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.