receptors and that nearly all of these neurons were immunoreactive for the NMDAR1 subunit of the NMDA receptor. These results demonstrate that activation of NK-1 receptors in the RVM enhances responses of on cells evoked by NMDA. It is suggested that activation of NK-1 receptors in the RVM and the ensuing sensitization of on cells may contribute to the development of central sensitization and hyperalgesia after tissue injury and inflammation.     

Anesthetic blockade of the dorsolateral funiculus enhances evoked activity of spinal cord dorsal horn neurons.

1. A previous study of cat lumbar dorsal horn neurons found reduced responsiveness to A-fiber stimulation 1.5-12 h after thoracic dorsolateral funiculus (DLF) lesions. The present study was undertaken to determine whether this was due to the loss of descending activity or to factors specifically associated with injury by examining the response properties of dorsal horn cells before and during lidocaine blockade of the ipsilateral DLF. Electric shocks applied to the dorsal columns were used to search for dorsal horn cells. Noxious and nonnoxious cutaneous mechanical stimuli and graded electrical stimuli applied to the tibial nerve were used to activate peripheral afferent fibers. Cells were classed as low threshold (LT), high threshold (HT), or multireceptive (MR), according to their responses to natural stimuli. Baseline data were collected from a total of 58 cells. Twelve of these were further studied after lidocaine injection of the DLF. All cells examined with lidocaine were in dorsal horn laminae III-V. 2. All cells responded to activation of tibial nerve A fibers. However, the median threshold for the HT and MR cells (200 microA) was significantly higher than that of the LT cells (75 microA). Some cells in each class were also activated by C fibers (10, 70, and 64% of the LT, HT, and MR cells, respectively). 3. For the cells that were further characterized by lidocaine blockade of the DLF, all LT cells (n = 3) responded only to A-fiber stimulation, and all HT (n = 3) and MR cells (n = 6) responded to both A- and C-fiber stimulation. 4. For LT cells, responses evoked by mechanical and electrical stimuli were unaltered by lidocaine blockade. 5. HT and MR cells showed enhanced responses to electrical stimulation of C fibers during DLF blockade. There was no consistent effect of the blockade on A-fiber-evoked responses. 6. Two of three HT and four of six MR cells studied with lidocaine had spontaneous activity, which exhibited a small but significant increase during DLF blockade. 7. Receptive fields for noxious stimulation expanded in two of six MR cells during DLF blockade. Two of three HT cells developed responses to tactile stimuli during the blockade. 8. In two additional cells (1 HT and 1 MR), spontaneous activity and responses to C-fiber input increased after the DLF was cut.(ABSTRACT TRUNCATED AT 400 WORDS)     

M1 and M4 receptors modulate hippocampal pyramidal neurons

Acetylcholine (ACh), acting at muscarinic ACh receptors (mAChRs), modulates the excitability and synaptic connectivity of hippocampal pyramidal neurons. CA1 pyramidal neurons respond to transient ("phasic") mAChR activation with biphasic responses in which inhibition is followed by excitation, whereas prolonged ("tonic") mAChR activation increases CA1 neuron excitability. Both phasic and tonic mAChR activation excites pyramidal neurons in the CA3 region, yet ACh suppresses glutamate release at the CA3-to-CA1 synapse (the Schaffer-collateral pathway). Using mice genetically lacking specific mAChRs (mAChR knockout mice), we identified the mAChR subtypes responsible for cholinergic modulation of hippocampal pyramidal neuron excitability and synaptic transmission. Knockout of M1 receptors significantly reduced, or eliminated, most phasic and tonic cholinergic responses in CA1 and CA3 pyramidal neurons. On the other hand, in the absence of other G(q)-linked mAChRs (M3 and M5), M1 receptors proved sufficient for all postsynaptic cholinergic effects on CA1 and CA3 pyramidal neuron excitability. M3 receptors were able to participate in tonic depolarization of CA1 neurons, but otherwise contributed little to cholinergic responses. At the Schaffer-collateral synapse, bath application of the cholinergic agonist carbachol suppressed stratum radiatum-evoked excitatory postsynaptic potentials (EPSPs) in wild-type CA1 neurons and in CA1 neurons from mice lacking M1 or M2 receptors. However, Schaffer-collateral EPSPs were not significantly suppressed by carbachol in neurons lacking M4 receptors. We therefore conclude that M1 and M4 receptors are the major mAChR subtypes responsible for direct cholinergic modulation of the excitatory hippocampal circuit.     

Postsynaptic dorsal column neurons express NK1 receptors following colon inflammation

Recent clinical and experimental studies have suggested that the dorsal column pathway and specifically postsynaptic dorsal column neurons play an important role in the transmission of visceral pain. In our study we have mapped the distribution of postsynaptic dorsal column neurons in thoracic, lumbar and sacral spinal cord segments. The presence of immunoreactivity for neurokinin 1 receptors on these postsynaptic dorsal column neurons was examined under control conditions and after colon inflammation. The largest number of postsynaptic dorsal column neurons was found in the lumbar enlargement. They were mostly located in laminae III-IV, but depending on the spinal segment, about 7-15% of them were in the deep medial dorsal horn and in the central canal area. Under control conditions none of the 1438 postsynaptic dorsal column neurons examined expressed neurokinin 1 receptors. However, after induction of colon inflammation about 1.4% of the 2015 postsynaptic dorsal column neurons observed in the experimental group showed immunoreactivity for neurokinin 1 receptors. These neurons were preferentially found in the lower thoracic and lumbosacral spinal segments where they represented about 3-4% of the total population of postsynaptic dorsal column neurons examined. The de novo expression of neurokinin1 receptors on postsynaptic dorsal column neurons after colon inflammation suggests that substance P released from visceral primary afferents under inflammatory conditions could help produce central sensitization of these neurons.     

Neurokinin-1 receptors modulate the excitability of expiratory neurons in the ventral respiratory group

We studied the role of neurokinin-1 receptors (NK1-R) on the excitability of expiratory (E) neurons (tonic discharge, E(TONIC); augmenting, E(AUG); decrementing, E(DEC)) throughout the ventral respiratory group, including B?tzinger Complex (B?tC) using extracellular single-unit recording combined with pressurized picoejection in decerebrate, arterially perfused juvenile rats. Responses evoked by picoejection of the NK1-R agonist, [Sar9-Met(O2)11]-substance P (SSP) were determined before and after the selective NK1-R antagonist, CP99,994. SSP excited 20 of 35 expiratory neurons by increasing the number of action potentials per burst (+33.7 +/- 6.5% of control), burst duration (+20.6 +/- 7.9% of control), and peak firing frequency (+16.2 +/- 4.8% of control; means +/- SE). Pretreatment with CP99,994 completely blocked SSP-evoked excitation in a subset of neurons tested, supporting the notion that SSP excitation was mediated through NK1-R activation. Because we had previously shown that E(AUG) neurons were crucial to locomotor-respiratory coupling (LRC), we reasoned that blockade of NK1-R would alter LRC by preventing somatic-evoked excitation of E(AUG) neurons. Blockade of NK1-Rs by CP99,994 in the B?tC severely disrupted LRC and prevented somatic-evoked excitation of E(AUG) neurons. These findings demonstrate that LRC is dependent on endogenous SP release acting via NK1-Rs on E(AUG) neurons of the B?tC. Taken together with our earlier finding that inspiratory off-switching by the Hering-Breuer Reflex requires endogenous activation of NK1-Rs through activation of NK1-Rs on E(DEC) neurons, we suggest that endogenous release of substance P in the B?tC provides a reflex pathway-dependent mechanism to selectively modulate respiratory rhythm.     

Synaptic strength between motoneurons and terminals of the dorsolateral funiculus is regulated by GABA receptors in the turtle spinal cord

The role of GABAA and GABAB receptors in modulation of excitatory synaptic transmission between motoneurons and terminals from dorsolateral funiculus (DLF) was studied in in vitro spinal cord slices of adult turtles. Muscimol--a GABAA receptor agonist--depressed the monosynaptic excitatory postsynaptic potential (EPSP) induced by stimulation of the DLF and shortened its duration. The input resistance and the membrane time constant also were strongly reduced. The input membrane resistance, the amplitude, and the half-width of the EPSP were reduced at the same rate in the presence of muscimol. Bicuculline--a GABAA receptor antagonist--increased the EPSPs amplitude and the input membrane resistance. The EPSP amplitude ratio elicited by a paired-pulse protocol did not change significantly. Our results suggest that muscimol acts mainly by activation of postsynaptic GABAA receptors located on the motoneuron and the synaptic strength on motoneurons may be modulated by tonic activation of postsynaptic GABAA receptors. Baclofen--a GABAB receptor agonist--also depressed DLF-motoneuron synaptic transmission. However, it did not affect the falling phase of the EPSPs or the motoneuron membrane time constant but induced a small decrement in input resistance. In the presence of baclofen, the amplitude ratio produced by a paired-pulse protocol increased significantly. This suggests that baclofen decreased the synaptic strength by inhibition of neurotransmitter release from the DLF terminals via activation of presynaptic GABAB receptors.     

Functional and in situ hybridization evidence that preganglionic sympathetic vasoconstrictor neurons express ghrelin receptors

Agonists of ghrelin receptors can lower or elevate blood pressure, and it has been suggested that the increases in blood pressure are caused by actions at receptors in the spinal cord. However, this has not been adequately investigated, and the locations of neurons in the spinal cord that express ghrelin receptors, through which blood pressure increases may be exerted, are not known. We investigated the effects within the spinal cord of two non-peptide ghrelin receptor agonists, GSK894490 and CP464709, and two peptide receptor agonists, ghrelin and des-acyl ghrelin, and we used polymerase chain reaction (PCR) and in situ hybridization to examine ghrelin receptor expression. I.v. application of the non-peptide ghrelin receptor agonists caused biphasic changes in blood pressure, a brief drop followed by a blood pressure increase that lasted several minutes. The blood pressure rise, but not the fall, was antagonized by i.v. hexamethonium. Application of these agonists or ghrelin peptide directly to the spinal cord caused only a blood pressure increase. Des-acyl ghrelin had no significant action. The maximum pressor effects of agonists occurred with application at spinal cord levels T9 to T12. Neither i.v. nor spinal cord application of the agonists had significant effect on heart rate or the electrocardiogram. Ghrelin receptor gene expression was detected by PCR and in situ hybridization. In situ hybridization localized expression to neurons, including autonomic preganglionic neurons of the intermediolateral cell columns at all levels from T3 to S2. The numbers of ghrelin receptor expressing neurons in the intermediolateral cell columns were similar to the numbers of nitric oxide synthase positive neurons, but there was little overlap between these two populations. We conclude that activation of excitatory ghrelin receptors on sympathetic preganglionic neurons increases blood pressure, and that decreases in blood pressure caused by ghrelin agonists are mediated through receptors on blood vessels.     

Superficial NK1-expressing neurons control spinal excitability through activation of descending pathways

The increase in pain sensitivity that follows injury is regulated by superficially located projection neurons in the dorsal horn of the spinal cord that express the neurokinin-1 (NK1) receptor. After selective ablation of these neurons in rats, we identified changes in receptive field size, mechanical and thermal coding and central sensitization of deeper dorsal horn neurons that are important for both pain sensations and reflexes. We were able to reproduce these changes by pharmacological block of descending serotonergic facilitatory pathways. Using Fos histochemistry, we found changes in the activation of serotonergic neurons in the brainstem as well as evidence for a loss of descending control of spinal excitability. We conclude that NK1-positive spinal projection neurons, activated by primary afferent input, project to higher brain areas that control spinal excitability--and therefore pain sensitivity--primarily through descending pathways from the brainstem.     

Subsets of retinal neurons and glia express P2Y1 receptors

Recent evidence suggests that extracellular ATP modulates retinal processing and could play a role in modulating glial cells during retinal diseases. Here, we evaluated the localization of P2Y₁ receptors in the rat retina using indirect immunofluorescence immunocytochemistry. We observed labeling within defined populations of inner retinal neurons and Müller cell processes and end feet. Double labeling of P2Y₁ receptor with choline acetyltransferase revealed extensive colocalization indicating the expression of this receptor by cholinergic amacrine cells. Ganglion cell labeling for P2Y₁ receptors was also observed. Having established the normal pattern of immunolabeling within the retina, we next examined whether immunolabeling was altered by retinal disease. P2Y₁ receptor immunolabeling of Müller cells was of greater intensity following light-induced retinal degeneration, suggesting that Müller cell gliosis is accompanied by changes in P2Y₁ receptor expression. Overall, these data provide further evidence for a role of extracellular ATP in retinal signaling within subsets of retinal neurons as well as glia.     

GABAergic neurons express mu-opioid receptors in the ventrolateral orbital cortex of the rat

Behavioral studies have indicated that GABAergic modulation is involved in the opioid-induced antinociception in the ventrolateral orbital cortex (VLO). The aim of the current study was to examine whether the GABAergic neurons in the rat VLO expressed mu-opioid receptor subtype 1 (MOR1). This study employed immunofluorescence histochemical double-staining technique and showed that a considerable amount of GABA- and MOR1-like immunoreactive neurons existed in layers II-VI in the VLO. Of these GABA-like immunoreactive neurons, 92.0% of them showed MOR1-like immunoreactivities. Similarly, 80.2% of MOR1-like immuoreactive neurons also exhibited GABA-like immunoreactivities. These results provide morphological evidence that opioid-induced antinociception in the VLO might be due to an inhibitory effect by opioid via MOR1 on GABAergic neurons, resulting in disinhibition of VLO projection neurons and leading to activation of the VLO-PAG brainstem descending pain control system to depress the nociceptive inputs at the spinal cord level.     

Purinergic actions on neurons that modulate nociception in the rostral ventromedial medulla

The rostral ventromedial medulla (RVM) serves as a critical link in bulbo-spinal nociceptive modulation. Within the RVM, 'off-cells' pause and 'on-cells' discharge immediately prior to a nocifensive reflex. These neurons are also activated and inactivated, respectively, by local or systemic application of opioids. Off-cell activation leads to behavioral anti-nociception and on-cell activation to hyperalgesia. Thus, on- and off-cell populations allow bi-directional modulation of nociception by the RVM. A third neuronal population, neutral cells, shows no reflex-related change in discharge. The role of neutral cells in nociception, if any, is unknown. We investigated the responses of on-, off- and neutral cells to the iontophoretic application of purinergic ligands in lightly anesthetized rats. On-cell firing increased rapidly in response to application of ATP and to the P2X-receptor agonist, alpha,beta-methylene ATP. Off-cell firing increased gradually in response to ATP and to the P2Y-receptor agonist, 2-methylthio-ATP. All of these responses were attenuated or reversed by the non-specific P2-receptor antagonists, suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). Activation of off-cells was preferentially antagonized by the relatively selective P2Y antagonist, MRS2179. By contrast with activation of on- and off-cells by ATP, neutral cell firing was depressed by ATP, adenosine and the P1-receptor agonist, 5'-(N-ethylcarboxamido) adenosine (NECA). Neutral cell responses to these agonists were at least partially reversed by the adenosine-receptor antagonist, 8-phenyltheophylline (8PT). These data imply that on-cells preferentially express P2X-receptors, off-cells P2Y-receptors and neutral cells P1-receptors. Immunohistochemical localization of purinergic receptors confirms the presence of some subtypes of P2X, P2Y and A1 receptors on neuronal cell bodies and fibers within the RVM. The differential responses of on-, off- and neutral-cells to purinergic ligands highlight the value of pharmacological signatures in further delineation of the anatomy, connectivity and function of this therapeutically important system.     

Muscarinic receptors differentially modulate the persistent potassium current in striatal spiny projection neurons

Cholinergic regulation of striatal spiny projection neuron activity is predominantly mediated through muscarinic receptor modulation of several subclasses of ion channels. Because of its critical role in governing the recurring episodes of hyperpolarization and depolarization characteristic of spiny neurons in vivo, the 4-aminopyridine-resistant, persistent potassium (K+) current, IKrp, would be a strategic target for modulation. The present results show that IKrp can be either suppressed or enhanced by muscarinic receptor stimulation. Biophysical analysis demonstrated that the depression of IKrp was associated with a hyperpolarizing shift in the voltage dependence of inactivation and a reduction in maximal conductance. By contrast, the enhancement of IKrp was linked to hyperpolarizing shifts in both activation and inactivation voltage dependencies. Viewed in the context of the natural activity of spiny neurons, muscarinic depression of IKrp should uniformly increase excitability in both hyperpolarized and depolarized states. In the hyperpolarized state, the reduction in maximal conductance should bolster the efficacy of impending excitatory input. Likewise, in the depolarized state, the decreased availability of IKrp produced by the shift in inactivation should enhance ongoing synaptic input. The alterations associated with enhancement of IKrp are predicted to have a more dynamic influence on spiny cell excitability. In the hyperpolarized state, the negative shift in activation should increase the flow of IKrp and attenuate subsequent excitatory synpatic input; whereas once the cell has traversed into the depolarized state, the negative shift in inactivation should reduce the availability of this current and diminish its influence on the existing excitatory barrage.     

5-Hydroxytryptamine-induced outward currents mediated via 5-HT(1A) receptors in neurons of the rat dorsolateral septal nucleus

Effects of 5-hydroxytryptamine (5-HT) on neurons of the rat dorsolateral septal nucleus (DLSN) were examined by intracellular and whole-cell patch-clamp recording techniques. An outward current was induced by 5-HT (1-100 microM) in a concentration-dependent manner. The EC(50) for 5-HT was 4.8 microM. Also, 8-OH-DPAT (10-100 microM) produced the outward current an EC(50) of 17 microM. Amplitudes of the outward currents produced by 5-HT (100 microM) and 8-OH-DPAT (100 microM) were 117+/-4 (n=6) and 58+/-8 pA (n=6), respectively. Fluvoxamine (200 nM), a specific serotonin re-uptake inhibitor, enhanced the 5-HT (1 microM)-induced outward current: the EC(50) for 5-HT was 0.5 microM in the presence of fluvoxamine (200 nM). L-694247 (100 microM) and CP 93129 (100 microM) also produced outward currents with amplitudes of 33+/-3 (n=4) and 18+/-5 pA (n=4), respectively in DLSN neurons. DOI (100 microM) and RS 67333 (100 microM) did not produce outward currents. NAN-190 shifted, in a parallel manner, the concentration-response relationship of 5-HT to the right. The Lineweaver-Burk plot of the concentration-response curve showed that NAN-190 depressed the 5-HT-induced current in a competitive manner. The current-voltage relationship indicates that the 5-HT-induced current reversed polarity at a potential close to the equilibrium potential of K(+). Ba(2+) (100 microM-1 mM) partially depressed the outward current produced by 5-HT. These results suggest that 5-HT induces multiple K(+) currents via 5-HT(1A) receptors in DLSN neurons.     

D2-like dopamine receptors modulate SKCa channel function in subthalamic nucleus neurons through inhibition of Cav2.2 channels

The activity patterns of subthalamic nucleus (STN) neurons are intimately related to motor function/dysfunction and modulated directly by dopaminergic neurons that degenerate in Parkinson's disease (PD). To understand how dopamine and dopamine depletion influence the activity of the STN, the functions/signaling pathways/substrates of D2-like dopamine receptors were studied using patch-clamp recording. In rat brain slices, D2-like dopamine receptor activation depolarized STN neurons, increased the frequency/irregularity of their autonomous activity, and linearized/enhanced their firing in response to current injection. Activation of D2-like receptors in acutely isolated neurons reduced transient outward currents evoked by suprathreshold voltage steps. Modulation was inhibited by a D2-like receptor antagonist and occluded by voltage-dependent Ca2+ (Cav) channel or small-conductance Ca2+-dependent K+ (SKCa) channel blockers or Ca2+-free media. Because Cav channels are targets of G(i/o)-linked receptors, actions on step- and action potential waveform-evoked Cav channel currents were studied. D2-like receptor activation reduced the conductance of Cav2.2 but not Cav1 channels. Modulation was mediated, in part, by direct binding of Gbetagamma subunits because it was attenuated by brief depolarization. D2 and/or D3 dopamine receptors may mediate modulation because a D4-selective agonist was ineffective and mRNA encoding D2 and D3 but not D4 dopamine receptors was detectable. Brain slice recordings confirmed that SKCa channel-mediated action potential afterhyperpolarization was attenuated by D2-like dopamine receptor activation. Together, these data suggest that D2-like dopamine receptors potently modulate the negative feedback control of firing that is mediated by the functional coupling of Cav2.2 and SKCa channels in STN neurons.     

NK-1, but not NK-2, tachykinin receptors mediate plasma extravasation induced by antidromic C-fiber stimulation in rat hindpaw: demonstrated with the NK-1 antagonist CP-96,345 and the NK-2 antagonist Men 10207.

The effects of intradermal injection of CP-96,345 and Men 10207, selective antagonists for NK-1 and NK-2 tachykinin receptors, respectively, on the extravasation of plasma protein induced by antidromic stimulation of unmyelinated sensory fibers in the sciatic nerve was studied in rat hindpaw. Activation of unmyelinated fibers by antidromic sciatic nerve stimulation (1 Hz, 5 min) consistently evoked a localized plasma extravasation of Evans blue on the skin area of the hindpaw innervated by the sciatic nerve, which was not inhibited by intradermal injection of saline or Men 10207 (9 and 35 nmol). In contrast, CP-96,345 (3 and 9 nmol, but not 1 nmol), injected intradermally 15 min prior to nerve stimulation dose-dependently inhibited this response. Plasma extravasation induced by intravenously injected substance P was also inhibited by CP-96,345. Since CP-96,345 is a highly selective antagonist for NK-1 tachykinin receptors, it is suggested that the plasma extravasation induced by antidromic C-fiber stimulation and by systemically applied tachykinins is mediated by NK-1 tachykinin receptors.     

Ultrastructural evidence that substance P neurons form synapses with noradrenergic neurons in the A7 catecholamine cell group that modulate nociception.

Potent antinociception can be produced by electrical stimulation of spinally projecting noradrenergic neurons in the A7 catecholamine cell group and this effect is blocked by intrathecal injection of alpha2-adrenoceptor antagonists. Microinjection of substance P near A7 neurons also produces antinociception that is blocked by intrathecal injection of alpha2-adrenoceptor antagonists. These observations suggest that substance P produces antinociception by activating noradrenergic A7 neurons. However, it is not known whether this effect of substance P is produced by a direct or an indirect action on A7 neurons. Although light microscopic studies have demonstrated the existence of both substance P-containing axon terminals and neurokinin-1 receptors in the region of the A7 cell group, it is not known whether substance P terminals form synapses with noradrenergic A7 neurons. These experiments used double-labeling immunocytochemical methods and electron microscopic analysis to determine whether substance P-containing axons form synapses with noradrenergic neurons in the A7 cell group. Pre-embedding immunocytochemistry, combined with light and electron microscopic analysis, was used to provide ultrastructural evidence for synaptic connections between substance P-immunoreactive terminals labeled with immunoperoxidase and tyrosine hydroxylase-immunoreactive A7 neurons labeled with silver-enhanced immunogold. Tyrosine hydroxylase labeling was found in perikarya and dendrites in the A7 region, and substance P labeling was found in axons and synaptic terminals. Substance P-labeled terminals formed asymmetric synapses with tyrosine hydroxylase-labeled dendrites, but only a few of these were present on tyrosine hydroxylase-labeled somata. Substance P-labeled terminals also formed asymmetric synapses with unlabeled dendrites, and many unlabeled terminals formed both symmetric and asymmetric synapses with tyrosine hydroxylase-labeled dendrites. These results demonstrate that substance P neurons form a significant number of synapses with the dendrites of noradrenergic A7 neurons and support the conclusion that microinjection of substance P in the A7 cell group produces antinociception by direct activation of spinally projecting noradrenergic neurons.     

Bidirectional modulation of nociception by GABA neurons in the dorsolateral pontine tegmentum that tonically inhibit spinally projecting noradrenergic A7 neurons

The A7 catecholamine cell group in the dorsolateral pontine tegmentum constitutes an important part of the descending pathways that modulate nociception. Evidence from immunocytochemical studies demonstrate that noradrenergic A7 neurons are densely innervated by GABA terminals arising from GABA neurons that are located in the dorsolateral pontine tegmentum medial to the A7 cell group. GABA(A) receptors are also located on the somata and dendrites of noradrenergic A7 neurons. These findings suggest that noradrenergic neurons in the A7 cell group may be under tonic inhibitory control by GABA neurons. To test this hypothesis, the GABA(A) antagonist bicuculline methiodide in doses of 0.2 or 1.0nmol was microinjected into sites located dorsal to the A7 cell group and the resulting effects on tail flick and nociceptive foot withdrawal responses were measured. Both doses of bicuculline produced significant increases in tail flick latencies and small, but significant, increases in foot withdrawal latencies. Intrathecal injection of the alpha(2)-adrenoceptor antagonist yohimbine, in a dose of 76.7nmol (30microg), attenuated the antinociceptive effect of bicuculline on both the tail and the feet. In contrast, the alpha(1)-adrenoceptor antagonist WB4101, in a nearly equimolar dose of 78.6nmol (30microg), increased the antinociceptive effect of bicuculline on both the tail and the feet. Intrathecal injection of the antagonists alone did not consistently alter nociceptive responses of either the feet or the tail.These findings suggest that noradrenergic neurons in the A7 cell group are tonically inhibited by local GABA neurons. Furthermore, these findings suggest that inhibition of GABA(A) receptors located on spinally-projecting A7 noradrenergic neurons disinhibits, or activates, two populations of A7 neurons that have opposing effects on nociception. One of these populations facilitates nociception by an action mediated by alpha(1)-adrenoceptors in the spinal cord dorsal horn and the other population inhibits nociception by an action mediated by alpha(2)-adrenoceptors.