Recording of mouse ventral tegmental area dopamine-containing neurons

We examined the electrophysiologic and pharmacologic properties of dopamine-containing ventral tegmental area neurons in the mouse using extracellular single-unit recording techniques in both chloral hydrate-anesthetized mice and in vitro mouse midbrain slices. In vivo the ventral tegmental area neurons had long-duration action potentials (2 to 5 ms) and discharged at 1 to 9 spikes/s with either a decremental burst pattern or a regular pattern. Systemic administration of the dopamine agonist, apomorphine, decreased their firing rate, and the dopamine receptor blocker, haloperidol, reversed this effect. Similarly, systemic administration of the dopamine-releasing agent, d-amphetamine, suppressed their discharge rate, an effect blocked by pretreatment of the animals with alpha-methyl-p-tyrosine. When recorded in vitro from midbrain slices, ventral tegmental area neurons showed electrophysiologic properties similar to those found in vivo; however, the neurons recorded in vitro fired at a significantly faster rate and their firing pattern tended to be more pacemaker-like, especially when recordings were made in an incubation medium that blocked synaptic transmission (i.e., low calcium/high magnesium). The activity of most of these neurons was suppressed by addition of apomorphine to the incubation medium, an effect reversed by haloperidol. Pretreatment with alpha-methyl-p-tyrosine produced no significant change in the discharge pattern or rate for cells recorded in vitro. These data indicate that mouse ventral tegmental area dopamine neurons in vivo exhibit the same electrophysiologic and pharmacologic properties as do rat and cat dopamine-containing neurons and that in vitro they fire with pacemaker regularity in a low-calcium/high-magnesium medium. The in vitro preparation offers an approach to examining the fundamental properties of ventral tegmental area dopamine-containing neurons in the absence of afferent inputs.     

Functional properties of presumed dopamine-containing and other ventral tegmental area neurons in conscious rats

To elucidate the functional significance of mesolimbocortical dopamine (DA)-containing neurons in animal adaptive activity, the properties of single units in ventral tegmental area (VTA) and adjacent regions of the midbrain were studied in conscious rats with strictly fixed skull. Analysis of spontaneous firing activity, its changes during polymodal activating and aversive stimulations and their interrelations was performed in electrophysiologically-identified presumed DA-containing (D-type, 48 cells) and other (A-B and C-type, 47 and 29 cells accordingly) neurons found in this brain area. A common feature of all cells was the dependence of their discharge changes on the biological significance of the stimulation used and the strong correlation between these firing changes and stimulation-induced or spontaneous movement activity and blood pressure oscillations. Moreover, a significant correlation between the rate of firing and its dispersion and constancy of directions of neuronal changes during experimental stimulation in single cells were found. Presumed DA-containing neurons of D-type had a high variability of all properties and heterogeneity in the pattern of their discharges and in direction changes (prevalent activations). In presumed acetylcholine (ACh)-containing A-B type cells strong tonic-like activations and in presumed GABA- or ACh-containing interneurons of C-type depressions of firing both correlated with animal movement activity were found. Present data were discussed in relation with mediator specifity of studied cells and the differences of their participation in avoidance behavior forming in aversive environment.     

Stimulation of the lateral habenula inhibits dopamine-containing neurons in the substantia nigra and ventral tegmental area of the rat

Neurons in the lateral habenula (LHb) of rats have efferent projections that terminate in the substantia nigra pars compacta (SNC) and ventral tegmental area (VTA), where cell bodies of dopamine-containing neurons are located. In order to study the influence of the habenula on dopaminergic activity, single-cell electrophysiological techniques were used to record unit discharge of dopamine-containing neurons in the SNC and VTA during electrical stimulation of the LHb or adjacent structures. Dopamine-containing neurons in the SNC and VTA were identified by their characteristic spike duration (greater than 2 msec), discharge rate (2-8 spikes/sec), and irregular firing pattern. Analysis of peristimulus time histograms showed that 85% of SNC cells and 91% of VTA neurons were inhibited after single pulse stimulation (0.25 mA, 0.1 msec) of the LHb. The mean time between stimulation and onset of inhibition was 11 msec (range, 2-22 msec) and mean duration of maximal suppression was 76 msec (range, 20-250 msec). Stimulation of structures adjacent to the LHb (hippocampus, lateral thalamus, medial dorsal thalamus, medial habenula) had little or no effect. Destruction of the fasciculus retroflexus, the fiber pathway that contains most habenular efferents, blocked the stimulation effects on dopamine-containing neurons. Destruction of the stria medullaris, which contains most habenular afferents, did not alter the inhibitory effect of habenular stimulation. Injection of a cytotoxin, kainic acid, in the LHb 1 week before recording sessions blocked the inhibitory consequences of habenular stimulation. These experiments show that activation of neuronal perikarya in the LHb causes orthodromic inhibition of dopamine-containing neurons in SNC and VTA via the fasciculus retroflexus.     

Responses of ventral tegmental area GABA neurons to brain stimulation reward

Dopamine neurons in the ventral tegmental area (VTA) have been implicated in rewarded behaviors, including intracranial self-stimulation (ICSS). We demonstrate, in unrestrained rats, that the discharge activity of a homogeneous population of presumed VTA GABA neurons, implicated in cortical arousal, increases before ICSS of the medial forebrain bundle (MFB). These findings suggest that VTA GABA neurons may be involved in the attentive processes related to brain stimulation reward (BSR).     

Intracellular recording of lamina X neurons in a horizontal slice preparation of rat lumbar spinal cord

A horizontal slice preparation of postnatal rat lumbar spinal cord has been developed which allows correlative observations of the morphology, electrophysiology, and receptor pharmacology of lamina X neurons. These slices better maintain afferent input and somatodendritic morphology and are amenable to subsequent immunohistochemical processing. Stable intracellular recordings obtained from postnatal day 14–45 animals reveal that a number of different intrinsic membrane conductances contribute to the regulation of excitability in lamina X neurons. In addition, lamina X neurons possess inhibitory GABAergic as well as excitatory glutamate and cholecystokinin receptors. This preparation will be useful in future studies designed to characterize developmental changes in the intrinsic membrane properties, synaptic profiles and neuropeptide responsiveness of lamina X neurons in the rat. Such a characterization is important given that lamina X represents a unique sexually dimorphic region that is a convergence site for somatic and visceral afferent inputs, which includes nociceptive information.     

Inhibition from ventral tegmental area of nucleus accumbens neurons in the rat

The role of the ventral tegmental area (VTA), which is rich in dopamine-containing cell bodies, on nucleus accumbens (Acc) neurons was examined. In Acc neurons receiving input from parafascicular nucleus (Pf) of thalamus, VTA conditioning stimulation produced an inhibition of spike generation with Pf stimulation. In contrast, VTA conditioning stimulation did not affect Acc neurons receiving input from limbic structures such as the amygdala nucleus and hippocampus.     

Neurotransmitter regulation of dopamine neurons in the ventral tegmental area

Over the last 10 years there has been important progress towards understanding how neurotransmitters regulate dopaminergic output. Reasonable estimates can be made of the synaptic arrangement of afferents to dopamine and non-dopamine cells in the ventral tegmental area (VTA). These models are derived from correlative findings using a variety of techniques. In addition to improved lesioning and pathway-tracing techniques, the capacity to measure mRNA in situ allows the localization of transmitters and receptors to neurons and/or axon terminals in the VTA. The application of intracellular electrophysiology to VTA tissue slices has permitted great strides towards understanding the influence of transmitters on dopamine cell function, as well as towards elucidating relative synaptic organization. Finally, the advent of in vivo dialysis has verified the effects of transmitters on dopamine and γ-aminobutyric acid transmission in the VTA. Although reasonable estimates can be made of a single transmitter's actions under largely pharmacological conditions, our knowledge of how transmitters work in concert in the VTA to regulate the functional state of dopamine cells is only just emerging. The fact that individual transmitters can have seemingly opposite effects on dopaminergic function demonstrates that the actions of neurotransmitters in the VTA are, to some extent, state-dependent. Thus, different transmitters perform similar functions or the same transmitter may perform opposing functions when environmental circumstances are altered. Understanding the dynamic range of a transmitter's action and how this couples in concert with other transmitters to modulate dopamine neurons in the VTA is essential to defining the role of dopamine cells in the etiology and maintenance of neuropsychiatrie disorders. Further, it will permit a more rational exploration of drugs possessing utility in treating disorders involving dopamine transmission.     

Organization of the projections from the ventral tegmental area of Tsai to the hippocampal formation in the rat.

The projection from the ventral tegmental area of Tsai (VTA-A10) to the hippocampal formation (HF) has been investigated in the rat by means of the Fink-Heimer technique, after VTA destruction by electrolytic lesions or local injections of 6-hydroxydopamine (6-OHDA, 1 microgram/0.5 microliters). Degenerated axons prevail in the ventral subiculum and adjacent CA1 field. Some degenerated fibers are also observed in the dorsal subiculum and a few in the stratum oriens of the CA3, in the hilus of fascia dentata and in the fimbria. The distribution of VTA neurons projecting to the HF has also been examined by injecting retrograde fluorescent tracers in different combinations (Fast Blue, 2% and Nuclear Yellow, 1%) in several hippocampal areas. The most abundant VTA-HF projections originate from the lower third, the upper and lower edges and the lower half of the VTA. The major terminal fields of VTA projections in the HF (i.e. the ventral subiculum, the adjacent CA1 field and the dorsal subiculum) match with the HF area projecting to the nucleus accumbens. Thus, the dopaminergic meso-limbic pathway could modulate the HF-striatal projection which provides a link between the limbic and motor systems.     

The cytoarchitecture of the interfascicular nucleus and ventral tegmental area of Tsai in the rat.

The cytoarchitecture of the ventral tegmental area of Tsai (VTA) has been studied in detail in the rat with the acid of both conventional techniques and glyoxylic acid-fluorescence histochemistry. Three main dopamine containing cells groups can be distinguished: nucleus paranigralis, nucleus parabrachialis pigmentosus, nucleus linearis raphe caudalis. Nucleus parabrachialis pigmentosus lies dorsally, and nucleus paranigaralis ventrally in the VTA, while nucleus linearis continues posteriorly and medially from VTA and extends dorsally in the midline up towards the dorsal raphe nucleus. Fluorescent neurons in these three groups correspond to the A10 group. In addition to these previously described findings, the present study shows evidence for further small but cytoarchitecturally distinct dopaminergic group called the interfascicular necleus. This lies anteriorly and ventrally in the ventral tegmentum in the midline, dorsal to the rostral portion of the interpeduncular nucleus and interpeduncular fossa. The significance of these cytoarchitectural findings is discussed in relation to the known connections of the region.     

A Golgi study of the ventral tegmental area of Tsai and interfascicular nucleus in the rat

The ventral tegmental area of Tsai (VTA) and interfascicular nucleus of the adult rat brain has been studied with two variants of the Golgi method in three planes of section. Neurons were studied in relation to the cytoarchitectural groupings of the VTA. Dendritic organisation and dendritic fields were mapped out for each cytoarchitectural subgroup and cell types within each subgroup were classified on the basis of cell size and dendritic morphology. In each subnucleus of VTA, neurons had distinct characteristics. In nucleus paranigralis neurons were small to medium in size and their dendritic fields organised in an approximately horizontal plane orientated in an anteromedial direction and slanting dorsally over the interpeduncular nucleus and fossa. Neurons of the parabrachial group were small to medium sized with no preferential orientation. In nucleus linearis raphe caudalis small neurons were strongly orientated in the plane of the nucleus in a dorso-ventral direction slanting forwards. Neurons in the interfascicular group were small to very small and their maximum dendritic extents were seen in the horizontal plane. In frontal section they formed a compact ball of cells in the midline and were separated on either side from the larger neurons in the medial edge of nucleus paranigralis. Ingeneral VTA neurons tended to fall into one of two morphological categories. Type 1 were small to medium, and had two to four primary dendrites which divided into varicose secondary dendrites. Type 2 were medium sized, with two to five primary dendrites. Both primary and secondary dendrites and the cell soma of Type 2 neurons were moderately spiny. Secondary dendrites were not varicose. Forms also occurred which were intermediate between Types 1 and 2. In the nucleus paranigralis, Type 1 was more common medially, while Type 2 was more common laterally, particularly in the ventrolateral paranigral region. Only neurons of Type 1 were seen in nucleus linearis raphe and interfascicular nuclei. Local axon circuits were observed to arise from the primary dendrites of Type 1 neurons and to ramify close to neighbouring neurons. Axon swellings from such circuits were observed to make apparent contact with primary dendrites of nearby neurons and clusters of axon swellings were observed near cell somas of neighbouring impregnated neurons of similar type. The results are discussed and particular attention is paid to the similarities and differences between VTA and the substantia nigra pars compacts (SNC). The major difference appears to be that, whereas in SNC dendrites are organised in vertical as well as horizontal planes, in the VTA no long ventrally directed dendrites were observed. Combining these results with the known cytoarchitecture and connections of VTA and SNC, it appears that fundamental differences occur between VTA and some neurons of the SNC, both in the nature of their morphology and intrinsic organisation, and in the organisation of their efferent and afferent connections.     

Glycine-activated chloride currents of neurons freshly isolated from the ventral tegmental area of rats

Properties of whole-cell glycine currents (I_Gly) of ventral tegmental area (VTA) neurons from 3- to 7-day old Sprague–Dawley rats were investigated with the patch-clamp technique. Ninety-three percent of the 126 neurons examined produced I_Gly in response to glycine. For 70% of these neurons, I_Gly did not decay in response to a threshold concentration of glycine (1–5 μM). At elevated glycine concentrations, I_Gly consistently decayed from a peak to a steady state (SS). I_Gly increased in amplitude sigmoidally as a function of the concentration of agonist with an EC₅₀ of 32 μM. Strychnine (STR), when co-applied with glycine after a prepulse of STR, suppressed both the peak and SS I_Gly noncompetitively. In the absence of a prepulse, STR had a smaller effect on peak I_Gly while increasing its decay rate; the SS amplitude decreased. These STR effects were concentration dependent with an IC₅₀ of 31 nM and 184 nM STR for the peak and SS I_Gly, with prepulse, respectively, and 732 nM and 193 nM for the peak and SS I_Gly, respectively, without prepulse. Picrotoxin (PTX) co-applied with glycine suppressed both the peak and the SS I_Gly with an IC₅₀ of 25 μM. In contrast to STR, 1 min preincubation with PTX had no effect on I_Gly. Thus, PTX acts on the open channel. The inhibitory effects of both STR and PTX on I_Gly did not depend on the membrane potential.     

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.     

TPH2 in the ventral tegmental area of the male rat brain

This study surveyed the distribution of tryptophan hydroxylase 2 (TPH2) mRNA, protein, and enzymatic activity throughout the male Sprague-Dawley rat brain.TPH2 is the genetic isoform of TPH that catalyzes the rate-limiting step in serotonin biosynthesis within the central nervous system. Although cell bodies of serotonergic neurons are located mainly in the raphe, serotonin-containing axons innervate many regions of the brain. In the present study, we assessed the levels of mRNA, protein expression, and enzyme activity of TPH2 in the rat raphe, ventral tegmental area (VTA), substantia nigra, hippocampus, cerebellum, dorsal striatum, nucleus accumbens, amygdala, and medial prefrontal cortex to more fully understand the distribution of this enzyme throughout the central nervous system. The pineal gland was used as a control tissue that expresses TPH1 (the peripheral enzyme), but not TPH2. As expected, the raphe showed the highest brain TPH2 activity and protein expression. In the contrast to other reports, however, the VTA followed the raphe as the region with the second-highest amount of TPH2 activity, mRNA and protein expression. There were significantly lower TPH activities and levels of TPH2 protein in the other regions. In addition, TPH2 immunocytochemistry demonstrated the presence of TPH-positive cell bodies within the VTA. The results of this study indicate that TPH2 and serotonergic signaling may play an important role in the mesolimbic/mesocortical reward pathway.     

Neurotensin neurons in the ventral tegmental area project to the medial nucleus accumbens

Opiate receptors measured in vitro or in vivo with [3H]lofentanil in the rat vagus nerve were found to accumulate on both sides of a ligature, thus indicating a bidirectional axoplasmic transport of these receptors. When rats were treated with capsaicin, the accumulation of opiate receptors was tremendously reduced in the vagus whereas muscarinic receptors in ligated sciatic nerves were unaffected. Since capsaicin is known to affect sensory neurones, mostly those containing substance P, the present results support the idea that the opiate receptors in the vagus are associated with substance P neurones.     

GABA-containing neurons in the rat ventral tegmental area project to the prefrontal cortex

Dopamine-containing projections from the ventral tegmental area (VTA) to the prefrontal cortex (PFC) have been extensively characterized since their discovery over 25 years ago. However, the VTA projection to the PFC also contains a substantial nondopamine component, whose neurochemical phenotype is unknown. To examine if a portion of this nondopamine VTA projection contains GABA, anterograde and retrograde tract-tracing in the rat was combined with GABA immunocytochemistry and electron microscopy. Following injections of Fluoro-Gold (FG) into the PFC, many VTA neurons were retrogradely labeled, as visualized by immunoperoxidase staining for FG. A large portion of FG-labeled somata (58%) and dendrites (33%) within the VTA also contained immunogold-silver labeling for GABA. These dually labeled profiles exhibited a morphology similar to dopamine-containing cells within the VTA. To confirm and extend these findings, anterograde transport of biotinylated dextran amine (BDA) from the VTA was combined with immunogold-silver labeling for GABA within the PFC. Consistent with the results obtained from retrograde tracing, a portion of BDA-labeled terminals in the PFC also contained immunoreactivity for GABA. These dually labeled terminals formed symmetric synapses onto small caliber dendrites and dendritic spines. Some PFC dendrites contacted by GABA-containing VTA terminals were themselves GABA-labeled. The results of this investigation have identified a substantial population of GABA-containing neurons in the VTA that send axons to the PFC where they synapse on the distal processes of both pyramidal and local circuit neurons. This GABA-containing mesocortical pathway may provide substrates for both inhibitory and disinhibitory influences on PFC neuronal activity.     

Afferent projections to the ventral tegmental area of Tsai and interfascicular nucleus: a horseradish peroxidase study in the rat.

Using the retrograde transport of horseradish peroxidase (HRP), a study has been made of projections to the ventral tegmental area of Tsai (VTA) and related dopaminergic cell groups (A 10). In order to minimise the possibility of damage to fibres of passage, a technique was evolved for the microiontophoresis of HRP such that minimal current strengths and durations were applied. In addition to a sham injection, control injections were also made to the medial lemnisuc, red nucleus, deep tegmental decussations, mesencephalic reticular formation and brachium conjunctivum. Following HRP injections confined to the areas of the VTA containing the dopamine cell groups, labelled neurons appeared in prefrontal cortex, dorsal bank of rhinal sulcus, nucleus accumbens, bed nucleus of stria terminalis, amygdala, diagonal band of Broca, substantis innominata, magnocellular preoptic area, medial and lateral preoptic areas, anterior, lateral and postero-dorsal hypothalamus, lateral habenular, nucleus parafascicular nucleus of thalamus, superior colliculus, nucleus raphe dorsalis, nucleus raphe nagnus and pontis, dorsal and ventral parabrachial nuclei, locus coeruleus and deep cerebellar nuclei. Regions containing catecholamine groups A 1, A 5, A 6, A 7, A 9, A 13 and the serotonin group B 7 corresponded to the topography of labeled cell groups. Injections of HRP to the interfascicular nucleus resulted in labeling predominantly confined to the medial habenular and median raphe nuclei. The results are discussed in relation to the known connections of these regions. Other regions of the brain labelled by VTA injections are assessed in relation to control injections and the limitations of the HRP technique. A review of the organisation of some of these afferents in relation to the known cortical-subcortical-mesencephalic projection systems, suggests that the VTA is in a position to recieve information from a massively convergent system derived ultimately from the entire archi-, paleo-, and neo-cerebral cortices. In addition A 10 dopaminergic neurons are known to project to restricted regions of both pre-frontal and entorhinal cortices, which themselves also recieve massively convergent association cortico-cortical connections. It would appear reasonable to propose that these neurons perform a correspondingly important integrative function.     

Dopamine D-1 receptor-mediated inhibition of nucleus accumbens neurons from the ventral tegmental area.

1. Spike generation by stimulation of the parafascicular nucleus of thalamus was extracellularly recorded in the nucleus accumbens of chloral hydrate-anesthetized adult Wistar rats using a silver-wire microelectrode attached along a seven-barreled micropipette, each of which was filled with dopamine, SKF 38393 (D-1 agonist), bromocriptine (D-2 agonist), haloperidol, SCH 23390 (D-1 antagonist) and domperidone (D-2 antagonist). The drugs were microiontophoretically applied to the target neurons recorded. 2. Effects of dopamine receptor antagonists on the inhibition of the spike generation by conditioning stimuli applied to the ventral tegmental area preceding the test stimulus to the parafascicular nucleus and those of dopamine agonists on the test stimulus-induced spikes were examined. 3. The parafascicular nucleus stimulation-induced spikes were inhibited by dopamine as well as D-1 and D-2 agonists and by the conditioning stimulation of the ventral tegmental area. The conditioning stimulation-induced inhibition was antagonized by haloperidol and SCH 23390, but not by domperidone. 4. Activation of D-1 receptors, which make probably synaptic contact with dopaminergic nerve terminals from the ventral tegmental area, is considered to result in inhibition of the neuronal activity of the nucleus accumbens neurons receiving input from the parafascicular nucleus of the thalamus. In addition, D-2 receptors located extrajunctionally may be involved in the inhibition of the same neurons in the nucleus accumbens.     

Topographical organization of GABAergic neurons within the ventral tegmental area of the rat

The ventral tegmental area (VTA), the origin of dopaminergic cell bodies that comprise the mesocorticolimibic DA system, is widely implicated in drug and natural reward, cognition, and several psychiatric disorders. In addition to dopaminergic neurons, this region is populated by GABAergic neurons, which both regulate the firing of their dopaminergic counterparts and send projections throughout the brain. Although the dopaminergic neurons of the VTA have been extensively characterized neuroanatomically, much less is known about the GABAergic neurons in this region. Recent data suggest that the rostro-caudal topographic organization of these GABAergic neurons may correspond to their ability to regulate drug reward. In the present study, we used immunohistochemical techniques to examine the frequency and topography of GABAergic neurons throughout the rostro-caudal axis of the VTA and the extent to which they coexpress other proteins, including tyrosine hydroxylase (a marker of DA neurons), cholecystokinin, parvalbumin, calretinin, and calbindin d 28k.