Synapses between corticotropin-releasing factor-containing axon terminals and dopaminergic neurons in the ventral tegmental area are predominantly glutamatergic

Interactions between stress and the mesocorticolimbic dopamine (DA) system have been suggested from behavioral and electrophysiological studies. Because corticotropin-releasing factor (CRF) plays a role in stress responses, we investigated possible interactions between neurons containing CRF and those producing DA in the ventral tegmental area (VTA). We first investigated the cellular distribution of CRF in the VTA by immunolabeling VTA sections with anti-CRF antibodies and analyzing these sections by electron microscopy. We found CRF immunoreactivity present mostly in axon terminals establishing either symmetric or asymmetric synapses with VTA dendrites. We established that nearly all CRF asymmetric synapses are glutamatergic, insofar as the CRF-immunolabeled axon terminals in these synapses coexpressed the vesicular glutamate transporter 2, and that the majority of CRF symmetric synapses are GABAergic, insofar as the CRF-immunolabeled axon terminals in these synapses coexpressed glutamic acid decarboxylase, findings that are of functional importance. We then looked for synaptic interactions between CRF- and DA-containing neurons, by using antibodies against CRF and tyrosine hydroxylase (TH; a marker for DA neurons). We found that most synapses between CRF-immunoreactive axon terminals and TH neurons are asymmetric (in the majority likely to be glutamatergic) and suggest that glutamatergic neurons containing CRF may be part of the neuronal circuitry that mediates stress responses involving the mesocorticolimbic DA system. The presence of CRF synapses in the VTA offers a mechanism for interactions between the stress-associated neuropeptide CRF and the mesocorticolimbic DA system.     

GIRK2 expression in dopamine neurons of the substantia nigra and ventral tegmental area

G-protein-regulated inward-rectifier potassium channel 2 (GIRK2) is reported to be expressed only within certain dopamine neurons of the substantia nigra (SN), although very limited data are available in humans. We examined the localization of GIRK2 in the SN and adjacent ventral tegmental area (VTA) of humans and mice by using either neuromelanin pigment or immunolabeling with tyrosine hydroxylase (TH) or calbindin. GIRK2 immunoreactivity was found in nearly every human pigmented neuron or mouse TH-immunoreactive neuron in both the SN and VTA, although considerable variability in the intensity of GIRK2 staining was observed. The relative intensity of GIRK2 immunoreactivity in TH-immunoreactive neurons was determined; in both species nearly all SN TH-immunoreactive neurons had strong GIRK2 immunoreactivity compared with only 50-60% of VTA neurons. Most paranigral VTA neurons also contained calbindin immunoreactivity, and approximately 25% of these and nearby VTA neurons also had strong GIRK2 immunoreactivity. These data show that high amounts of GIRK2 protein are found in most SN neurons as well as in a proportion of nearby VTA neurons. The single previous human study may have been compromised by the fixation method used and the postmortem delay of their controls, whereas other studies suggesting that GIRK2 is located only in limited neuronal groups within the SN have erroneously included VTA regions as part of the SN. In particular, the dorsal layer of dopamine neurons directly underneath the red nucleus is considered a VTA region in humans but is commonly considered the dorsal tier of the SN in laboratory species.     

Low doses of ethanol activate dopaminergic neurons in the ventral tegmental area

In unanesthetized rats the intravenous administration of low doses of ethanol (0.125-0.5 g/kg) produced a dose-dependent increase (30-80%) in the firing rate of dopaminergic (DA) neurons in the Ventral Tegmental Area (VTA). In agreement with previous observations, a dose range between 0.5 and 2 g/mg of ethanol was needed to produce comparable stimulant responses in DA neurons of the Substantia Nigra Pars Compacta. However, in anesthetized rats, doses of ethanol up to 1 g/kg failed to activate VTA-DA neurons. The high sensitivity of VTA-DA neurons to ethanol activation suggests that they might be involved in the reinforcing properties of the drug.     

Activity of neurons in the ventral tegmental region of the behaving monkey

To investigate the functions of neurons in the ventral tegmental area, recordings were made of the activity of 257 single neurons in this area in the behaving monkey. Four main types of neuronal response were found in the ventral part of the tegmentum. First, neurons with activity phasically related to mouth or arm movements were found. Most of these were located relatively far lateral, close to the junction of the midbrain reticular formation with the zona incerta, or were in the substantia nigra, pars reticulata. Second, neurons were found which responded differentially in a visual discrimination task on trials on which the monkey had to initiate a licking response compared with trials on which he did not, and which also altered their firing rate tonically while mouth movements were being made in other situations (differential motor neurons). These were found mainly in the midbrain reticular formation, consistent with the view that populations of neurons in these regions are involved in the execution of movements. Third, neurons which also responded differentially in the visual discrimination task, but did not respond when the same movements were made in other situations, were found in the ventral tegmental area, in a region medial to and in some cases immediately dorsal to the substantia nigra pars compacta. Fourth, neurons which responded to cues such as a tone which enabled the monkey to prepare for performance on each trial of the visual discrimination task were found in the ventral tegmental area close to the midline. These third and fourth types of neurons were thus found in the region where neurons of the mesocortical and mesolimbic pathways are located. Their responses are similar to those of neurons found in the striatum, and it is suggested that they are important in enabling the animal to prepare for and then to engage in particular behavioral responses.     

GABA(A) receptors in the ventral tegmental area control bidirectional reward signalling between dopaminergic and non-dopaminergic neural motivational systems

In the midbrain ventral tegmental area (VTA), both dopaminergic and nondopaminergic neural substrates mediate various behavioural reward phenomena. VTA GABAergic neurons are anatomically positioned to influence the activity of both the mesolimbic dopamine system and nondopamine efferents from the VTA. In order to examine the possible functional role of VTA GABA(A) receptors in neural reward processes, we performed discrete, bilateral microinjections of the GABA(A) receptor agonist, muscimol, or the GABA(A) receptor antagonist, bicuculline, into the VTA. Using a fully counterbalanced, unbiased conditioned place-preference paradigm, we demonstrate that activation of VTA GABA(A) receptors, with the GABA(A) receptor agonist muscimol (5--50 ng/microL), or inhibition of VTA GABA(A) receptors, with the GABA(A) receptor antagonist bicuculline (5--50 ng/microL), both produce robust rewarding effects. Furthermore, these rewarding effects can be pharmacologically dissociated: blockade of dopamine receptors with a dopamine receptor antagonist, alpha-flupenthixol (0.8 mg/kg; i.p.), or concurrent activation of VTA GABA(B) receptors with a GABA(B) receptor agonist, baclofen (70 ng/microL), blocked the rewarding properties of the GABA(A) receptor agonist, but had no effect on the rewarding properties of the GABA(A) receptor antagonist. These results suggest that, within the VTA, a single GABA(A) receptor substrate controls bidirectional reward signalling between dopaminergic and nondopaminergic brain reward systems.     

A topographic localization of enkephalin on the dopamine neurons of the rat substantia nigra and ventral tegmental area demonstrated by combined histofluorescence-immunocytochemistry

The concentration of choline acetyltransferase, a specific marker for cholinergic neurons, was determined in the supraoptic nucleus after a variety of lesions. Surgical lesions immediately rostral as well as medial and lateral to the nucleus did not affect the concentration of the enzyme. Only lesions which separated the nucleus from the posterior part of the lateral hypothalamus slightly decreased its concentration in choline acetyltransferase. It is concluded that the bulk of the cholinergic neurons is in the supraoptic nucleus or its immediate vicinity.     

Phasic activation of substantia nigra and the ventral tegmental area by chemical stimulation of the superior colliculus: an electrophysiological investigation in the rat

The source of short-latency visual input to midbrain dopaminergic (DA) neurons is not currently known; however, the superior colliculus (SC) is a subcortical visual structure which has response latencies consistently shorter than those recorded for DA neurons in substantia nigra and the ventral tegmental area. To test whether the SC represents a plausible route by which visual information may gain short latency access to the ventral midbrain, the present study examined whether experimental stimulation of the SC can influence the activity of midbrain DA neurons. In urethane-anaesthetized rats, 63 pairs of extracellular recordings were obtained from neurons in the SC and ipsilateral ventral midbrain, before and after local disinhibitory injections of the GABA antagonist bicuculline (20-40 ng/200-400 nL saline) into the SC. Neurons recorded from substantia nigra and the ventral tegmental area were classified as putative DA (25/63, 39.7%) or putative non-DA (38/63, 60.3%). In nearly half the cases (27/63, 42.8%), chemical stimulation of the SC evoked a corresponding increase in neural activity in the ventral midbrain. This excitatory effect did not distinguish between DA and non-DA neurons. In 6/63 cases (9.5%), SC activation elicited a reliable suppression of activity, while the remaining 30/63 cases (47.6%) were unaffected. In almost a third of cases (16/57, 28.1%) intense phasic activation of the SC was associated with correlated phasic activation of neurons in substantia nigra and the ventral tegmental area. These data suggest that the SC is in a position to play an important role in discriminating the appropriate stimulus qualities required to activate DA cells at short latency.     

The effect of long-term amphetamine administration on the activity of dopaminergic neurons of the substantia nigra

Spontaneously active neurons from the pars compacta region of the substantia nigra were studied in chloral hydrate anesthetized, immobilized rats which had been pretreated with daily intraperitoneal saline or d-amphetamine injections according to several schedules. The spontaneous firing rates of these neurons were not altered by any of the pretreatment regimens. In addition, the intravenous dose of d-amphetamine sulfate which was required to reduce the spontaneous firing rate of these cells by at least 50% (threshold dose) was not modified by amphetamine pretreatment. There was a significant positive linear correlation between the threshold dose and the initial spontaneous firing rate of the neurons from animals pretreated with multiple saline injections, but not from those receiving long-term d-amphetamine. These results demonstrate that there are no alterations in the sensitivity of dopaminergic neurons to amphetamine under conditions known to produce the enhanced effects of the drug on motor behaviors which develop during multiple injections.     

Plasmalemmal mu-opioid receptor distribution mainly in nondopaminergic neurons in the rat ventral tegmental area

Opiate-evoked reward and motivated behaviors reflect, in part, the enhanced release of dopamine produced by activation of the mu-opioid receptor (muOR) in the ventral tegmental area (VTA). We examined the functional sites for muOR activation and potential interactions with dopaminergic neurons within the rat VTA by using electron microscopy for the immunocytochemical localization of antipeptide antisera raised against muOR and tyrosine hydroxylase (TH), the synthesizing enzyme for catecholamines. The cellular and subcellular distribution of muOR was remarkably similar in the two major VTA subdivisions, the paranigral (VTApn) and parabrachial (VTApb) nuclei. In each region, somatodendritic profiles comprised over 50% of the labeled structures. MuOR immunolabeling was often seen at extrasynaptic/perisynaptic sites on dendritic plasma membranes, and 10% of these dendrites contained TH. MuOR-immunoreactivity was also localized to plasma membranes of axon terminals and small unmyelinated axons, none of which contained TH. The muOR-immunoreactive axon terminals formed either symmetric or asymmetric synapses that are typically associated with inhibitory and excitatory amino acid transmitters. Their targets included unlabeled (30%), muOR-labeled (25%), and TH-labeled (45%) dendrites. Our results suggest that muOR agonists in the VTA affect dopaminergic transmission mainly indirectly through changes in the postsynaptic responsivity and/or presynaptic release from neurons containing other neurotransmitters. They also indicate, however, that muOR agonists directly affect a small population of dopaminergic neurons expressing muOR on their dendrites in VTA and/or terminals in target regions.     

Dopaminergic and non-dopaminergic projections to amygdala from substantia nigra and ventral tegmental area

The projections from the substantia nigra (SN) and ventral tegmental area (VTA) to the amygdala of the rat were examined by simultaneous visualization of catecholamine (CA) histofluorescence and retrograde tracer. CA-containing cells in lateral VTA, medial SN and the dorsal edge of SN pars compacta were labeled by injections of propidium iodide (PI) into the amygdala. While CA-containing cells were present in SN pars lateralis (SNl), those cells which were labeled by injections into the amygdala did not contain CA. There is, thus, a significant non-DA projection from SNl to the amygdala.     

Ultrastructural localization of the serotonin 2A receptor in dopaminergic neurons in the ventral tegmental area

Serotonin (5-hydroxytryptamine, 5-HT) 2A receptor antagonists are clinically effective antipsychotics that may differentially target mesocortical and mesolimbic dopaminergic neurons having partially segregated distribution in the parabrachial (PB) and paranigral (PN) ventral tegmental area (VTA). We examined the ultrastructural immunocytochemical localization of the 5-HT2A receptor in these subdivisions of rat VTA, to determine (1) the functional sites for receptor activation, and (2) cellular associations between the receptor and dopaminergic neurons identified by their content of tyrosine hydroxylase (TH). The mean area density of neuronal profiles containing 5-HT2A receptor labeling was not significantly different in the PB and PN VTA. In each region approximately 44% of the 5-HT2A labeled profiles were dendrites while the remainder were mainly axons. Dendritic 5-HT2A-immunoreactivity was often localized to membranous cytoplasmic organelles resembling smooth endoplasmic reticulum, and to more rarely to segments of the plasma membrane beneath contacts from unlabeled axon terminals. 5-HT2A labeling was also seen within the cytoplasm of a few axon initial segments and many small unmyelinated axons. Approximately 40% of the 5-HT2A-labeled dendritic profiles contained TH in either PB or PN VTA. Our results suggest that 5-HT2A receptors in VTA are largely cytoplasmic and play an equally important role in modulating dopaminergic neurons in PB and PN VTA. These results also implicate 5-HT2A receptors in the postsynaptic activation of non-dopaminergic neurons and possibly the presynaptic release from terminals of axons originating in, or passing through, these regions.     

Targeting of serotonin 1A receptors to dopaminergic neurons within the parabrachial subdivision of the ventral tegmental area in rat brain

Serotonin (5-hydroxytryptamine [5-HT]) modulates dopamine-related cognitive functions and motor activity through activation of selective receptor subtypes including 5-HT1A. Potential targets for these 5-HT1A-mediated actions of 5-HT include mesocortical and mesolimbic dopaminergic neurons having partially segregated distribution in the parabrachial and paranigral subdivisions of the ventral tegmental area (VTA), respectively. We therefore examined the ultrastructural immunocytochemical localization of the 5-HT1A receptor in the parabrachial (VTApb) and paranigral (VTApn) subdivisions of rat VTA, to determine 1) the functional sites for receptor activation, and 2) the cellular associations between this receptor and dopaminergic neurons identified by their tyrosine hydroxylase (TH) content. In each region, 5-HT1A immunoreactivity was mainly observed in somatodendritic profiles, but it was also present in small unmyelinated axons and in a few axon terminals and glia, suggesting a role for 5-HT1A receptors in presynaptic and glial functions, as well as postsynaptic neuronal activation, in VTA. In somatodendritic profiles, 5-HT1A gold particles were mainly localized to tubulovesicles presumed to be smooth endoplasmic reticulum. In addition, however, in distal dendrites receiving multiple inputs the receptor was targeted to selective postsynaptic junctions, or more randomly distributed on nonsynaptic portions of the plasma membrane. Of the 5-HT1A-labeled dendrites, 64% in VTApb and 44% in VTApn contained TH. These findings suggest a reserve of cytoplasmic 5-HT1A receptors that are mobilized to functional postsynaptic sites on the plasma membrane by afferent input to distal dendrites in the VTA. They also indicate that 5-HT1A activation may affect a larger population of dopaminergic neurons in VTApb compared with VTApn, thus having a potentially greater impact on cognitive functions modulated by mesocortical dopaminergic neurons.     

Cholinergic axons in the rat ventral tegmental area synapse preferentially onto mesoaccumbens dopamine neurons

Cholinergic afferents to the ventral tegmental area (VTA) contribute substantially to the regulation of motivated behaviors and the rewarding properties of nicotine. These actions are believed to involve connections with dopamine (DA) neurons projecting to the nucleus accumbens (NAc). However, this direct synaptic link has never been investigated, nor is it known whether cholinergic inputs innervate other populations of DA and gamma-aminobutyric acid (GABA) neurons, including those projecting to the prefrontal cortex (PFC). We addressed these questions by using electron microscopic analysis of retrograde tract-tracing and immunocytochemistry for the vesicular acetylcholine transporter (VAChT) and for tyrosine hydroxylase (TH) and GABA. In tissue labeled for TH, VAChT(+) terminals frequently synapsed onto DA mesoaccumbens neurons but only seldom contacted DA mesoprefrontal cells. In tissue labeled for GABA, one-third of VAChT(+) terminals innervated GABA-labeled dendrites, including both mesoaccumbens and mesoprefrontal populations. VAChT(+) synapses onto DA and mesoaccumbens neurons were more commonly of the asymmetric (presumed excitatory) morphological type, whereas VAChT(+) synapses onto GABA cells were more frequently symmetric (presumed inhibitory or modulatory). These findings suggest that cholinergic inputs to the VTA mediate complex synaptic actions, with a major portion of this effect likely to involve an excitatory influence on DA mesoaccumbens neurons. As such, the results suggest that natural and drug rewards operating through cholinergic afferents to the VTA have a direct synaptic link to the mesoaccumbens DA neurons that modulate approach behaviors.     

Effects of kainic acid lesions of the striatum on self-stimulation in the substantia nigra and ventral tegmental area

Unilateral kainic acid lesions of the dorsal striatum provided evidence for a dissociation of neural substrates of brain-stimulation reward at sites in the ventral tegmental area and substantia nigra. The lesions caused a significant increase in current intensity thresholds at substantia nigra placements, whereas similar lesions had no effect on self-stimulation thresholds at sites in the ventral tegmentum. In addition, the rate-increasing effects of d-amphetamine (0.1–1.0 mg/kg) on self-stimulation were determined before and after lesions to the dorsal striatum. No significant changes in dose-response curves were observed at either loci. Amphetamine-induced rotation was used to confirm damage to the dorsal striatum and lesioned animals were observed to rotate towards the side of the lesion. In contrast, sham-lesioned animals showed turning away from the side stimulated electrically in previous tests. The results of the self-stimulation and rotation experiments are discussed in the context of neural substrates of reward and motor activity.     

Intracerebroventricular administration of NMDA-R1 antisense oligodeoxynucleotide significantly alters the activity of ventral tegmental area dopamine neurons: an electrophysiological study

In this study, we determined the activity of midbrain dopamine (DA) neurons in male albino rats following the intracerebroventricular (i.c.v.) administration of antisense oligodeoxynucleotide (aODN) against the mRNA for the NR1 subunit of the NMDA receptor. In addition, the effect of aODN on the specific binding of the NMDA receptor ligand [(3)H]MK-801 was also examined in various brain areas, including the midbrain. Antisense ODN against the NR1 mRNA, the corresponding sense ODN (sODN) or saline was continuously administered into the right ventricle of rats by osmotic minipumps for 7 days (20 nmol/day). Autoradiographic binding studies indicated that aODN significantly reduced the density of [(3)H]MK-801 binding by an average of 20-30% in several forebrain regions, including the anterior cingulate cortex, caudate putamen, and nucleus accumbens. However, [(3)H]MK-801 binding was not significantly altered in the ventral tegmental area (VTA) or substantia nigra pars compacta (SNC). Subsequently, using the technique of extracellular single-unit recording, the number, as well as the firing pattern, of spontaneously active DA neurons was determined in the VTA and SNC. The administration of aODN did not significantly alter the number of spontaneously active VTA and SNC DA neurons compared to saline- of sODN-treated animals. Furthermore, the firing pattern of spontaneously active SNC DA neurons was not significantly altered. However, for spontaneously active VTA DA neurons, the administration of aODN significantly decreased the percent events in bursts, number of bursts, and percentage of DA neurons exhibiting a bursting pattern compared to saline- and sODN-treated animals, i.e., neurons show less bursting activity. The present results suggest that subchronic aODN treatment against the mRNA for the NR1 subunit of the NMDA receptors can reduce NMDA receptor number and can result in an altered activity of spontaneously active VTA DA neurons in anesthetized rats.     

Optogenetic excitation in the ventral tegmental area of glutamatergic or cholinergic inputs from the laterodorsal tegmental area drives reward

Converging evidence shows that ventral tegmental area (VTA) dopamine neurons receive laterodorsal tegmental nucleus (LDTg) cholinergic and glutamatergic inputs. To test the behavioral consequences of selectively driving the two sources of excitatory LDTg input to the VTA, channelrhodopsin‐2 (ChR2) was expressed in LDTg cholinergic neurons of ChAT::Cre mice (ChAT‐ChR2 mice) or in LDTg glutamatergic neurons of VGluT2::Cre mice (VGluT2‐ChR2 mice). Mice were tested in a 3‐chamber place preference apparatus where entry into a light‐paired chamber resulted in VTA light stimulation of LDTg‐cholinergic or LDTg‐glutamatergic axons for the duration of a chamber stay. ChAT‐ChR2 mice spent more time in the light‐paired chamber and subsequently showed conditioned place preference for the light‐paired chamber in the absence of light. VGluT2‐ChR2 mice, entered the light‐paired chamber significantly more times than a light‐unpaired chamber, but remained in the light‐paired chamber for short time periods and did not show a conditioned place preference. When each entry into the light‐paired chamber resulted in a single train of VTA light stimulation, VGluT2‐ChR2 mice entered the light‐paired chamber significantly more times than the light‐unpaired chamber, but spent approximately equal amounts of time in the two chambers. VTA excitation of LDTg‐glutamatergic inputs may be more important for reinforcement of initial chamber entry while VTA excitation of LDTg‐cholinergic inputs may be more important for the rewarding effects of chamber stays. We suggest that LDTg‐cholinergic and LDTg‐glutamatergic inputs to the VTA each contribute to the net rewarding effects of exciting LDTg axons in the VTA.     

铜离子引起大鼠黑质多巴胺能神经元的退行性变

目的探讨黑质（substantia nigra,SN）内注射不同剂量的CuSO45H2O对大鼠黑质纹状体系统多巴胺能神经元的影响。方法实验用Wistar大鼠,分成对照组和左侧SN内分别注射10nmol、50nmol、200nmol CuSO。组,7天后采用高效液相色谱法（high performance lipid chromotophotography,HPLC）检测纹状体内多巴胺（dopamine,DA）及其代谢产物的含量;酪氨酸羟化酶（tyrosine hydroxylase,TH）免疫组织化学法检测纹状体内TH免疫阳性纤维的改变;半定量RT-PCR法检测黑质内TH,Caspase-3mRNA的表达量：用生化试剂盒分析大鼠中脑内超氧化物岐化酶（superoxide dismutase,SOD）活性的改变。结果在10nmol CuSO4注射组中,DA及其代谢产物的含量与对照组相比没有统计学差别。但是从50nmol组开始,损毁侧纹状体内DA含量随注射CuSO4剂量的增加而逐渐减少,显示出明显的剂量依赖关系（F=34．16,P〈0．01）。注射50nmol CuSO4组大鼠纹状体内TH免疫阳性纤维明显少于对照组和未损毁侧（F=121．9,P〈0．01）。注射50nmol CuSO4组大鼠SN内THmRNA的表达与对照组相比下降（t=3．12,P〈0．01）,但Caspase-3mRNA的表达量与对照组相比却明显增加（t=8．96,P〈0．01）。在注射50nmolCuSO4组中,大鼠损伤侧中脑内SOD的活性与对照组相比下降（t=2．33,P〈0．01）。结论铜离子可以导致大鼠黑质内多巴胺能神经元的损伤,该损伤作用可能是通过破坏抗氧化保护系统和促进细胞凋亡而实现的。