Scott C. Steffensen

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.

Ventral Tegmental Area BDNF Induces an Opiate-Dependent–Like Reward State in Naïve Rats

BDNF and Drug Dependence Brain-derived neurotrophic factor (BDNF) is a growth factor involved in neuronal plasticity that is expressed after chronic administration of drugs of abuse and may play a crucial role in chronic opiate effects. Vargas-Perez et al. (p. 1732, published online 28 May) found that BDNF was directly involved in the switching mechanism in the ventral tegmental area, from an opiate-naïve, dopamine-independent drug reward substrate to an opiate-dependent, dopamine-dependent motivational substrate. In the ventral tegmental area, BDNF changed the action of GABA-A receptors from inhibitory to excitatory. This change led to behavioral changes that defined a neurobiological boundary between the acute phase of drug-taking and addiction. A growth factor involved in neuronal plasticity alters neurons in a specific area of the brain after chronic exposure to opioid drugs. The neural mechanisms underlying the transition from a drug-nondependent to a drug-dependent state remain elusive. Chronic exposure to drugs has been shown to increase brain-derived neurotrophic factor (BDNF) levels in ventral tegmental area (VTA) neurons. BDNF infusions into the VTA potentiate several behavioral effects of drugs, including psychomotor sensitization and cue-induced drug seeking. We found that a single infusion of BDNF into the VTA promotes a shift from a dopamine-independent to a dopamine-dependent opiate reward system, identical to that seen when an opiate-naïve rat becomes dependent and withdrawn. This shift involves a switch in the γ-aminobutyric acid type A (GABAA) receptors of VTA GABAergic neurons, from inhibitory to excitatory signaling.

Structural and functional neuropathology in transgenic mice with CNS expression of IFN-α1

Abstract Cytokines belonging to the type I interferon (e.g. interferon-α) family are important in the host response to infection and may have complex and broad ranging actions in the central nervous system (CNS) that may be beneficial or harmful. To better understand the impact of the CNS expression of the type I interferons (IFN), transgenic mice were developed that produce IFN-α1 chronically from astrocytes. In two independent transgenic lines with moderate and low levels of astrocyte IFN-α mRNA expression respectively, a spectrum of transgene dose- and age-dependent structural and functional neurological alterations are induced. Structural changes include neurodegeneration with loss of cholinergic neurons, gliosis, angiopathy with mononuclear cell cuffing, progressive calcification affecting basal ganglia and cerebellum and the up-regulation of a number of IFN-α-regulated genes. At a functional level, in vivo and in vitro electrophysiological studies revealed impaired neuronal function and disturbed synaptic plasticity with pronounced hippocampal hyperexcitability. Severe behavioral alterations were also evident in higher expressor GFAP-IFNα mice which developed fatal seizures around 13 weeks of age precluding their further behavioral assessment. Modest impairments in discrimination learning were measured in lower expressor GFAP-IFNα mice at various ages (7–42 weeks). The behavioral and electrophysiological findings suggest regional changes in hippocampal excitability which may be linked to abnormal calcium metabolism and loss of cholinergic neurons in the GIFN mice. Thus, these transgenic mice provide a novel animal model in which to further evaluate the mechanisms that underlie the diverse actions of type I interferons in the intact CNS and to link specific structural changes with functional impairments.

The Role of Length of Nerve Exposed to Local Anesthetics in Impulse Blocking Action

The quantitative relation between the concentration of local anesthetic (LA), the length of nerve exposed, and severity of conduction blockade was studied with use of a chamber where exposure length was varied as the concentration of lidocaine was held constant. Recordings of the compound action potential and of single axons established that small variations in the length of nerve exposed to LA strongly modulate conduction block even at exposure lengths in excess of 2 cm. Therefore, exposure length is a significant factor in determining blocking potency, and only at very high concentrations of LA, where voltage-dependent Na conductance is almost completely blocked, is the critical exposure length less than three nodes of Ranvier. The concentration required for 50% block of impulses in single fibers (that is, where 50% of the impulses would fail to propagate through the exposed region of the nerve) diminished as the exposed length of nerve increased, approximately halving as exposure length was changed from 6 mm to 15--25 mm. Conduction latency increased with the exposure length becoming sharply more variable as the critical exposure length for conduction block was approached. The results are consistent with the hypothesis of decremental conduction, where a partial active response in nodes exposed to marginal blocking concentrations extends the decay of the action potential along the axon, and do not support the interpretation that lengths of several centimeters affect blocking concentration because such distances increase the probability that three nodes will be blocked in succession. This study contradicts the broader common assumption that beyond three nodes, the length of nerve exposed is not a factor in nerve block with local anesthetics.

C10 Is a Novel Chemokine Expressed in Experimental Inflammatory Demyelinating Disorders that Promotes Recruitment of Macrophages to the Central Nervous System

Chemokines may be important in the control of leukocytosis in inflammatory disorders of the central nervous system. We studied cerebral chemokine expression during the evolution of diverse neuroinflammatory disorders in transgenic mice with astrocyte glial fibrillary acidic protein-targeted expression of the cytokines IL-3, IL-6, or IFN-alpha and in mice with experimental autoimmune encephalomyelitis. Distinct chemokine gene expression patterns were observed in the different central nervous system inflammatory models that may determine the phenotype and perhaps the functions of the leukocytes that traffic into the brain. Notably, high expression of C10 and C10-related genes was found in the cerebellum and spinal cord of GFAP-IL3 mice with inflammatory demyelinating disease and in mice with experimental autoimmune encephalomyelitis. In both these neuroinflammatory models, C10 RNA and protein expressing cells were predominantly macrophage/microglia and foamy macrophages present within demyelinating lesions as well as in perivascular infiltrates and meninges. Intracerebroventricular injection of recombinant C10 protein promoted the recruitment of large numbers of Mac-1(+) cells and, to a much lesser extent, CD4(+) lymphocytes into the meninges, choroid plexus, ventricles, and parenchyma of the brain. Thus, C10 is a prominent chemokine expressed in the central nervous system in experimental inflammatory demyelinating disease that, we show, also acts as a potent chemotactic factor for the migration of these leukocytes to the brain.

Effects of baclofen and bicuculline on inhibition in the fascia dentata and hippocampus regio superior

The effects of microiontophoretically applied baclofen, bicuculline and phaclofen were studied on evoked field responses, paired-pulse (PP) plasticity and single-unit activity of dentate granule cells (DGCs) and CA1 pyramidal cells (PCs) in anesthetized rats. The GABAB agonist, baclofen, increased population spike (PS) amplitudes in the dentate evoked by perforant path stimulation but decreased PS amplitudes in CA1 evoked by Schaffer collateral stimulation, whereas the GABAA antagonist, bicuculline, increased PS amplitudes in both regions. Neither baclofen nor bicuculline had significant effects on dendritically recorded population excitatory postsynaptic potentials (EPSPs) in the dentate or CA1 evoked by stimulation of their respective afferents. Control PP curves in the dentate revealed a triphasic response of inhibition/potentiation/inhibition, whereas control PP curves in CA1 manifested a biphasic response of inhibition/potentiation of test/conditioned PS amplitudes. Baclofen and bicuculline reversed the early and late phases of PP inhibition in the dentate and the early phase of PP inhibition in CA1. The GABAB antagonist, phaclofen, selectively reversed the effects of baclofen on PP inhibition in both the dentate and CA1. Whereas baclofen had no effect, bicuculline incre sed and phaclofen decreased DGC single-unit spontaneous firing rate, while baclofen decreased and bicuculline and phaclofen increased PC firing rate. These results support and extend studies suggesting that GABAergic feedback inhibition of DGCs and PCs is mediated by postsynaptic GABAA receptors and feedback inhibition of PCs is mediated by postsynaptic GABAB receptors. Our results also provide significant new evidence suggesting that postsynaptic inhibition in the dentate is not regulated by GABAB receptors and that feedback and feedforward inhibition of DGCs and PCs is regulated by presynaptic GABAB receptors located on GABAergic interneurons.

Site-specific hippocampal pathophysiology due to cerebral overexpression of interleukin-6 in transgenic mice

Transgenic mice expressing the cytokine interleukin-6 exhibit distinctive hippocampal interneuron pathology and behavioral seizures. Electroencephalographic recordings from these mice revealed anomalous hippocampal paroxysmal discharges and suppressed theta rhythm. Analysis of hippocampal field responses evoked by monosynaptic afferent stimulation revealed a site-specific increase in recurrent inhibition in the dentate gyrus. In addition, the cholinergic component of septohippocampal conditioning of dentate-evoked activity was absent in the transgenic mice. These results indicate that overexpression of interleukin-6 selectively disrupts cholinergic transmission by inducing a functional pathophysiology of hippocampal cholinoceptive target neurons.

Transgenic models to assess the pathogenic actions of cytokines in the central nervous system

In order to better understand the actions of proinflammatory cytokines in the mammalian CNS, a transgenic approach was employed in which the expression of IL-6, IL-3 or TNF-α was targeted to astrocytes in the intact CNS of mice. Transgenic mice exhibited distinct chronic-progressive neurological disorders with neurodegeneration and cognitive decline due to IL-6 expression, macrophage/microglial-mediated primary demyelination with motor impairment due to IL-3 expression and lymphocytic meningoencephalomyelitis with paralysis induced by TNF-αexpression. Thus, expression of specific cytokines alone in the intact CNS results in unique neuropathological alterations and functional impairments, thereby directly implicating these mediators in the pathogenesis of CNS disease.

Brain stimulation reward is integrated by a network of electrically coupled GABA neurons

The neural substrate of brain stimulation reward (BSR) has eluded identification since its discovery more than a half-century ago. Notwithstanding the difficulties in identifying the neuronal integrator of BSR, the mesocorticolimbic dopamine (DA) system originating in the ventral tegmental area (VTA) of the midbrain has been implicated. We have previously demonstrated that the firing rate of a subpopulation of gamma-aminobutyric acid (GABA) neurons in the VTA increases in anticipation of BSR. We show here that GABA neurons in the VTA, midbrain, hypothalamus, and thalamus of rats express connexin-36 (Cx36) gap junctions (GJs) and couple electrically upon DA application or by stimulation of the internal capsule (IC), which also supports self-stimulation. The threshold for responding for IC self-stimulation was the threshold for electrical coupling between GABA neurons, the degree of responding for IC self-stimulation was proportional to the magnitude of electrical coupling between GABA neurons, and GJ blockers increased the threshold for IC self-stimulation without affecting performance. Thus, a network of electrically coupled GABA neurons in the ventral brain may form the elusive neural integrator of BSR.

Enduring Effects of Chronic Ethanol in the CNS: Basis for Alcoholism

This symposium focused on functional alterations in the mesolimbic dopamine system during the abstinence phase after chronic alcohol intake. Mark Brodie first described his recordings from midbrain slices prepared after chronic alcohol treatment in vivo by daily injection in C57BL/6J mice. No changes were found in the baseline firing frequency of dopaminergic neurones in the VTA (ventral tegmental area), but the excitation produced in these neurones by an acute ethanol challenge was significantly increased in neurons from ethanol-treated mice compared with those from the saline-treated controls. There was also a significant decrease in the inhibitory response to GABA by the dopamine neurones following the chronic ethanol treatment. These data suggest that the timing pattern and mode of ethanol administration may determine the types of changes observed in dopaminergic reward area neurons. Annalisa Muntoni lectured on the relationship between electrophysiological and biochemical in vivo evidence supporting a reduction in tonic activity of dopamine neurons projecting to the nucleus accumbens at various times after suspension of chronic ethanol treatment and morphological changes affecting dopamine neurons in rat VTA. Hilary J. Little then described changes in dopaminergic neurone function in the VTA during the abstinence phase. Decreases in baseline firing were seen at 6 days after withdrawal of mice from chronic ethanol treatment but were not apparent after 2 months abstinence. Increases in the affinity of D1 receptors in the striatum, but not in the cerebral cortex, were seen however up to 2 months after withdrawal. Scott Steffensen then described his studies recording in vivo from GABA containing neurones in the VTA in freely moving rats. Chronic ethanol administration enhanced the baseline activity of these neurones and resulted in tolerance to the inhibition by ethanol of these neurones. His results demonstrated selective adaptive circuit responses within the VTA or in extrategmental structures that regulate VTA-GABA neurone activity.