17beta-estradiol inhibits excitatory amino acid-induced activity of neurons of the nucleus tractus solitarius

The effects of 17beta-estradiol (17betaE2) on spontaneous and excitatory amino acid (EAA) induced nucleus tractus solitarius (NTS) neuronal activity were investigated by electrophysiological and immunohistochemical experiments in ovariectomized female Sprague-Dawley rats. Out of 62 NTS neurons tested, 42 were inhibited (68%) following iontophoretic application of 17betaE2 in a current-dependent manner. The averaged firing rate decreased from 3.06+/-0.40 to 0.78+/-0.17 Hz. The inhibitory responses were rapid in onset (within 1 min) and variable in duration (2-4 min). The inhibitory effects of 17betaE2 were blocked by simultaneously applied 17betaE2 antagonist ICI182,780, but not by GABA antagonist, bicuculline and phaclofen. L-Glutamate, AMPA or NMDA enhanced the activity of 71, 73 or 69% of NTS cells tested, respectively. The excitatory effects of EAA were significantly inhibited in the presence of 17betaE2. Fluorescent immunohistochemistry revealed that all subnuclei of the NTS contained high levels of estrogen receptors (ERs) immunoreactivity. These results suggest that 17betaE2 inhibits spontaneous and EAA-induced NTS neuronal activity through 17betaE2 activation of ERs.     

Dissociation of antinociceptive and motor effects of supraspinal opioid agonists in the rat

This study compared the antinociceptive and motor effects produced by intracerebroventricular administration of selective mu- (DAMGO) and delta- (DPDPE) opioid receptor agonists in the rat. Changes in nociceptive thresholds were measured using the Randall-Selitto paw-withdrawal test and changes in motor coordination were evaluated using the rotarod treadmill test. Both DAMGO and DPDPE produced statistically significant, dose-dependent increases in mechanical nociceptive thresholds compared to vehicle controls. However, in the motor coordination studies, neither opioid agonist produced statistically significant changes in rotarod performance scores. The dissociation of antinociceptive and motor effects at this supraspinal site differs from the strong association between antinociceptive and motor effects produced by intrathecal administration of the same opioid agonists.     

Tonic stimulation of GABAB receptors in the nucleus tractus solitarius modulates the baroreceptor reflex

Previous studies have indicated that tonic stimulation of GABA_B receptors in the nucleus tractus solitarius (NTS) contributes to the regulation of arterial blood pressure (AP). The present studies examined the hypotheses that (1) tonic stimulation of GABA_B receptors in the NTS provides a tonic attenuation of the baroreceptor reflex and (2) enhanced stimulation of these GABA_B receptors markedly attenuates the baroreceptor reflex resulting in an increase in AP. In chloralose-anesthetized rats electrical stimulation of the aortic depressor nerve elicited frequency-dependent decreases in AP and heart rate (HR). These responses were markedly attenuated, but not eliminated, by injection of the GABA_B receptor agonist baclofen into the ipsilateral NTS. In contrast, the GABA_A receptor agonist muscimol completely inhibited aortic depressor nerve-evoked responses. Blockade of GABA_B receptors in the NTS by local injection of CGP-35348 elicited a dose-dependent decrease in AP, and a dose-dependent blockade of the pressor response elicited by injection of baclofen into the NTS. These results support the hypothesis that GABA acts tonically on GABA_B receptors in the NTS to attenuate the baroreceptor reflex, thereby contributing to the regulation of AP.     

Microinjection of orexin into the rat nucleus tractus solitarius causes increases in blood pressure

Orexin A (OX-A) and orexin B (OX-B), also known as hypocretin-1 and hypocretin-2, have been suggested to play a role cardiovascular control. The nucleus tractus solitarius (NTS), located in the dorsal medulla plays an essential role in neural control of the cardiovascular system. Orexin-immunoreactive axons have been demonstrated within this nucleus suggesting that NTS may be a site through which OX acts to influence cardiovascular control. We report here that microinjection of OX-A into the NTS of urethane anesthetized rats causes increases in blood pressure (10⁻⁹ M, mean AUC=607.1±65.65 mmHg s, n=5) and heart rate (10⁻⁹ M, mean AUC=16.15±3.3 beats, n=5) which returns to baseline within 90 s. We show that these effects are dose related and site specific. Microinjection of OX-B into NTS elicited similar increases in BP (mean AUC=680.8±128.5 mmHg s, n=4) to that of OX-A suggesting specific actions at the OX₂R receptor. These observations support the conclusion that orexins act as chemical messengers in the NTS likely influencing the excitability of cardiovascular neurons in this region and thus regulating global cardiovascular function.     

Heterogeneous distribution and upregulation of μ, δ and κ opioid receptors in the amygdala

Levels of μ, δ and κ opioid receptors in 4 subnuclei of the rat amygdala were determined by quantitative autoradiography following chronic treatment with naloxone or saline. A different distribution of each receptor subtype was observed, with μ binding greatest in the lateral nucleus (La), δ greatest in the basolateral (B1), and κ greatest in the medial (Me). Levels of all 3 receptors were very low in the central nucleus. Receptor upregulation following chronic naloxone treatment was also anatomically heterogeneous. Increases in μ receptors were statistically significant in the Me, Bl and La, while increases in δ and κ receptors were significant only in the Bl.     

Ultrastructure of glutamate and GABA immunoreactive axon terminals of the rat nucleus tractus solitarius, with a note on infralimbic cortex afferents

The principal fast neurotransmitters in the CNS are glutamate and GABA. Our aim was to provide a baseline account on the ultrastructure of the axon terminals immunoreactive to glutamate or GABA present in the nucleus tractus solitarius (NTS) of the rat. In addition, we wanted to complete our study of cortico-solitary afferents at the electron microscopic level, by analyzing the inputs from the infralimbic cortex. Using post-embedding immunogold, we found that nearly 61% of the axon terminals were glutamatergic, and 36% were GABAergic in the rat visceral NTS. In general, axons making asymmetric synaptic contacts were enriched in glutamate, compared to axons involved in symmetric synapses. In contrast, the vast majority of the GABAergic axon terminals made symmetric synaptic contacts. We could discern five types of glutamatergic and two types of GABAergic axon terminals that differed in their fine structure. Afferents from the infralimbic cortex were small, with clear synaptic vesicles and no dense core vesicles; they made asymmetric contacts with fine dendrites, and were glutamatergic. We conclude that most axon terminals in the NTS use glutamate or GABA as fast transmitters, in addition to being a heterogeneous population of morphological types.     

Opioid receptor subtypes in the rat spinal cord: electrophysiological studies with μ- and δ-opioid receptor agonists in the control of nociception

We have compared the ability of selective mu- and delta-opiate agonists to modulate nociceptive transmission at the level of the rat dorsal horn using electrophysiological approaches. Single-unit extracellular recordings were made from neurones in the lumbar dorsal horn of the intact rat under halothane anaesthesia. Neurones could be activated by both A- and C-fibre electrical stimulation (and by natural innocuous and noxious stimuli). Agonists were applied directly onto the cord in a volume of 50 microliters. The intrathecal administration of 3 agonists, Tyr-D-Ala-Gly-MePhe-Gly-ol (DAGO) (mu-selective) (2 X 10(-3)-10 nmol) Tyr-D-Thr-Gly-Phe-Leu-Thr (DTLET) (mu/delta) (7 X 10(-4)-70 nmol), and cyclic Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE) (delta) (2 X 10(-2)-100 nmol) produced dose-dependent inhibitions of C-fibre-evoked neuronal activity whilst A-fibre activity was relatively unchanged. DAGO produced near-maximal inhibitions which could be completely reversed by naloxone (1.5 nmol) whilst DPDPE causes less marked inhibitions which could only be partially reversed by naloxone (1.5-13.5 nmol). DTLET produced effects intermediate to those of DAGO and DPDPE. The results suggest that both mu- and delta-opioid receptors can modulate the transmission of nociceptive information in the rat spinal cord.     

Choline acetyltransferase activity in the nucleus tractus solitarius: Regulation by the afferent vagus nerve

The influence of nodose ganglionectomy or transection of the peripheral branches of the afferent vagus nerve on choline acetyltransferase (ChAT) activity in the nucleus tractus solitarius (NTS) was studied. ChAT activity was reduced in the microdissected caudal and intermediate portions of the NTS in vagotomized as well as ganglionectomized rats. However, only the ganglionectomy resulted in the degeneration of medullary nerve fibers. These results suggest that the changes in ChAT activity in the NTS are independent of neuronal degeneration and may be due to transynaptic modulation of ChAT activity by afferent vagal impulses. The presence of ChAT in the sensory nodose projection to the NTS, however, cannot be ruled out.     

Multiple opioid receptors mediate feeding elicited by mu and delta opioid receptor subtype agonists in the nucleus accumbens shell in rats

The nucleus accumbens, and particularly its shell region, is a critical site at which feeding responses can be elicited following direct administration of opiate drugs as well as micro-selective and delta-selective, but not kappa-selective opioid receptor subtype agonists. In contrast to observations of selective and receptor-specific opioid antagonist effects upon corresponding agonist-induced actions in analgesic studies, ventricular administration of opioid receptor subtype antagonists blocks feeding induced by multiple opioid receptor subtype agonists. The present study examined whether feeding responses elicited by either putative mu ([D-Ala(2), NMe-Phe(4), Gly-ol(5)]-enkephalin (DAMGO)), delta(1) ([D-Pen(2), D-Pen(5)]-enkephalin (DPDPE)) or delta(2) ([D-Ala(2), Glu(4)]-deltorphin (Deltorphin)) opioid receptor subtype agonists administered into the nucleus accumbens shell were altered by accumbens pretreatment with either selective mu (beta-funaltrexamine), mu(1) (naloxonazine), delta(1) ([D-Ala(2), Leu(5), Cys(6)]-enkephalin (DALCE)), delta(2) (naltrindole isothiocyanate) or kappa(1) (nor-binaltorphamine) opioid receptor subtype antagonists. Similar magnitudes and durations of feeding responses were elicited by bilateral accumbens administration of either DAMGO (2.5 microg), DPDPE (5 microg) or Deltorphin (5 microg). DAMGO-induced feeding in the nucleus accumbens shell was significantly reduced by accumbens pretreatment of mu, delta(1), delta(2) and kappa(1), but not mu(1) opioid receptor subtype antagonists. DPDPE-induced feeding in the accumbens was significantly reduced by accumbens pretreatment of mu, delta(1), delta(2) and kappa(1), but not mu(1) opioid receptor subtype antagonists. Deltorphin-induced feeding in the accumbens was largely unaffected by accumbens delta(2) antagonist pretreatment, and was significantly enhanced by accumbens mu or kappa(1) antagonist pretreatment. These data indicate different opioid pharmacological profiles for feeding induced by putative mu, delta(1) and delta(2) opioid agonists in the nucleus accumbens shell, as well as the participation of multiple opioid receptor subtypes in the elicitation and maintenance of feeding by these agonists in the nucleus accumbens shell.     

The types of neuron which contain protein kinase C gamma in rat spinal cord

Protein kinase C (PKC) is thought to have a role in sensitization of dorsal horn neurons in certain pain states, and a recent study has reported that mice which lack the γ isoform (PKCγ) show reduced neuropathic pain after peripheral nerve injury. Although PKCγ is present at high levels in the ventral part of lamina II we have limited information concerning the types of neuron in which it is located. In this study we have used immunocytochemistry to characterise the neurons which contain PKCγ. Immunoreactive neurons were concentrated in ventral lamina II, but were also present in lamina III. Some weakly-immunoreactive neurons were located in the dorsal part of lamina II and in lamina I. The great majority (92%) of cells with PKCγ were not GABA-immunoreactive, and these cells are likely to be excitatory interneurons. Dual-immunofluorescence labelling showed that PKCγ was not randomly distributed amongst non-GABAergic neurons, since it was present in 76% of cells with neurotensin and 45% of those with somatostatin, but only 5% of those with the μ-opioid receptor (MOR-1). Cells with the neurokinin 1 receptor are found in lamina I and lamina III, and PKCγ was present in 22% and 37% of these populations, respectively. These results suggest that excitatory interneurons in laminae II and III which lack the μ-opioid receptor may have a significant role in generating neuropathic pain.     

Evidence for opioid and non-opioid forms of stress-induced analgesia in the snail, Cepaea nemoralis

Exposure to either cold or warm stress increased the thermal nociceptive thresholds of the terrestrial snail, Cepaea nemoralis. The warm stress-induced 'analgesia' was blocked by the prototypic opiate antagonist, naloxone, and the delta-opiate antagonist, ICI 154,129, and was suppressed by a 24-h pretreatment with the irreversible opiate antagonist, beta-funaltrexamine (B-FNA). In contrast, cold stress-induced analgesia was unaffected by either naloxone, ICI 154,129 or B-FNA. These results indicate that this mollusc displays both opioid and non-opioid forms of stress-induced analgesia in a manner analogous to that reported for mammals. These findings suggest an early evolutionary development and phylogenetic continuity of opioid and non-opioid mediated stress responses to aversive environmental stimuli.     

Effects of enkephalin and morphine on rat globus pallidus neurons

This study was conducted in order to compare the effects of microiontophoretically-applied morphine and met-enkephalin (met-ENK) on spontaneous and/or glutamate-evoked activity of single globus pallidus (GP) neurons in locally anesthetized, paralyzed rats. The predominant effect of both morphine and met-ENK was a depression of pallidal neuronal activity. While very few GP neurons were excited by morphine (2/89), a small population of neurons was excited by met-ENK (16/89). Both the inhibitory and excitatory responses produced by morphine and met-ENK could be attenuated by the microiontophoretic application of naloxone. It was also found that morphine and met-ENK did not affect all GP neurons in a similar manner. When applied to the same neurons, morphine elicited depression in 11 of 16 GP neurons which were excited by the application of met-ENK. In contrast, neither of two GP neurons excited by morphine in this study displayed inhibition upon application of met-ENK. Thus the microiontophoresis of morphine and met-ENK to single GP neurons has demonstrated that these two substances can produce opposite effects when applied to the same neurons and suggests that two functionally distinct types of opiate receptor may exist within rat GP, one which mediates the inhibitory effects of morphine and met-ENK, possibly the classical mu (mu) receptor, and one that is preferentially selective to met-ENK and which mediates the excitatory effects of opiates within this region, possibly the classical delta (delta) receptor.     

Pharmacological and anatomical evidence of selective μ, δ, and χ opioid receptor binding in rat brain

While the distribution of opioid receptors can be differentiated in the rat central nervous system, their precise localization has remained controversial, due, in part, to the previous lack of selective ligands and insensitive assaying conditions. The present study analyzed this issue further by examining the receptor selectivity of [³H]DAGO (Tyr-d-Ala-Gly-MePhe-Gly-ol), [³H]DPDPE (2-d-penicillamine-5-d-penicillamine-enkephalin), [³H]DSLET (Tyr-d-Ser-Gly-Phe-Leu-Thr) and [³H](−)bremazocine, and their suitability in autoradiographically labelling selective subpopulations of opoiod receprtors in rat brain. The results from saturation, competitions, and autoradiographic experiments indicated that the three opioid receptor subtypes can be differentiated in the rat brain and that [³H]-DAGO and [³H]DPDPE selectively labelled μ and δ binding sites, respectively. In contrast, [³H]DSLET was found to be relatively non-selective, and labelled both μ and δ sites. [³H]Bremazocine was similarly non-selective in the absence of μ and δ ligands and labelled all three opioid receptor subtypes. However, in the presence of 100 nM DAGO and DPDPE, concentrations sufficient to saturate the μ and δ sites, [³H]bremazocine did label χ sites selectively. The affinity [³H]bremazocine binding sites showed a unique distribution with relatively dense χ labelling in the hypothalamus and median eminence, areas with extremely low μ and δ binding. These results point to the selectivity, under appropriate conditions, of [³H]DAGO, [³H]DPDPE and [³H]bremazocine and provide evidence for the differential distribution of μ, δ, and χ opioid receptors in rat brain.     

Antinociception produced by receptor selective opioids: modulation of spinal antinociceptive effects by supraspinal opioids

The effect of intracerebroventricular administration of low-antinociceptive doses of selective μ-(DAMGO) or δ-(DPDPE) opioid agonists on the dose-dependent antinociceptive effects produced by intrathecal administration of sequentially increasing doses of selective μ-, δ-, or κ- (U50, 488H) opioid agonists was evaluated, in the rat, using the Randall-Selitto paw-withdrawal test. When DPDPE or U50,488H was administered intrathecally, the low doses of both intracerebroventricular DAMGO and intracerebroventricular DPDPE markedly enhanced the antinociceptive effects of both intrathecal opiods. In contrast, when DAMGO was administered intrathecally, both intracerebroventricular DAMGO and intracerebroventricular DPDPE, administered in low doses, markedly antagonized the antinociceptive effects of the intrathecal opioid. In addition, the intracerebroventricular administration of low-antinociceptive dose of a second μ-opioid agonist, morphiceptin, antagonized the antinociceptive effects of intrathecal morphiceptin. The antagonism of the antinociceptive effects observed with spinal administration of DAMGO is dose-dependent, with the effect observed only at low doses. Furthermore, the antagonism cannot be explained by a reduction in motor deficits produced by intrathecal administration of DAMGO, because there were no differences in motor deficits, measured with an accelerating Rotarod treadmill, between intrathecal DAMGO administered as a single agent or as part of a combination regimen. The differences in antinociceptive effects obtained with the various supraspinal and spinal combinations are discussed in terms of the interactions that may occur between brainstem and spinal opioid receptor sites.     

Effects of neonatal cocaine treatment and gender on opioid agonist-stimulated [S]GTPγS binding in the striatum and nucleus accumbens

Prenatal cocaine exposure increases μ-opioid receptor binding in dopaminergic terminal areas and enhances behavioral responsiveness to μ-opioid agonists. We investigated the influence of early postnatal cocaine treatment on in vitro μ- and δ-opioid receptor activation in male and female weanling rats. Pups received subcutaneous injections of either 20 mg/kg cocaine HCl or saline once daily on postnatal days 1 through 5. On postnatal day 25, animals were decapitated and their brains were removed and frozen for later sectioning. Opioid receptor activation was assessed in the striatum and the shell of the nucleus accumbens by autoradiographic analysis of agonist-stimulated [³⁵S]GTPγS binding. Brain sections were incubated in the presence of [³⁵S]GTPγS, GDP, and either the μ-opioid agonist [ -Ala²-N-MePhe⁴-Gly⁵-ol]enkephalin (DAMGO) or the δ-opioid agonist -Pen²-D-Pen⁵-enkephalin (DPDPE). Baseline binding was assessed in the absence of agonist, and nonspecific binding was determined by the addition of unlabeled GTPγS. Film images were quantified using brain mash-calibrated [¹⁴C] standards. Neonatal cocaine treatment had no effect on either baseline or agonist-stimulated [³⁵S]GTPγS binding. However, males exhibited significantly greater activation than females of δ-opioid receptors in both striatum and accumbens shell, regardless of neonatal treatment. These findings indicate a gender difference in δ-opioid receptor function that could mediate behavioral differences in response to opioid agonists.     

Inhibitory effect of 17β-estradiol in the parabrachial nucleus is mediated by GABA

In the present investigation, electrophysiological recordings of thalamic relay neurons were used to investigate the role of estrogen as a modulator of visceral afferent information through the PBN to forebrain structures. Experiments were done in anaesthetized (sodium thiobutabarbitol; 100 mg/kg) male and ovariectomized female rats supplemented for 7 days prior with either 17β-estradiol (OVX-E₂) or saline (OVX-S). A portion of the right cervical vagus was isolated for the electrical activation (0.8 Hz, 2 ms duration) of visceral afferents. The evoked single and multi-unit activity was recorded via a recording electrode in the ventrobasal thalamus. Exogenous microinjection of 17β-estradiol (0.1, 0.25 and 0.5 μM; 200 nl) into the parabrachial nucleus (PBN) produced a significant, dose-dependent attenuation in the magnitude of visceral afferent activation-evoked responses of neurons recorded in the thalamus in both male and OVX-E₂ groups. No effect on evoked thalamic activity was observed following injection of estrogen into the PBN of OVX-S animals. Co-injection of estrogen with the GABA_A receptor antagonist, bicuculine (0.1 μM; 200 nl) but not phaclofen (GABA_B; 0.1, 0.5 or 1 μM; 200 nl) resulted in an increase in the evoked thalamic response in males (55±11%) and OVX-E₂ female (68±15%) rats. These studies suggest that estrogen inhibits neurotransmission in the PBN via an interaction with the GABA_A receptor to modulate the flow of visceral information to the thalamus.     

Lack of change in high affinity glutamate uptake in nucleus tractus solitarius following removal of the nodose ganglion

High affinity glutamate uptake in the intermediate portion of the nucleus tractus solitarius (NTS) was not altered by prior removal of one nodose ganglion or by prior midline transection at the level of the NTS. In contrast, glutamate uptake in a tissue punch of the septal nucleus was significantly reduced by prior transection of the fornix. Studies also compared septal glutamate uptake in crude homogenate, supernatant resulting from centrifugation at 3000 X g, and the pellet resulting from centrifugation at 17,000 X g. The results show quantitative differences between measures of glutamate uptake in these different fractions, but qualitatively the results were similar. (These results, do, however, suggest that glutamate uptake might be most appropriately measured in the pellet resulting from centrifugation at 17,000 X g.) The observation that removal of the nodose ganglion did not effect glutamate uptake in the NTS is therefore not due to the method by which glutamate uptake was assessed. These studies fail to replicate the previous observation that glutamate uptake in the NTS decreases following removal of the nodose ganglion, and thus question the hypothesis that glutamate is the neurotransmitter of vagal afferent fibers.     

Taste responses in the nucleus tractus solitarius of the chronic decerebrate rat

The ingestive behavior of decerebrate rats has been studied for some time, yet little is known of its neural substrates. While taste fibers in rats proceed from hindbrain to thalamus and ventral forebrain, these regions return centrifugal fibers to the hindbrain by which lower-order taste activity may be influenced. We examined the functional characteristics of taste neurons in the nucleus tractus solitarii (NTS) of chronic decerebrate rats in which this reciprocal communication was disrupted and compared them with those of intact controls. Nine Wistar rats were decerebrated at the supracollicular level. After a minimum of one week recovery, they were immobilized with Flaxedil, anesthetized locally and prepared for recording. The responses of 50 taste cells were isolated bilaterally from the NTS of these animals, while the activity of 50 additional neurons was recorded from 12 intact rats under the same conditions. Taste stimuli included 7 Na-Li salts, 3 sugars, HCl and citric acids, quinine HCl and NaSaccharin. Mean spontaneous activity in decerebrates was 6.5 spikes/s, 36.0% lower than the level in intact animals. Mean evoked activity was reduced by 32.6%. Analyses of the effects of stimulus quality, intensity and time course of the responses all indicated that the decrease in activity was attributable to the inability of taste cells in decerebrate rats to respond to demands for high discharge rates. This deficit could be responsible for the failure of these animals to develop conditioned taste aversions. Neurons from decerebrate preparations did, however, retain the broad sensitivity across stimuli that characterized taste cells in intact preparations. It was also typical that most neuron response profiles from decerebrates could be grouped into 3 loose clusters with peak sensitivities to acid-salt, salt or sugar. An analysis of similarities among stimulus activity profiles indicated that Na-Li salts, sugars and an acid-quinine complex represented 3 groups of stimulus quality; in intact animals, the primary distinction was between sweet and non-sweet stimuli. Moreover, the response to sodium saccharin lost its bitter component in decerebrates. These findings were in general agreement with those derived from acute decerebrate rats.     

Characterization of stimulation-produced analgesia from the nucleus tractus solitarius in the rat

Electrical stimulation of the commissural region of the nucleus tractus solitarius (NTS) inhibits the tail-flick reflex evoked by noxious heat. This antinociception can be measured in the awake or pentobarbital anesthetized rat at current intensities that do not induce overt behavioral side effects. Glutamate microinjections into the NTS, but not immediately surrounding the NTS, also inhibit the tail-flick reflex, demonstrating that activation of NTS cell bodies, and not fibers of passage, mediates antinociception from this region. In contrast, morphine microinjections into the NTS have no effect on the tail-flick reflex in anesthetized rats. These findings provide further evidence that the NTS is involved in the modulation of nociception.