Adena L. Svingos

Electrophysiological Characterization of GABAergic Neurons in the Ventral Tegmental Area

GABAergic neurons in the ventral tegmental area (VTA) play a primary role in local inhibition of mesocorticolimbic dopamine (DA) neurons but are not physiologically or anatomically well characterized. We used in vivo extracellular and intracellular recordings in the rat VTA to identify a homogeneous population of neurons that were distinguished from DA neurons by their rapid-firing, nonbursting activity (19.1 ± 1.4 Hz), short-duration action potentials (310 ± 10 μsec), EPSP-dependent spontaneous spikes, and lack of spike accommodation to depolarizing current pulses. These non-DA neurons were activated both antidromically and orthodromically by stimulation of the internal capsule (IC; conduction velocity, 2.4 ± 0.2 m/sec; refractory period, 0.6 ± 0.1 msec) and were inhibited by stimulation of the nucleus accumbens septi (NAcc). Their firing rate was moderately reduced, and their IC-driven activity was suppressed by microelectrophoretic application or systemic administration of NMDA receptor antagonists. VTA non-DA neurons were recorded intracellularly and showed relatively depolarized resting membrane potentials (−61.9 ± 1.8 mV) and small action potentials (68.3 ± 2.1 mV). They were injected with neurobiotin and shown by light microscopic immunocytochemistry to be multipolar cells and by electron microscopy to contain GABA but not the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH). Neurobiotin-filled dendrites containing GABA received asymmetric excitatory-type synapses from unlabeled terminals and symmetric synapses from terminals that also contained GABA. These findings indicate that VTA non-DA neurons are GABAergic, project to the cortex, and are controlled, in part, by a physiologically relevant NMDA receptor-mediated input from cortical structures and by GABAergic inhibition.

Regulated endocytosis of opioid receptors: cellular mechanisms and proposed roles in physiological adaptation to opiate drugs.

Opiate drugs such as morphine and heroin are among the most effective analgesics known. Prolonged or repeated administration of opiates produces adaptive changes in the nervous system that lead to reduced drug potency or efficacy (tolerance), as well as physiological withdrawal symptoms and behavioral manifestations such as craving when drug use is terminated (dependence). These adaptations limit the therapeutic utility of opiate drugs, particularly in the treatment of chronically painful conditions, and are thought to contribute to the highly addictive nature of opiates. For many years it has been proposed that physiological tolerance to opiate drugs is associated with a modification of the number or functional activity of opioid receptors in specific neurons. We now understand certain mechanisms of opioid receptor desensitization and endocytosis in considerable detail. However, the functional roles that these mechanisms play in the complex physiological adaptation of the intact nervous system to opiates are only beginning to be explored.

μ Opiate receptor immunoreactivity in rat central nervous system

Immunoreactivity corresponding to the C-terminus of the rat μ opiate receptor can be detected by light microscopy in fiber- and terminal-like patterns in a number of rat brain and spinal cord regions, and in immunoreactive perikarya in several of these regions. Especially abundant fiber- and terminal-like patterns were localized to superficial layers of the spinal cord dorsal horn and nucleus caudalis of the spinal tract of the trigeminal, the nucleus of the solitary tract, nucleus ambiguous, locus coeruleus, interpeduncular nucleus, medial aspect of the lateral habenular nucleus, presumed “striasomes” of the caudate-putamen and nucleus accumbens. Moderate fiber and terminal densities were found in the ventral tegmental area, more medial aspects of the thalamus and hypothalamus, and several amygdaloid nuclei. Immunostained perikarya were prominent in the nucleus accumbens and also observed in the middle layers of the cerebral cortex, septum and diagonal band, preoptic area, medial thalamic and habenular nuclei, locus coeruleus, nucleus ambiguous, nucleus of the solitary tract, trigeminal nucleus caudalis and spinal cord substantia gelatinosa zones. Many of these localizations correspond well with the previously-determined autoradiographic distributions of μ opiate receptor ligand binding, and with reports of μ opiate receptor immunoreactivity determined using other antisera. Electron microscopic immunohistochemical studies reveal details of the membrane distribution of the μ receptor in nucleus accumbens, caudate/putamen, locus coeruleus, and spinal cord. These results suggest largely neuronal and largely extrasynaptic distributions of μ receptors that show differential patterns of perikaryal, dendritic, and/or axonal immunostaining in different central nervous system zones. Identification of these distributions adds substantially to data identifying the cellular localization of the principal opiate receptor involved in both analgesic and addictive processes.

Localization of the δ-opioid receptor and dopamine transporter in the nucleus accumbens shell: Implications for opiate and psychostimulant cross-sensitization

Opiate‐ and psychostimulant‐induced modulation of dopamine transmission in the nucleus accumbens shell (AcbSh) is thought to play a key role in their potent reinforcing and locomotor effects. To investigate the cellular basis for potential functional interactions involving opiates active at the δ‐opioid receptor (DOR) and psychostimulants that bind selectively to the dopamine transporter (DAT), we examined the electron microscopic localization of their respective antisera in rat AcbSh. DOR immunoperoxidase labeling was seen primarily, and DAT immunogold particles exclusively, in axon terminals. In these terminals, DOR immunoreactivity was prominently associated with discrete segments of the plasma membrane and the membranes of nearby small synaptic and large dense core vesicles. DAT immunogold particles were almost exclusively distributed along nonsynaptic axonal plasma membranes. Thirty‐nine percent DOR‐labeled profiles (221/566) either apposed DAT‐immunoreactive terminals or also contained DAT. Of these 221 DOR‐labeled profiles, 13% were axon terminals containing DAT and 15% were dendritic spines apposed to DAT‐immunoreactive terminals. In contrast, 70% were morphologically heterogeneous axon terminals and small axons apposed to DAT‐immunoreactive terminals. Our results indicate that DOR agonists in the AcbSh can directly modulate the release of dopamine, as well as postsynaptic responses in spiny neurons that receive dopaminergic input, but act principally to control the presynaptic secretion of other neurotransmitters whose release may influence or be influenced by extracellular dopamine. Thus, while opiates and psychostimulants mainly have differential sites of action, cross‐sensitization of their addictive properties may occur through common neuronal targets. Synapse 34:1–10, 1999.

Presynaptic dopamine D4 receptor localization in the rat nucleus accumbens shell

Dopamine D4 receptors in the nucleus accumbens shell (AcbSh) are thought to play a key role in mediating the locomotor and sensitizing affects of psychostimulants, as well as the therapeutic efficacy of atypical antipsychotic drugs. We used electron microscopic immunocytochemistry to determine the functional sites for endogenous and exogenous D4 receptor activation in this region. Of 1,090 D4 receptor‐labeled profiles observed in the AcbSh of rat brain, 65% were axons and axon terminals, while 22% were dendrites and dendritic spines. Within axons and terminals, D4 receptor immunoreactivity was localized to segments of the plasma membrane and membranes of nearby vesicles. The axon terminals were morphologically heterogenous, varying in size and content of either all small synaptic vesicles (ssv), or ssv and large dense‐core vesicles. The labeled terminals occasionally formed asymmetric excitatory‐type axospinous synapses, but the majority were without recognizable synaptic specializations. In a separate series of tissue sections that were processed for dual‐labeling of the D4 receptor and the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH), 56% of all observed associations were appositions between differentially labeled axonal profiles, and 17% were terminals that contained immunoreactivity for both antigens. Dendritic spines containing D4 receptor‐labeling also received convergent input from TH‐immunoreactive terminals and unlabeled terminals forming asymmetric synapses. These results provide the first ultrastructural evidence for a major presynaptic, and a more minor postsynaptic, involvement of D4 receptors in dopaminergic modulation of excitatory transmission in the AcbSh. Synapse 36:222–232, 2000.

Ultrastructural localization of δ-opioid receptor and Met5-enkephalin immunoreactivity in rat insular cortex

The insular cortex has been implicated in the reinforcing properties of opiates as well as in the integration of responses to sensory-motor stimulation. Moreover, the delta-opioid receptor (DOR) and the endogenous opioid ligand, Met5-enkephalin (ENK) are known to be prominently distributed in insular limbic cortex. To examine the anatomical sites for opioid activation of DOR in rat insular cortex, we used immunoperoxidase for detection of an antiserum raised against a peptide sequence unique to the DOR alone, and in combination with immunogold-silver labeling for ENK. Light microscopy showed intense DOR-like immunoreactivity (DOR-LI) in pyramidal cells and interneurons in deep laminae, and in varicose processes in both superficial and deep layers of the insular cortex. Ultrastructural analysis of layers V and VI in insular cortex showed that the most prominent immunoperoxidase labeling for DOR was in dendrites. This labeling was associated with asymmetric excitatory-type junctions postsynaptic to unlabeled terminals. Dendritic DOR-LI was also distributed along selective portions of non-synaptic plasma membranes and subsurface organelles. In dually labeled sections, dendrites containing DOR-LI sometimes received synaptic input from ENK-labeled terminals or more infrequently colocalized with ENK. Other axon terminals were exclusively immunolabeled for DOR or more rarely contained both DOR and ENK immunoreactivity. Within labeled axon terminals, distinct segments of the plasma membrane and membranes of immediately adjacent synaptic vesicles showed the largest accumulation of the peroxidase reaction product for DOR. These results indicate that in rat insular cortex DOR is primarily heteroreceptive, but also serves an autoreceptive function on certain ENK-containing neurons. Our results also provide the first ultrastructural evidence that in rat insular cortex endogenous opioids interact through the DOR (1) to modulate the postsynaptic responses to other excitatory afferents and (2) to presynaptically regulate the release of other neurotransmitters. The modulatory actions on both ENK-containing and non-ENK-containing neurons may contribute significantly to the reinforcing properties of exogenous opiates acting on the DOR in limbic cortex.

κ-Opioid and NMDA glutamate receptors are differentially targeted within rat medial prefrontal cortex

Activation of κ-opioid receptors (KOR) in the medial prefrontal cortex (mPFC) modulates excitatory transmission, which may involve interactions with N-methyl-d-aspartate (NMDA) glutamate receptors. We investigated possible anatomical correlates of this modulation by using dual labeling electron microscopy to examine the cellular distributions of antibodies raised against KOR and the R1 subunit of the NMDA receptor (NR1). KOR immunoreactivity primarily was localized to plasma and vesicular membranes of axons and axon terminals that were morphologically heterogeneous. A small proportion of KOR immunoreactivity was associated with cytosolic compartments of dendrites and membranes of glial processes. NR1 labeling was mainly postsynaptic, associated most often with membranes of cytoplasmic organelles in cell bodies and large dendrites and plasmalemmal surfaces of distal dendrites. The remaining NR1-labeled profiles were axonal profiles and glial processes. Of all cellular associations between labeled profiles, the majority were KOR-labeled axons that contacted NR1-immunoreactive dendrites or cell bodies. Occasionally the two antigens were colocalized in axon terminals that formed either asymmetric synapses or displayed varicose morphology. KOR and NR1 also were colocalized within dendrites, and rarely were observed in the same cell bodies. Occasionally glial processes coursing adjacent to axo-spinous appositions expressed both KOR and NR1 immunoreactivity. These results indicate that ligand activation of KOR or NMDA receptors differentially modulates excitatory transmission in the mPFC through pre- and postsynaptic mechanisms, respectively. The data also suggest more minor roles for colocalized KOR and NMDA receptors in shared regulation of presynaptic transmitter release, postsynaptic responsivity, and glial function.

Topographical distribution and synaptic localization of δ-opiate receptor immunoreactivity in rat prefrontal cortex

The rewarding effects of opioid-induced euphoria are thought to be mediated through dopiate receptors in cortical-limbic structures. We utilized antipeptide antisera shown to be specific for the d-opiate receptor to determine whether the cortical topographical or ultrastructural localization could demonstrate a potential cellular substrate for these actions. Guinea pig polyclonal antisera raised against a amino acid fragments from the d-opiate receptor were used for immunocytochemical examination. Detailed analysis was carried out using one extracellular fragment selectively found in the cloned delta receptor. Morphologically heterogeneous cell body labeling was seen in laminae I, II, III, V, VI, with deep laminae pyramidal and interneurons being the most heavily labeled. In contrast to other cortical areas, limbic associated prefrontal cortex was distinguished by a lack of superficial laminar labeling, heavier deep laminar labeling and sparse to moderate staining of varicosityladen fibers. Electron microscopic examination of deep insular cortex showed diffuse labeling within dendrites. However, the most intense localization was seen at selective postsynaptic densities on smaller dendrites and spines. Dense reaction product also was seen on plasma membranes and vesicles of numerous unmyelinated axons and terminals. The distinctive distribution of d-opiate receptor immunoreactivity observed in limbic prefrontal cortex may provide anatomical and cellular substrata for the reinforcing effects of opioid use.

Presynaptic location of δ-opioid receptor immunoreactivity in dorsal horn of the rat spinal cord

Abstract Endogenous opioid peptides, Met5- and Leu5-enkephalin elicit many physiological effects in the CNS through activation of δ-opioid receptors. To determine the potential cellular sites for the δ-opioid receptor mediated opioid actions in nociception, we examined the ultrastructural immunoperoxidase localization of guinea pig antisera against a peptide sequence located within the extracellular region of the cloned δ-opioid receptor, corresponding to amino acids 34–47 (p34). Additionally, we combined the labeling of this peptide with a rabbit anti Met5-enkephalin. Light microscopy showed a selective localization of δ-opioid receptor-like immunoreactivity (δ-ORLI) in layers I and II of the rat cervical spinal cord. This labeling was removed by preadsorption with excess peptide. Ultrastructural localization of the antibody revealed the most prominent localization along the plasma membrane and dense core vesicles along unmyelinated axons and axon terminals. However, selective postsynaptic junctions were also labeled. In sections dually labeled for the δ-opioid receptor (peroxidase) and Met5-enkephalin(immunogold/silver), δ-ORLI was present in axon terminals both with and without detectable Met5-enkephalin (ME). In addition, δ-ORLI was observed at the synaptic specializations on dendrites, postsynaptic to axon terminals containing ME. These results provide the first ultrastructural evidence for a prominent presynaptic, but also postsynaptic, action of Met5-enkephalin acting at δ-opioid receptors in nociceptive regions of the rat spinal cord.

Dopamine D4 Receptors Are Strategically Localized for Primary Involvement in the Presynaptic Effects of Dopamine in the Rat Nucleus Accumbens Shell

Clozapine, an atypical neuroleptic, ameliorates the symptoms of schizophrenia without causing the motor impairments associated with typical antipsychotic drugs.4 Clozapine also has a relatively higher affinity for D4 receptors, as compared to other dopamine receptors.9 The therapeutic efficacy of clozapine may be associated with D4 receptor-mediated modulation of dopamine transmission in distinct anatomical areas, including the nucleus accumbens shell (AcbSh).8 The present study used immunoperoxidase labeling of the D4 receptor and immunogold-silver labeling of tyrosine hydroxylase (TH) to examine (1) the cellular sites for D4 receptor activation, and (2) whether D4 receptors are localized to modulate the presynaptic release of dopamine in this brain region.