SUMMARY

The interplay between sensory, cognitive and motor functions defines many aspects of behaviour and its interaction with the environment. Behaviour is often driven by motivational and emotional states, such as hunger, pleasure, fear, etc., and the assembly of actions required to comply with such states is planned and organized within the brain.
The limbic cortico-striatal system (LCS) is a network of several interconnected structures of the brain that are thought to drive and regulate motivated behaviour. Because of its central position in the LCS, the nucleus accumbens (Nac), a relatively extended area within the ventral striatum, is thought to subserve important functions in motivated behaviour.
The Nac plays a major role in integrating converging inputs that originate in the prefrontal cortex, amygdala, hippocampus and thalamus. The projecting cells of the Nac, the medium-sized spiny neurons (MSNs), provide the output to downstream areas such as the ventral pallidum and the substantia nigra pars reticulata. Different cell types of the Nac constitute an intricate network, with largely unknown functional characteristics. The disclosure of some important aspects of the synaptic organization of the Nac microcircuits was the aim of the work presented in this thesis.
To study various electrophysiological properties of this network, we performed whole-cell patch clamp recordings from neurons and interneurons in a slice preparation of the rat Nac. The modulatory role of metabotropic glutamate receptors (mGluRs) on excitatory synapses is documented in chapter 2. Glutamatergic inputs provide the main excitatory drive to MSNs. Glutamate released into the synaptic cleft binds to ionotropic (AMPA/Kainate and NMDA) glutamate receptors, which in turn cause depolarization of the membrane potential due to their permeability to Na+ and Ca2+ ions. Metabotropic glutamate receptors (mGluRs) constitute a third class of glutamate receptors that modulate neuronal excitability through interaction with intracellular biochemical pathways. During episodes of strong glutamate release, each type of receptor may be activated and, hypothetically, mGluRs (located pre- and/or postsynaptically) may interfere with the activation of AMPA and NMDA receptors. Flash-photolysis of "caged" glutamate was used to activate postsynaptic glutamate receptors located on visually selected MSNs (see fig. 6 of the Introduction). Application of a specific blocker of one subfamily of mGluRs (Group III) was able to transiently potentiate both AMPA- and NMDA-receptor currents, indicating that postsynaptic Group III mGluRs exert an inhibitory effect on ionotropic receptors. Conversely, an antagonist of Group I mGluRs, as well as the neurotransmitter dopamine, did not exert any significant effect.
The importance of glutamatergic synapses in the striatum is underscored by several considerations. For example, AMPA receptors mediate membrane potential transitions to near-threshold depolarized levels (up-states), whereas the activation of NMDA receptors mediates the expression of synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD). Modulation of ionotropic receptors may therefore play important roles such as a negative feedback in the case of group III mGluRs, and this may at times prevent the occurrence of synaptic changes or up-state transitions. Group III mGluRs-mediated inhibition may also underlie a potential form of neuroprotection against glutamate-mediated excitotoxicity. Furthermore, agonists of Group III mGluRs seem to be promising therapeutical agents, since several studies have indicated that the function of striatal ionotropic receptors may constitute a potential target for drugs intended to treat syndromes such as Parkinson's disease, Huntington's disease, schizophrenia and epilepsy.
Chapter 3 illustrates results that demonstrate the existence of functional GABAergic synapses between MSNs in the Nac. Although anatomical data have previously indicated that MSNs possess an axonal arborization that may synaptically target neighbouring cells, convincing electrophysiological evidence has long been lacking. By using simultaneous dual recordings (fig. 7 of the Introduction), we found that MSNs are functionally interconnected by unidirectional, GABAergic synapses acting through bicuculline-sensitive GABAA receptors. Presynaptic action potentials elicited depolarizing postsynaptic potentials (dPSPs), which were mediated by outward Cl- currents induced by a high Cl- concentration present in our recording pipettes and recorded cells. Trains of dPSPs showed marked kinetics depending on the firing rate of the presynaptic neuron. Furthermore, paired pulse facilitation and depression occurred depending on the size of the first-elicited IPSC, suggesting that short-time dynamics depended on both pre- and postsynaptic factors.
The probability of finding a connection in slices was relatively high (>30%), suggesting that striatal lateral inhibition in vivo may be remarkably dense, although our recordings were confined in terms of spatial extent. Recent anatomical tracing data support the existence of long-range projections in the Nac as well (Van Dongen, Pennartz and Groenewegen, unpublished data), but their existence and functioning has not been examined in the present study. Our findings support the view of the striatum as a network of functionally competing ensembles of MSNs, which may be able to subserve input selection functions and to provide appropriate output for guiding or modulating behavior, avoiding redundancy. The precise functions of the (mainly unidirectional) synapses between MSNs, as well as the functional difference between interneuron- and MSN-mediated GABAergic inhibition in the NAc, remain unresolved issues so far. Alternatives besides the 'competitive-ensembles' hypothesis should be considered, such as a model in which lateral inhibition primarily serves to neutralize the excitatory impact of glutamatergic inputs localized on small dendritic segments of an inhibited MSN, and to prevent synaptic changes from occurring in those inputs.
The question of whether the neurotransmitter dopamine modulates lateral inhibition between MSNs was addressed in chapter 4. The striatum forms a main target for dopaminergic fibers originating in ventral midbrain areas, and various effects of dopamine on intrinsic and synaptic properties of MSNs have been described in recent years. Yet, the role of dopamine in vivo, and in particular its impact on learned reward-related behaviour, is still controversial. It has been suggested that transient release of dopamine underlies a behavioural "switch", possibly due to selective activation of striatal sub-networks at any given time according to environmental demands. We tested this hypothesis by applying dopamine to slices during dual-cell recordings by which the presence of GABAergic inhibition between two individual MSNs was established before the application. The amplitude of dPSPs elicited by presynaptic action potentials was significantly attenuated by dopamine, and a similar effect was found when a D1-receptor agonist was applied. In view of these data, we propose that dopamine may attenuate lateral inhibition exerted by given cells or ensembles onto competing units and allow recruitment of the latter as a response to salient environmental changes.
In chapter 5 we showed novel electrophysiological properties of fast-spiking interneurons (FSIs), a class of cells that may play important roles in striatal functions such as feed-forward GABAergic inhibition and network synchronization. By using simultaneous dual-cell recordings, we found that a fraction of FSIs were able to fire bursts of action potentials in response to prolonged steady depolarization, and that the bursting interneurons were synaptically connected to individual MSNs, probably by way of GABAergic neurotransmission. MSNs responded to action potential bursts of presynaptic FSIs with barrages of dPSPs that showed properties of short-term facilitation and depression. Furthermore, one pair of FSIs was reciprocally interconnected by means of both GABAergic synapses and gap junctions, which enabled synchronous entrainment of the two cells into coherent rhythmic firing. We also recorded neuronal spiking activity in vivo after implantation of microelectrode arrays (tetrodes) in the ventral striatum of freely moving rats, and performed off-line analysis of kinetic properties of individual action potential waveforms. Recognition of FSI-like and MSN-like waveforms was achieved after comparison with spike waveforms recorded in vitro. Cross-correlation analysis of pairs of neurons simultaneously recorded in vivo revealed short-latency temporal interactions between assorted pairs (i.e. FSI vs. MSN, FSI vs. FSI, MSN vs. MSN). These data were discussed in view of emerging evidence for functional rhythmic population activity originating in the basal ganglia, the putative role of FSIs in initiating and orchestrating striatal electrical oscillations, and behavioural correlates underlying basal ganglia network rhythms.
In chapter 6 (Discussion), several aspects of the synaptic microcircuitry of the Nac presented in this thesis were discussed and integrated, in part on the basis of pre-existing theories that were or are not yet substantiated by experimental data. Moreover, many new questions and perspectives emerged that require further work to be fulfilled. For example, the hypothesis that lateral inhibition between MSNs constitutes a substrate for functional competition between ensembles may be questioned by the almost exclusive unidirectionality of the GABAergic synapses. Moreover, does the putative dopamine-dependent switch of ensemble activation benefit from a local transient release of dopamine, or a generalized release across much of the striatum? What is the impact of bursting-firing of FSIs and the putative reciprocal, GABA- and gap junction-mediated interconnectivity between FSIs on the striatal network?
New viewpoints concerning the role of the ventral striatum in behaviour emerge now, and understanding the dynamic integration of the action of different neurotransmitters becomes more and more important for a detailed comprehension of the physiology and pathology of the basal ganglia.