Ethanol dually modulates GABAergic synaptic transmission onto dopaminergic neurons in ventral tegmental area: role of mu-opioid receptors

The mesolimbic dopaminergic system, originating from the ventral tegmental area (VTA) is implicated in the rewarding properties of ethanol. VTA dopaminergic neurons are under the tonic control of GABAergic innervations. Application of GABAergic agents changes ethanol consumption. However, it is unclear how acute ethanol modulates GABAergic inputs to dopaminergic neurons in the VTA. This report describes ethanol at clinically relevant concentrations (10-40 mM) dually modulates inhibitory postsynaptic currents (IPSCs). IPSCs were mediated by GABA(A) receptors and were recorded from VTA dopaminergic neurons in acute midbrain slices of rats. Acute application of ethanol reduced the amplitude and increased the paired pulse ratio of evoked IPSCs. Ethanol lowered the frequency but not the amplitude of spontaneous IPSCs. Nevertheless, ethanol had no effect on miniature IPSCs recorded in the presence of tetrodotoxin. These data indicate that ethanol inhibits GABAergic synaptic transmission to dopaminergic neurons by presynaptic mechanisms, and that ethanol inhibition depends on the firing of GABAergic neurons. Application of CGP 52432, a GABA(B) receptor antagonist, did not change ethanol inhibition of IPSCs. Tyr-d-Ala-Gly-N-Me-Phe-Gly-ol enkephalin (DAMGO), a mu-opioid receptor agonist, conversely, silenced VTA GABAergic neurons and inhibited IPSCs. Of note, in the presence of a saturating concentration of DAMGO (3 microM), ethanol potentiated the remaining IPSCs. Thus, ethanol dually modulates GABAergic transmission to dopaminergic neurons in the VTA. Ethanol modulation depends on the activity of VTA GABAergic neurons, which were inhibited by the activation of mu-opioid receptors. This dual modulation of GABAergic transmission by ethanol may be an important mechanism underlying alcohol addiction.     

GABA(A) receptor facilitation of neurokinin release from primary afferent terminals in the rat spinal cord

Our goal was to test the following hypotheses: 1) GABA_A receptors facilitate neurokinin release from primary afferent terminals; 2) they do this by suppressing an inhibitory effect of GABA_B receptors; 3) the activation of these two receptors is controlled by the firing frequency of primary afferents. We evoked neurokinin release by stimulating the dorsal root attached to spinal cord slices, and measured it using neurokinin 1 receptor (NK1R) internalization. Internalization evoked by root stimulation at 1 Hz (but not at 100 Hz) was increased by the GABA_A receptor agonists muscimol (effective concentration of drug for 50% of the increase [EC₅₀] 3 μM) and isoguvacine (EC₅₀ 4.5 μM). Internalization evoked by root stimulation at 100 Hz was inhibited by the GABA_A receptor antagonists bicuculline (effective concentration of drug for 50% of the inhibition [IC₅₀] 2 μM) and picrotoxin (IC₅₀ 243 nM). Internalization evoked by incubating the root with capsaicin (to selectively recruit nociceptive fibers) was increased by isoguvacine and abolished by picrotoxin. Therefore, GABA_A receptors facilitate neurokinin release. Isoguvacine-facilitated neurokinin release was inhibited by picrotoxin, low Cl⁻, low Ca²⁺, Ca²⁺ channel blockers and N-methyl-d-aspartate receptor antagonists. Bumetanide, an inhibitor of the Na⁺-K⁺-2Cl⁻ cotransporter, inhibited isoguvacine-facilitated neurokinin release, but this could be attributed to a direct inhibition of GABA_A receptors. The GABA_B agonist baclofen inhibited NK1R internalization evoked by 100 Hz root stimulation (IC₅₀ 1.5 μM), whereas the GABA_B receptor antagonist (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl) phosphinic acid (CGP-55845) increased NK1R internalization evoked by 1 Hz root stimulation (EC₅₀ 21 nM). Importantly, baclofen inhibited isoguvacine-facilitated neurokinin release, and CGP-55845 reversed the inhibition of neurokinin release by bicuculline. In conclusion, 1) GABA_B receptors located presynaptically in primary afferent terminals inhibit neurokinin release; 2) GABA_A receptors located in GABAergic interneurons facilitate neurokinin release by suppressing GABA release onto these GABA_B receptors; 3) high frequency firing of C-fibers stimulates neurokinin release by activating GABA_A receptors and inhibiting GABA_B receptors, whereas low frequency firing inhibits neurokinin release by the converse mechanisms.     

组胺对星形胶质细胞Egr-1表达的调节作用

 目的: 探讨组胺对星形胶质细胞早期反应生长因子-1(Egr-1)表达是否具有调节作用。方法: 将野生型(WT)和组氨酸脱羧酶敲除(HDC-KO)小鼠及组胺处理的HDC-KO小鼠取脑,并提取皮层组织总RNA。将原代培养的大鼠皮层星形胶质细胞分别给予不同浓度的组胺(10^-8、10^-7、10^-6、10^-5或10^-4 mol/L)处理15、30、60、120或240 min。组胺H₁、H₂受体拮抗剂分别于组胺给药前15 min加入。组胺处理完毕后,提取细胞总RNA或蛋白。利用real-time PCR和Western blot测定Egr-1的表达。结果: 与WT小鼠相比,HDC-KO小鼠大脑皮层Egr-1的mRNA表达量显著降低,而外源性给予组胺则能促进其Egr-1的mRNA表达。在培养的星形胶质细胞上,组胺可促进Egr-1的mRNA表达,其中10^-5 mol/L的组胺作用最强,而组胺(10^-5 mol/L)处理30 min时Egr-1的mRNA表达量达到峰值,相应的Egr-1蛋白表达于60 min时显著增高,该作用可被组胺H₁受体拮抗剂而非H₂受体拮抗剂显著抑制。结论: 组胺对大脑皮层组织及培养的星形胶质细胞Egr-1表达具有上调作用,该作用与激动组胺H₁受体有关。     

The mechanism of ethanol action on midbrain dopaminergic neuron firing: a dynamic-clamp study of the role of I(h) and GABAergic synaptic integration

Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels are expressed in dopaminergic (DA) neurons of the ventral tegmental area (VTA) as well as in DA and GABAergic neurons of the substantia nigra (SN). The excitation of DA neurons induced by ethanol has been proposed to result from its enhancing HCN channel current, I(h). Using perforated patch-clamp recordings in rat midbrain slices, we isolated I(h) in these neurons by voltage clamp. We showed that ethanol reversibly increased the amplitude and accelerated the activation kinetics of I(h) and caused a depolarizing shift in its voltage dependence. Using dynamic-clamp conductance injection, we injected artificial I(h) and fluctuating GABAergic synaptic conductance inputs into neurons following block of intrinsic I(h). This demonstrated directly a major role of I(h) in promoting rebound spiking following phasic inhibition, which was enhanced as the kinetics and amplitude of I(h) were changed in the manner induced by ethanol. Similar effects of ethanol were observed on I(h) and firing rate in non-DA, putatively GABAergic interneurons, indicating that in addition to its direct effects on firing, ethanol will produce large changes in the inhibition and disinhibition (via GABAergic interneurons) converging on DA neurons. Thus the overall effects of ethanol on firing of DA cells of the VTA and SN in vivo, and hence on phasic dopamine release in the striatum, appear to be determined substantially by its action on I(h) in both DA cells and GABAergic interneurons.     

Requirement of neural activity for the maintenance of dopaminergic neurons in rat midbrain slice cultures

Chronic treatment of organotypic midbrain slice cultures with L-type Ca²⁺ channel blocker nicardipine (3–10 μM) or verapamil (10 μM) for 18 days resulted in a drastic decrease in the number of dopaminergic neurons. A voltage-dependent Na⁺ channel blocker tetrodotoxin (1 μM) was also effective in decreasing the number of dopaminergic neurons. Concurrent application of forskolin (20 μM) or dibutyryl cyclic AMP (1 mM) counteracted the effects of nicardipine and tetrodotoxin. These results suggest that spontaneous neuronal activity within midbrain slice cultures, causing Ca²⁺ influx through L-type Ca²⁺ channels that maintains intracellular cyclic AMP levels, is required for the maintenance of dopaminergic neurons.     

Induction of endothelin-1 expression by oxidative stress in vascular smooth muscle cells

Atherosclerosis is based on endothelial dysfunction leading to impaired vasomotor function. This is partially due to nitric oxide (NO) depletion caused by oxidative stress. Since the vasoconstrictor endothelin-1 (ET-1) might also be involved in endothelial dysfunction, we investigated whether oxidative stress regulates ET-1 expression in vascular smooth muscle cells (VSMC). Human aortic VSMC were treated with H₂O₂ (200 μM) for up to 8 h. mRNA expression of preproendothelin (prepro-ET) was analyzed by RT-PCR. ET-1 protein and the marker for oxidative stress, 8-isoprostane, were determined by ELISA. Activity of cytosolic phospholipase A2 (cPLA₂) as an indicator of ET-1 autocrine activity was measured photometrically. Stimulation of VSMC with H₂O₂ resulted in increased expression of prepro-ET mRNA after 1 h with a maximum after 6 h (fourfold), similar to treatment with angiotensin II. ET-1 protein was significantly increased by H₂O₂ treatment with a maximum after 8 h (P<.05). This effect was inhibited by the antioxidants resveratrol (100 μM) and quercetin (50 μM). In quiesced VSMC, incubation with H₂O₂-conditioned medium resulted in increased cPLA₂ activity compared to the controls (P<.05). This activity was partially inhibited by the ET_A-receptor antagonist, PD 142893 (10 μM), indicating functional ET-1 in the conditioned medium. The presence of oxidative stress in H₂O₂-treated VSMC was associated by significantly increased formation of 8-isoprostane (P<.05). The data indicate for the first time that oxidative stress increases ET-1 generation and autocrine ET-1 activity in VSMC, a mechanism that might contribute to endothelial dysfunction in atherosclerosis.     

SIGNAL TRANSDUCTION OF NITRIC OXIDE DONOR-INDUCED PROTECTION IN HYDROGEN PEROXIDE-MEDIATED APOPTOSIS IN H9C2 CARDIOMYOBLASTS

Nitric oxide (NO) attenuates hydrogen peroxide (H₂O₂)-mediated injury to H9C2 cardiomyoblasts. To examine the role of nitric oxide, cultured H9C2 cardiomyoblasts were treated with H₂O₂ for 2 h in the presence or absence of the NO donor, diethylamine nitric oxide (DEANO). DEANO (30 μM) attenuated H₂O₂-induced apoptosis in H9C2 cells. H₂O₂-exposed H9C2 cells resulted in apoptosis in a time-dependent manner estimated by DNA fragmentation assay, nuclear morphology stained with fluorescent dye, Hoechst 33258 and Annexin V staining. Pretreatment with z-VAD-FMK, a pancaspase inhibitor, or z-DEVD-CHO, a specific caspase-3 inhibitor, completely suppressed the DNA ladder in response to H₂O₂. An increase in caspase-3-like protease (DEVDase) activity was observed during apoptosis, but no caspase-1 activity (YVADase) was detected. Treatment of H9C2 cells with 100 μM H₂O₂, resulted in a strong activation of JNK/SAPK. However, the activation of JNK/SAPK was clearly attenuated by 30 μM DEANO. Furthermore, the dominant negative JNK and SEK1-expressing cells displayed a marked decrease in a number of apoptotic cells. This inhibition of JNK1 in the system is involved in the protection of H₂O₂-induced apoptosis in H9C2 cardiomyoblasts.     

Catecholaminergic neuronal loss in locus coeruleus of aged female dtg APP/PS1 mice

Alzheimer's disease (AD) is the most common type of dementia afflicting the elderly. In addition to the presence of cortical senile plaques and neurofibrillary tangles, AD is characterized at autopsy by extensive degeneration of brainstem locus coeruleus (LC) neurons that provide noradrenergic innervation to cortical neuropil, together with relative stability of dopaminergic neuron number in substantia nigra (SN) and ventral tegmental area (VTA). The present study used design-based stereological methods to assess catecholaminergic neuronal loss in brains of double transgenic female mice that co-express two human mutations associated with familial AD, amyloid precursor protein (APP_swe) and presenilin-1 (PS1_ΔE9). Mice were analyzed at two age groups, 3–6 months and 16–23 months, when deposition of AD-type β-amyloid (Aβ) plaques occurs in cortical brain regions. Blocks of brain tissue containing the noradrenergic LC nucleus and two nuclei of dopaminergic neurons, the SN and VTA, were sectioned and sampled in a systematic-random manner and immunostained for tyrosine hydroxylase (TH), a specific marker for catecholaminergic neurons. Using the optical fractionator method we found a 24% reduction in the total number of TH-positive neurons in LC with no changes in SN-VTA of aged dtg APP/PS1 mice compared with non-transgenic controls. No significant differences were observed in numbers of TH-positive neurons in LC or SN-VTA in brains of young female dtg APP/PS1 mice compared to their age-matched controls. The findings of selective neurodegeneration of LC neurons in the brains of aged female dtg APP/PS1 mice mimic the neuropathology in the brains of AD patients at autopsy. These findings support the use of murine models of Aβ deposition to develop novel strategies for the therapeutic management of patients afflicted with AD.     

Intracellular studies on the dopamine-induced firing inhibition of neostriatal neurons in vitro: evidence for D1 receptor involvement

Intracellular recordings were obtained from rat neostriatal slices. Bath-applied dopamine (1–10 μM) produced a reversible inhibition of the action potentials evoked by direct stimulation and a decrease in the amplitude of the intrastriatally evoked depolarizing postsynaptic potentials. No change in membrane potential was detected during the application of 1–10 μM dopamine. Dopamine application also produced a decrease in anomalous rectification in the depolarizing direction. This subthreshold inward rectification was abolished by tetrodotoxin, but not by calcium-free and cadmium (0.1–1 mM)-containing solutions. The dopamine-induced decrease in excitatory postsynaptic potential amplitude was evident at resting membrane potential or at more positive levels, but was absent at hyperpolarized values of the membrane potential. Addition of bicuculline (50–500 μM) to the medium did not affect the inhibitory action of dopamine. The inhibitory action of dopamina also persisted in calcium-free and cadmium-containing solutions. The adenosine 3′,5′-cyclic monophosphate analogue, 8-bromo-adenosine 3′,5′-cyclic monophosphate (0.1–1 mM), mimicked the effects produced by D1 receptor activation. Bath application of 2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine (SKF 38393) (1–10 μM), a selective D1 dopaminergic agonist, mimicked the effects of micromolar concentrations of dopamine. The D2 dopaminergic agonists,4,4a,5,6,7,8,8a,9-octahydro-5-n-propyl-2H-pyrazolo-3,4-g-quinoline (LY 171555) and bromocriptine (both at 10 nM-10 μM), had no effects on neostriatal cells. The inhibition induced by micromolar doses of dopamine or SKF 38393 was antagonized by bath applications of R-( + )-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol (SCH 23390; 0.1–10 μM), a D1-selective antagonist, but not by sulpiride (10nM–10μM), a D2 antagonist.

We conclude that the inhibitory effect of dopamine on rat striatal neurons is postsynaptically mediated by the activation of D1 dopaminergic receptors via the reduction of a voltage-dependent tetrodotoxin sensitive inward conductance.     

Ethanol effects on dopaminergic ventral tegmental area neurons during block of Ih: involvement of barium-sensitive potassium currents

The dopaminergic neurons of the ventral tegmental area (DA VTA neurons) are important for the rewarding and reinforcing properties of drugs of abuse, including ethanol. Ethanol increases the firing frequency of DA VTA neurons from rats and mice. Because of a recent report on block of ethanol excitation in mouse DA VTA neurons with ZD7288, a selective blocker of the hyperpolarization-activated cationic current I(h), we examined the effect of ZD7288 on ethanol excitation in DA VTA neurons from C57Bl/6J and DBA/2J mice and Fisher 344 rats. Ethanol (80 mM) caused only increases in firing rate in mouse DA VTA neurons in the absence of ZD7288, but in the presence of ZD7288 (30 muM), ethanol produced a more transient excitation followed by a decrease of firing. This same biphasic phenomenon was observed in DA VTA neurons from rats in the presence of ZD7288 only at very high ethanol concentrations (160-240 mM) but not at lower pharmacologically relevant concentrations. The longer latency ethanol-induced inhibition was not observed in DA VTA neurons from mice or rats in the presence of barium (100 muM), which blocks G protein-linked potassium channels (GIRKs) and other inwardly rectifying potassium channels. Ethanol may have a direct effect to increase an inhibitory potassium conductance, but this effect of ethanol can only decrease the firing rate if I(h) is blocked.     

Mapping dopaminergic deficiencies in the substantia nigra/ventral tegmental area in schizophrenia

Previous work from our laboratory showed deficits in tyrosine hydroxylase protein expression within the substantia nigra/ventral tegmental area (SN/VTA) in schizophrenia. However, little is known about the nature and specific location of these deficits within the SN/VTA. The present study had two aims: (1) test if tyrosine hydroxylase deficits could be explained as the result of neuronal loss; (2) assess if deficits in tyrosine hydroxylase are sub-region specific within the SN/VTA, and thus, could affect specific dopaminergic pathways. To achieve these objectives: (1) we obtained estimates of the number of dopaminergic neurons, total number of neurons, and their ratio in matched SN/VTA schizophrenia and control samples; (2) we performed a qualitative assessment in SN/VTA schizophrenia and control matched samples that were processed simultaneously for tyrosine hydroxylase immunohistochemistry. We did not find any significant differences in the total number of neurons, dopaminergic neurons, or their ratio. Our qualitative study of TH expression showed a conspicuous decrease in labeling of neuronal processes and cell bodies within the SN/VTA, which was sub-region specific. Dorsal diencephalic dopaminergic populations of the SN/VTA presented the most conspicuous decrease in TH labeling. These data support the existence of pathway-specific dopaminergic deficits that would affect the dopamine input to the cortex without significant neuronal loss. Interestingly, these findings support earlier reports of decreases in tyrosine hydroxylase labeling in the target areas for this dopaminergic input in the prefrontal and entorhinal cortex. Finally, our findings support that tyrosine hydroxylase deficits could contribute to the hypodopaminergic state observed in cortical areas in schizophrenia.     

Lack of an interaction between orexinergic and opioid/nociceptinergic systems in rat cerebrocortical slices

We have recently reported that orexins (OXs) selectively evoke norepinephrine release from rat cerebrocortical slices. In the present study, we have examined orexin–opioid interactions in OXA (100 nM) and K⁺ (40 mM)-evoked norepinephrine release. OXA-evoked norepinephrine release was reversed 90% by SB-334867 (OX₁-receptor antagonist) (10 μM) but not naloxone (10 μM). [D-Pen²,D-Pen⁵]-enkephalin (DPDPE) (DOP-agonist) and nociceptin/orphanin-FQ (N/OFQ) also failed to affect OXA-evoked release. [D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin (DAMGO) (MOP-agonist) and spiradoline (KOP-agonist) significantly reduced OXA-evoked release with the concentration producing 50% of the maximal inhibition (EC₅₀) [maximal inhibition (E_max)] of 3.2 μM [41.8%] and 4.3 μM [54.9%] respectively. The effects of DAMGO and spiradoline were naloxone (10 μM)-insensitive. In contrast, naloxone significantly antagonized the inhibitory effects of DAMGO and spiradoline on K⁺-evoked release. We conclude that opioid receptors (DOP and KOP) are involved in K⁺ but not OXA-evoked release. Moreover, we have failed to demonstrate an interaction between orexinergic and opioid/N/OFQ-ergic systems in this system.     

Tyrosine hydroxylase mRNA in the dopaminergic neurons of young adult and aged mice by in situ hybridization

Tyrosine hydroxylase (TH) mRNA in the dopaminergic neurons of the substantia nigra (SN) and the ventral tegmental area (VTA) in young adult and aged mice was detected and quantitated using in situ hybridization. Using 3H-labeled antisense RNA complementary to TH mRNA, these studies demonstrate the presence of TH mRNA in dopaminergic neurons of the SN and the VTA. Alternate sections stained immunocytochemically using TH-specific antiserum demonstrated that the neurons containing TH mRNA also contained TH protein. Quantitative analysis of the number of silver grains present over the dopaminergic neurons of the SN and VTA revealed no statistically significant difference between the two age groups. The results suggest that TH gene expression in dopaminergic neurons of the SN and VTA is not different in young adult and aged mice.     

Histamine innervation and activation of septohippocampal GABAergic neurones: involvement of local ACh release

Recent studies indicate that the histaminergic system, which is critical for wakefulness, also influences learning and memory by interacting with cholinergic systems in the brain. Histamine-containing neurones of the tuberomammillary nucleus densely innervate the cholinergic and GABAergic nucleus of the medial septum/diagonal band of Broca (MSDB) which projects to the hippocampus and sustains hippocampal theta rhythm and associated learning and memory functions. Here we demonstrate that histamine, acting via H₁ and/or H₂ receptor subtypes, utilizes direct and indirect mechanisms to excite septohippocampal GABA-type neurones in a reversible, reproducible and concentration-dependent manner. The indirect mechanism involves local ACh release, is potentiated by acetylcholinesterase inhibitors and blocked by atropine methylbromide and 4-DAMP mustard, an M₃ muscarinic receptor selective antagonist. This indirect effect, presumably, results from a direct histamine-induced activation of septohippocampal cholinergic neurones and a subsequent indirect activation of the septohippocampal GABAergic neurones. In double-immunolabelling studies, histamine fibres were found in the vicinity of both septohippocampal cholinergic and GABAergic cell types. These findings have significance for Alzheimer's disease and other neurodegenerative disorders involving a loss of septohippocampal cholinergic neurones as such a loss would also obtund histamine effects on septohippocampal cholinergic and GABAergic functions and further compromise hippocampal arousal and associated cognitive functions.     

GABAergic control of rat substantia nigra dopaminergic neurons: role of globus pallidus and substantia nigra pars reticulata

Dopaminergic neurons in vivo fire spontaneously in three distinct patterns or modes. It has previously been shown that the firing pattern of substantia nigra dopaminergic neurons can be differentially modulated by local application of GABA(A) and GABA(B) receptor antagonists. The GABA(A) antagonists, bicuculline or picrotoxin, greatly increase burst firing in dopaminergic neurons whereas GABA(B) antagonists cause a modest shift away from burst firing towards pacemaker-like firing. The three principal GABAergic inputs to nigral dopaminergic neurons arise from striatum, globus pallidus and from the axon collaterals of nigral pars reticulata projection neurons, each of which appear to act in vivo primarily on GABA(A) receptors (see preceding paper). In this study we attempted to determine on which afferent pathway(s) GABA(A) antagonists were acting to cause burst firing. Substantia nigra dopaminergic neurons were studied by single unit extracellular recordings in urethane anesthetized rats during pharmacologically induced inhibition and excitation of globus pallidus. Muscimol-induced inhibition of pallidal neurons produced an increase in the regularity of firing of nigral dopaminergic neurons together with a slight decrease in firing rate. Bicuculline-induced excitation of globus pallidus neurons produced a marked increase in burst firing together with a modest increase in firing rate. These changes in firing rate were in the opposite direction to what would be expected for a monosynaptic GABAergic pallidonigral input. Examination of the response of pars reticulata GABAergic neurons to similar manipulations of globus pallidus revealed that the firing rates of these neurons were much more sensitive to changes in globus pallidus neuron firing rate than dopaminergic neurons and that they responded in the opposite direction. Pallidal inhibition produced a dramatic increase in the firing rate of pars reticulata GABAergic neurons while pallidal excitation suppressed the spontaneous activity of pars reticulata GABAergic neurons. These data suggest that globus pallidus exerts significant control over the firing rate and pattern of substantia nigra dopaminergic neurons through a disynaptic pathway involving nigral pars reticulata GABAergic neurons and that at least one important way in which local application of bicuculline induces burst firing of dopaminergic neurons is by disinhibition of this tonic inhibitory input.     

A null mutation in TGF-alpha leads to a reduction in midbrain dopaminergic neurons in the substantia nigra

Transforming growth factor (TGF)-alpha is neurotrophic for midbrain dopaminergic neurons in vitro. Here I investigated whether a null mutation in the TGF-alpha gene affects the normal development or survival of dopaminergic neurons in either the substantial nigra (SN) or the ventral tegmental area (VTA). The SN of TGF-alpha knockout mice contained 50% fewer dopaminergic neurons than the control SN, but VTA neuron number was unchanged. In addition, the overall volume of the dorsal striatum was reduced by 20%. Newborn mice showed a similar decrease in the number of SN dopaminergic neurons, suggesting that TGF-alpha is unlikely to regulate developmental neuron death. These studies indicate that TGF-alpha is required for the normal proliferation or differentiation of a select population of dopaminergic neurons within the SN.     

Possible roles of kainate receptors on GABAergic nerve terminals projecting to rat substantia nigra dopaminergic neurons

GABAergic afferent inputs are thought to play an important role in the control of the firing pattern of substantia nigra pars compacta (SNc) dopaminergic neurons. We report here the actions of presynaptic kainite (KA) receptors in GABAergic transmission of rat SNc dopaminergic neurons. In mechanically dissociated rat SNc dopaminergic neurons attached with native presynaptic nerve terminals, GABAergic miniature inhibitory postsynaptic currents (mIPSCs) were recorded by use of conventional whole cell patch recording mode. In the voltage-clamp condition, KA (3 microM) significantly increased GABAergic mIPSC frequency without affecting the current amplitude. This facilitatory effect of KA was not affected in the presence of 20 microM GYKI52466, a selective AMPA receptor antagonist, but was completely inhibited in the presence of 20 microM CNQX, an AMPA/KA receptor antagonist. Presynaptic KA receptors on GABAergic terminals were mainly permeable to Na+ but impermeable to Ca2+ because KA-induced facilitation of mIPSC frequency was completely suppressed in either Na+-free or Ca2+-free external solutions, and in the presence of 200 microM Cd2+, a general voltage-dependent Ca2+ channel blocker. In the slice preparation, KA increased GABAergic spontaneous mIPSC frequency, but significantly suppressed evoked IPSC (eIPSC) amplitude. However, this inhibitory action on eIPSCs was reversed by 10 microM CGP55845, a selective GABAB receptor antagonist, implicating the possible involvement of GABAB autoreceptors in KA-induced modulation of GABAergic transmission. Thus presynaptic KA receptors on GABAergic nerve terminals synapsing onto SNc neurons may play functional roles contributing the fine control of neuronal excitability and firing pattern of SNc.     

GABAA and GABAB antagonists prevent the opioid inhibition of endogenous acetylcholine release evoked by glutamate from rat neostriatal slices

We have examined the role of the GABAergic system in the opioid inhibition of endogenous acetylcholine (ACh) release from rat neostriatal slices by blocking either γ-aminobutyric acid-A (GABA_A) or GABA_B receptors. GABAergic antagonists (bicuculline or phaclofen) completely blocked μ- (morphine or DAGO) and δ-opioid (DPDPE) inhibition of glutamate-evoked endogenous ACh release in a concentration-dependent manner. However, GABA antagonists were ineffective in blocking the opioid inhibition of potassium-evoked endogenous ACh release. These findings point to the important role of the GABAergic system in the regulation of μ- and δ-opioid inhibition of cholinergic neurons stimulated by glutamate.     

Histamine-induced excitatory responses in mouse ventromedial hypothalamic neurons: ionic mechanisms and estrogenic regulation

Histamine is capable of modulating CNS arousal states by regulating neuronal excitability. In the current study, histamine action in the ventromedial hypothalamus (VMH), its related ionic mechanisms, and its possible facilitation by estrogen were investigated using whole cell patch-clamp recording in brain slices from ovariectomized female mice. Under current clamp, a bath application of histamine (20 microM) caused membrane depolarization, associated with an increased membrane resistance. In some cells, the depolarization was accompanied by action potentials. Histamine application also significantly reduced the latency of action potential evoked by current steps. Histamine-induced depolarization was not affected by either tetrodotoxin or Cd(2+). However, after blocking K(+) channels with tetraethylammonium, 4-aminopyridine, and Cs(+), depolarization was significantly decreased. Under voltage clamp, histamine-induced depolarization was associated with an inward current. The current-voltage relationship revealed that this inward current reversed near E(K). The histamine effect was mimicked by a histamine receptor 1 (H(1)) agonist, but not a histamine receptor 2 (H(2)) agonist. An H(1) antagonist, but not H(2) antagonist, abolished histamine responses. When ovariectomized mice were treated with estradiol benzoate (E2), histamine-induced depolarization was significantly enhanced with an increased percentage of cells showing action potential firing. These results suggest that histamine depolarized VMH neurons by attenuating a K(+) leakage current and this effect was mediated by H(1) receptor. E2 facilitated histamine-induced excitation of VMH neurons. This histamine effect may present a potential mechanism by which estrogens modulate the impact of generalized CNS arousal on a sexual arousal-related neuronal group.     

γ-Aminobutyrate as an autonomic neurotransmitter: Release and uptake of [H]γ-Aminobutyrate in guinea pig large intestine and cultured enteric neurons using physiological methods and electron microscopic autoradiography

To provide further evidence that some enteric neurons use γ-aminobutyrate (GABA) as a neurotransmitter, we have demonstrated a depolarization-induced release of [³H]GABA from isolated myenteric ganglia in culture, and from segments of large intestine containing the myenteric plexus. In addition, light and electron microscopic autoradiography has been employed to visualize the putative GABAergic neurons and their projections, both in cultured ganglia and in sections from the gut wall.

Explant cultures of the guinea-pig myenteric plexus, containing only neurons and glia intrinsic to the gut, were incubated with 0.14μ M [³H]GABA, washed and then repeatedly depolarized by 62 mM K⁺. The depolarizations always evoked release of [³H]GABA. The evoked release was reversibly blocked by 5mM Co²⁺, suggesting a dependence on Ca²⁺ influx, a characteristic of neuronal transmitter release. Strips of the guinea-pig taenia coli, containing the myenteric plexus, were incubated with 0.14 or 0.7μM [³H]GABA, washed and subjected to electrical field stimulation. This caused release of [³H]GABA, which could be evoked successively on repeated stimulation. The release was of neuronal origin and Ca²⁺ dependent, since it was abolished by 3μ M tetrodotoxin and reversibly blocked by 10 mM Co²⁺. By combined electrophoresis and chromatography the released tritium was identified as being attached to GABA.

Autoradiography, following incubation with low concentrations of [³H]GABA, was used to identify specifically putative GABAergic neurons. Light microscopic autoradiography of cultured ganglia, and electron microscopic autoradiography of sections from the taenia coli including the myenteric plexus, were in good agreement, showing a selective and heavy labelling over a sub-population of neurons, and light labelling over glial cells. The majority of neurons and the non-neural cells were unlabelled. The electron microscopic autoradiographs also showed heavy labelling over some, but not all, axons in the fine axon bundles that innervate the longitudinal muscle of the taenia.

These results strongly support our previous suggestion that a population of myenteric neurons are GABAergic, although more work is needed before this is fully established. Our data also suggest that some of the putative GABAergic neurons project out to the gut musculature, where their axons, together with axons from other neurons, run in bundles to form the intramuscular nerves. We therefore propose that one of the roles of these neurons in the gut is the presynaptic modulation of transmitter released from neighbouring axons in these nerves.