Angiotensin II inhibits GABAergic synaptic transmission in dorsolateral periaqueductal gray neurons

The purpose of this study was to determine the role of angiotensin II (Ang II) in modulating inhibitory and excitatory synaptic inputs to the dorsolateral periaqueductal gray (dl-PAG). The whole cell voltage-clamp recording was performed to examine inhibitory and excitatory postsynaptic currents (IPSCs and EPSCs) of the dl-PAG neurons. Ang II, at the concentration of 2 μM, decreased the frequency of miniature IPSCs from 0.83 ± 0.02 to 0.45 ± 0.03 Hz (P < 0.05) in 10 tested neurons. This did not significantly affect the amplitude and decay time constant. The effect of Ang II on miniature IPSCs was blocked by the prior application of Ang II AT1 receptor antagonist losartan, but not by AT2 receptor antagonist PD123319. Additionally, Ang II decreased the amplitude of evoked IPSCs from 148 ± 15 to 89 ± 7 pA (P < 0.05), and increased the paired-pulse ratio from 96 ± 5% to 125 ± 7% (P < 0.05) in eight tested neurons. In contrast, Ang II had no distinct effects on the EPSCs. Our data suggest that Ang II inhibits GABAergic synaptic inputs to the dl-PAG through activation of presynaptic AT1 receptors.     

Developmental change of GABAergic postsynaptic current in rat periaqueductal gray

The present study was designed to examine developmental changes of GABAergic spontaneous miniature inhibitory postsynaptic currents (mIPSCs) in periaqueductal gray (PAG) neurons mechanically isolated from young (12- to 18-day) and adult (8- to 12-week) rats. While the frequency of mIPSCs was similar, the current amplitude in adult rats was significantly smaller than in young rats. In the study of mIPSC kinetics, all kinetic parameters except for the fast decay time in adult rats were smaller or shorter than in the case of young rats. The present study demonstrates that a decrease in the amplitude of GABAergic mIPSC during development may result from a decrease in the GABA contents of synaptic vesicles and from changes in the kinetics of postsynaptic GABA-activated Cl- channels.     

Behavioral evidence linking opioid-sensitive GABAergic neurons in the ventrolateral periaqueductal gray to morphine tolerance

Tolerance develops to the antinociceptive effects of morphine with repeated microinjections into the ventrolateral periaqueductal gray (PAG). This tolerance could be caused by adaptations within the PAG or anywhere along the descending pathway (rostral ventromedial medulla to spinal cord). If tolerance is caused by a change along the descending pathway, then tolerance should develop to direct activation of PAG output neurons. However, if tolerance is caused by a change to neurons within the PAG, then tolerance will not occur with repeated direct activation of PAG output neurons. This hypothesis was tested by assessing antinociception following repeated microinjections of the GABA antagonist bicuculline and the excitatory amino acid kainate into the ventrolateral PAG. Microinjection of bicuculline and kainate produces antinociception by disinhibition and direct excitation of ventrolateral PAG output neurons, respectively. Repeated administration of these drugs into the ventrolateral PAG produced antinociception with no evidence of tolerance. That is, the hot-plate latency and responsiveness to intraplantar formalin administration was comparable whether rats received the drug for the first or fifth time. Moreover, microinjection of bicuculline or kainate produced comparable antinociception in rats pretreated with these drugs and saline-treated control rats.These data demonstrate that repeated activation of ventrolateral PAG output neurons is not sufficient to produce tolerance. Thus, tolerance must be caused by a change in neurons preceding output neurons in this circuit, presumably opioid-sensitive GABAergic neurons.     

c-Fos expression in the periaqueductal gray is induced by electroacupuncture in the rat, with possible reference to GABAergic neurons

Electroacupuncture (EA) delivered to the acupoint (AP) called Zusanli (ST36) was administered on the bilateral hindlimb. This experiment resulted in strong expression of c-Fos immunoreactivity in the ventrolateral to lateral subdivision throughout the periaqueductal gray (PAG) compared to the non-AP and sham cases. On the other hand, it was of particular interest in the experiment of the AP that strong expression of gamma aminobutylic acid (GABA) frequently showed similar pattern of distribution to that of c-Fos in the PAG. This overlapped pattern of distribution, demonstrated in the present study, suggests that the PAG neurons activated by EA at the AP might play an important role in the descending pain control system involving the GABA since the PAG has special reference to the dorsal horn of the spinal cord and function of pain control.     

Glutamate decarboxylase-immunoreactive neurons and terminals in the periaqueductal gray of the rat

The periaqueductal gray of 5 rats was processed for immunocytochemistry using an antiserum to glutamate decarboxylase. In both colchicine-pretreated (4 rats) and untreated (1 rat) animals, glutamate decarboxylase-positive cell bodies were present in all periaqueductal gray subdivisions, especially in the dorsal and ventrolateral subdivision. The perikaryal cross-sectional area of labelled neurons was smaller than that of periaqueductal gray projecting neurons retrogradely labelled with horseradish peroxidase in separate experiments. The morphology of glutamate decarboxylase-containing neurons resembled that of small polygonal, triangular and fusiform cells described in previous Golgi studies. Glutamate decarboxylase immunoreactivity was also observed in a large number of terminal-like structures, most of which were distributed close to the somata and dendrites of both glutamate decarboxylate-positive and -negative neurons. At all rostrocaudal levels the highest concentration of these elements was observed around the aqueduct. These results suggest that two sub-populations of neurons are present in the periaqueductal gray of rats, one consisting of small-sized glutamate decarboxylase-positive neurons (intrinsic neurons) and the other of large-sized glutamate decarboxylase-negative neurons (projecting neurons). Intrinsic circuits could be present between glutamate decarboxylase-positive and -negative neurons and between glutamate decarboxylase-positive neurons.     

Neurotensin inhibition of GABAergic transmission via mGluR-induced endocannabinoid signalling in rat periaqueductal grey

Neurotensin modulates pain via its actions within descending analgesic pathways which include brain regions such as the midbrain periaqueductal grey (PAG). The aim of this study was to examine the cellular actions of neurotensin on PAG neurons. Whole cell patch clamp recordings were made from rat midbrain PAG slices in vitro to examine the postsynaptic effects of neurotensin and its effects on GABA_A mediated inhibitory postsynaptic currents (IPSCs). Neurotensin (100–300 n m ) produced an inward current in subpopulations of opioid sensitive and insensitive PAG neurons which did not reverse over membrane potentials between –50 and –130 mV. The neurotensin induced current was abolished by the NTS1 and NTS1/2 antagonists SR48692 (300 n m ) and SR142948A (300 n m ). Neurotensin also produced a reduction in the amplitude of evoked IPSCs, but had no effect on the rate and amplitude of TTX-resistant miniature IPSCs. The neurotensin induced inhibition of evoked IPSCs was reduced by the mGluR5 antagonist MPEP (5μ m ) and abolished by the cannabinoid CB₁ receptor antagonist AM251 (3μ m ). These results suggest that neurotensin produces direct neuronal depolarisation via NTS1 receptors and inhibits GABAergic synaptic transmission within the PAG. The inhibition of synaptic transmission is mediated by neuronal excitation and action potential dependent release of glutamate, leading to mGluR5 mediated production of endocannabinoids which activate presynaptic CB₁ receptors. Thus, neurotensin has cellular actions within the PAG which are consistent with both algesic and analgesic activity, some of which are mediated via the endocannabinoid system.     

Involvement of protein kinase c in responses of rat dorsal horn neurons to mechanical stimuli and periaqueductal gray descending inhibition

 The effects of a protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), on the activity and periaqueductal gray (PAG)-induced inhibition of rat dorsal horn neurons of the lumbar spinal cord were tested. A microdialysis fiber was placed through the dorsal horn for the purpose of local application of pharmacological agents. Extracellular single-unit recordings from dorsal horn neurons were made near the microdialysis fiber. TPA was tested on nociceptive dorsal horn cells. There was a significant increase in the background activity and responses to ”brush”, with no changes in responses to pressure and pinch stimuli. TPA also significantly blocked the PAG-induced inhibition of responses to brush, press, and pinch. These effects were eliminated by coadministration of the PKC inhibitor NPC-15437. The solvent, which contained dimethyl sulfoxide, was also tested for its effect on the responses to peripheral mechanical stimuli and PAG-induced inhibition of the dorsal horn neurons. There were no significant changes. This experiment suggests that activation of the PKC second messenger system might increase the activity of dorsal horn neurons and their responses to peripheral stimuli; in addition, the phorbol ester attenuated the PAG-induced descending inhibition of the dorsal horn neuron activity. Received: 15 May 1996 / Accepted: 14 November 1996     

Mu-opioid agonist-induced activation of G-protein-coupled inwardly rectifying potassium current in rat periaqueductal gray neurons

The characteristics of the inwardly rectifying K+ current activated by a mu-type opioid agonist, D-Ala2,N-MePhe4,Gly5-ol-enkephalin (DAMGO), were examined in the acutely dissociated rat periaqueductal gray neurons using the nystatin-perforated and the conventional whole-cell recording modes under voltage-clamp conditions. DAMGO activated inward currents in a concentration- and voltage-dependent manner. The DAMGO-induced current was an inwardly rectifying K+ current (I(DAMGO)) which was sensitive to K+ channel blockers, quinine and Ba2+ but insensitive to Cs+ and tetraethylammonium. In the conventional whole-cell clamp mode, guanosine 5'-O-(2-thiodiphosphate) trilithium salt (GDPbetas, 0.4 mM) inhibited the amplitude of I(DAMGO) to 28% of that of the initial current. After the intracellular perfusion with guanosine 5'-O-(3-thiotriphosphate) tetralithium salt (GTPgammas, 0.4 mM) for 1 min, the first application of DAMGO irreversibly activated I(DAMGO). By the extracellular application of N-ethylmaleimide at a concentration of 50 microM for 2 min, I(DAMGO) was completely abolished. When a conventional whole-cell patch was made with a patch-pipette containing 1 microg/ml of pertussis toxin together with 1 mM of beta-nicotinamide adenine dinucleotide, I(DAMGO) gradually declined to about 41% of its initial amplitude. The extracellular application of second messenger modulators including protein kinase inhibitor (staurosporin), protein kinase A activators (forskolin, 3-isobutyl-l-methyl-xanthine and dibutyryladenosine 3'5'-cyclic monophosphate) and protein kinase C activators (phorbol-12-myristate-13-acetate and 1-oleoyl-2-acetyl-sn-glycerol) had no effect on I(DAMGO). These results suggest that (i) DAMGO-activated inwardly rectifying K+ current is mediated by pertussis toxin-sensitive guanine nucleotide binding proteins (G-proteins); (ii) the types of G protein involved in I(DAMGO) are Gi and/or Go; and (iii) the G-proteins exert their roles in I(DAMGO) without any mediation of the second messenger systems.     

Differential expression of NMDA receptors in serotonergic and/or GABAergic neurons in the midbrain periaqueductal gray of the mouse

N-methyl-d-aspartate (NMDA) receptors expressed in the midbrain periaqueductal gray (PAG) exert various physiological functions. The PAG contains various neurotransmitter phenotypes, which include GABAergic neurons and serotonergic neurons. In the present experiments, we made tight-seal whole-cell recordings from GABAergic and/or serotonergic neurons in mouse PAG slices and analyzed NMDA and non-NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) evoked by electrical stimulation. The NMDA/non-NMDA ratio of EPSC amplitude was high and the decay time course of NMDA-EPSC was slow in non-serotonergic/GABAergic neurons. In contrast, serotonergic neurons exhibited a low NMDA/non-NMDA ratio and a fast decay time course of NMDA-EPSC. Peripheral nerve ligation-induced chronic pain was associated with an increased NMDA/non-NMDA ratio in serotonergic neurons. Additionally, single-cell real-time RT-PCR analysis showed that peripheral nerve ligation up-regulated NR2B subunit expression in non-serotonergic/non-GABAergic neurons. Such changes in NMDA receptor expression in the PAG result in an alteration of the descending modulation of nociception, which might be an underlying mechanism for peripheral nerve injury-evoked persistent pain. Finally, the expression of NMDA receptors seems differentially regulated among neurons of different neurotransmitter phenotypes in the PAG.     

Zinc modulates GABAergic neurotransmission in rat globus pallidus

The globus pallidus plays a critical role in the regulation of movement, and abnormal activity of its neurons is associated with some basal ganglia motor diseases. A relatively high level of zinc has been reported in the globus pallidus, which is increased significantly after 6-OHDA treatments. To elucidate the action of zinc on GABAergic neurotransmission in the globus pallidus, whole-cell patch-clamp recordings were made from rat globus pallidus neurons. Superfusion of zinc significantly reduced both spontaneous and miniature inhibitory postsynaptic currents. The inhibition was selective to the amplitude with no change in the frequency, decay time and rise time. Furthermore, the reduction of spontaneous inhibitory postsynaptic currents (34.1 ± 4.0%) was stronger than that of miniature inhibitory postsynaptic currents (19.7 ± 3.2%). These results suggest that spontaneous inhibitory postsynaptic currents generated mainly by axonal collaterals and miniature inhibitory postsynaptic currents generated mainly by striatopallidal inputs may be mediated by different GABA_A receptor combinations.     

alpha 2-Adrenoceptor-mediated presynaptic modulation of GABAergic transmission in mechanically dissociated rat ventrolateral preoptic neurons

The ventrolateral preoptic nucleus (VLPO) is a key nucleus involved in the homeostatic regulation of sleep-wakefulness. Little is known, however, about the cellular mechanisms underlying its role in sleep regulation and how the neurotransmitters, such as GABA and noradrenaline (NA), are involved. In the present study we investigated GABAergic transmission to acutely dissociated VLPO neurons using an enzyme-free, mechanical dissociation procedure in which functional terminals remained adherent and we investigated how this GABAergic transmission was modulated by NA. As previously reported in slices, NA hyperpolarized multipolar VLPO neurons and depolarized bipolar VLPO neurons. NA also inhibited the release of GABA onto multipolar VLPO neurons but had no effect on GABAergic transmission to bipolar neurons. The inhibition of release was mediated by presynaptic alpha(2) adrenoceptors coupled to N-ethylmaleimide (NEM)-sensitive G-proteins which appeared to act via inhibition of adenylate cyclase and subsequent decreases in protein kinase A activity. The inhibition of GABA release did not, however, involve an inhibition of external Ca(2+) influx. The results indicate that all VLPO neurons contain GABAergic inputs and that the different morphological subgroups of VLPO neurons are correlated not only to different postsynaptic responses to NA but also to different presynaptic NA responses. Furthermore our results demonstrate an additional mechanism by which NA can modulate the excitability of multipolar VLPO neurons which may have important implications for its role in regulating sleep/wakefulness.     

Vasopressin increases GABAergic inhibition of rat hypothalamic paraventricular nucleus neurons in vitro

This investigation used an in vitro hypothalamic brain slice preparation and whole cell and perforated-patch recording to examine the response of magnocellular neurons in hypothalamic paraventricular nucleus (PVN) to bath applications of vasopressin (VP; 100-500 nM). In 22/38 cells, responses were characterized by an increase in the frequency of bicuculline-sensitive inhibitory postsynaptic potentials or currents with no detectable influence on excitatory postsynaptic events. Perforated-patch recordings confirmed that VP did not have an effect on intrinsic membrane properties of magnocellular PVN neurons (n = 17). Analysis of intrinsic membrane properties obtained with perforated-patch recording (n = 23) demonstrated that all of nine VP-sensitive neurons showed a rebound depolarization after transient membrane hyperpolarization from rest. By contrast, 12/14 nonresponding neurons displayed a delayed return to resting membrane potentials. Recordings of reversed inhibitory postsynaptic currents with chloride-loaded electrodes showed that responses to VP persisted in media containing glutamate receptor antagonists but were abolished in the presence of tetrodotoxin. In addition, responses were mimicked by vasotocin [Phe(2), Orn(8)], a selective V(1a) receptor agonist, and blocked by [beta-Mercapto-beta, beta-cyclopentamethylenepropionyl(1),O-Me-Tyr(2), Arg(8)]-VP (Manning compound), a V(1a)/OT receptor antagonist. Neither [deamino-Cys(1),Val(4),D-Arg(8)]-VP, a selective V(2) receptor agonist, nor oxytocin were effective. Collectively, the results imply that VP acts at V(1a) receptors to excite GABAergic neurons that are presynaptic to a population of magnocellular PVN neurons the identity of which features a unique rebound depolarization. Endogenous sources of VP may be VP-synthesizing neurons in suprachiasmatic nucleus, known to project toward the perinuclear regions of PVN, and/or the magnocellular neurons within PVN.     

Intranuclear bodies in neurons of the periaqueductal gray matter in the cat

The nucleoplasm of neurons in the nucleus lateralis of the periaqueductal gray matter in the cat contains fibrillar structures which have no limiting membranes. These intranuclear bodies are associated with neither the nucleolus nor the nuclear membrane and have two characteristic forms. The first, the rodlet, is a compact bundle of fibrils 2 to 8 nm in diameter. It is usually elongated in shape although it appears spherical when sectioned transversely. This rodlike structure appears to correspond to Roncoroni's rodlet or the accessory body of Cajal in light microscopy. The second and more commonly observed form is a long slender bundle of five rows of parallel fibrils. Although similar intranuclear structures have frequently been observed in the highly differentiated neurons of the sympathetic ganglia and the retina, this is the first report of their presence in the undifferentiated neurons of the isodendritic core of the brainstem.     

Opioid receptors in the midbrain periaqueductal gray regulate prediction errors during pavlovian fear conditioning

The authors used a within-subject blocking design to study the role of ventrolateral periaqueductal gray (v1PAG) opioid receptors in regulating prediction errors during Pavlovian fear conditioning. In Stage I, the authors trained rats to fear conditioned stimulus (CS) A by pairing it with shock. In Stage II, CSA and CSB were co-presented and followed with shock. Two novel stimuli, CSC and CSD, were also co-presented and followed with shock in Stage II. CSA blocked fear from accruing to CSB. Blocking was prevented by systemic pretreatment with naloxone. Blocking was also prevented in a dose-dependent and neuroanatomically specific fashion by vlPAG infusions of the micro-opioid receptor antagonist CTAP. These experiments show that v1PAG micro-opioid receptors contribute to Pavlovian fear learning by regulating predictive error.     

Opioid mediation of the antiaversive and hyperalgesic actions of bradykinin injected into the dorsal periaqueductal gray of the rat.

Reported evidence indicates that the dorsal region of the periaqueductal gray matter (PAG) is involved in the modulation of both pain and aversion, and that opioid mechanisms, among others, participate in their modulation. Since many central actions of bradykinin (BK) have been shown to be similar to those of morphine, the present was undertaken to measure the effects of microinjection of BK into the PAG on the thresholds of aversive electrical stimulation of the same brain area and of dental pulp electrical stimulation. Bradykinin, injected into the dorsal PAG, induced a dose-dependent increase in the aversive threshold, an effect similar to that reported by others for morphine. Also, as reported for morphine, the antiaversive effect of BK was antagonized by naloxone injected intraperitoneally. Whereas subcutaneously administered morphine induced marked analgesia, intra-PAG administration of BK caused a small but significant hyperalgesia. Similarly, morphine injected into the dorsal PAG tended to cause hyperalgesia instead of analgesia. Furthermore, the hyperalgesic effect of BK also appears to involve opioid mechanisms since it was blocked by naloxone. As in previously reported studies, intracerebroventricularly injected BK raised the pain threshold. These results indicate that BK mobilizes opioid mechanisms in the dorsal PAG that inhibit aversion but not pain.     

Hemorrhagic sympathoinhibition mediated through the periaqueductal gray in the rat

In response to hemorrhage in the anesthetized rat, an initial renal sympathoexcitation is followed by profound sympathoinhibition and hypotension with increasing blood loss. Microinjection of the gamma-aminobutyric acid(A) agonist muscimol to block neurotransmission through the sympathoinhibitory region of the ventrolateral periaqueductal gray matter (vlPAG) did not alter resting sympathetic nerve activity or blood pressure. However, the response to hemorrhage was converted to a maintained renal sympathoexcitation with a delayed and attenuated accompanying hypotension. These data indicate that neurons in the vlPAG mediate the sympathetic and cardiovascular responses to severe hemorrhage.     

Layer- and cell-type-specific tonic GABAergic inhibition of pyramidal neurons in the rat visual cortex

Tonic inhibition mediated by persistent activation of γ-aminobutyric acid_A (GABA_A) receptors by ambient GABA plays a crucial role in the regulation of network excitability and neuronal signal processing. Varying degrees in the strength of tonic inhibition were detected across different cell types throughout the brain. Since sensory information flows through cortical layers in a specific order, the characteristics of tonic inhibition in different cortical layers are of interest. Therefore, we examined the properties of tonic inhibition in pyramidal neurons (PyNs) throughout the rat visual cortex. Layer 2/3 PyNs and burst-spiking PyNs in layers 5 and 6 showed prominent tonic GABA_A currents. Tonic GABA_A currents in layer 4 star PyNs and regular-spiking PyNs in layers 5 and 6 were much weaker. The magnitude of tonic currents correlated well with the inhibition of spike generation. The amplitude of tonic GABA_A currents measured with bicuculline and gabazine, the two different GABA_A receptor blockers, did not differ. The differences in the expression levels of extrasynaptic GABA_A receptors might be the major contributor to the differences in tonic GABA_A currents among cell types. Furthermore, α5 subunits might contribute significantly to tonic currents in infragranular burst-spiking PyNs, especially in layer 5. These results suggest that ambient GABA might exert differential effects on the neuronal integration in a layer- and cell-type-specific manner and thus contribute to the processing of sensory properties by selectively tuning the signals flowing through the visual cortex.