GABA neurons in the substantia nigra pars reticulata receive input from GABAergic fibers originating in the forebrain. The role of dopaminergic D1 receptors located on these fibers was investigated using tight-seal whole-cell recordings from visually identified pars reticulata neurons of rat substantia nigra slices. Nondopaminergic pars reticulata neurons were characterized by their electrophysiological properties. Postsynaptic currents evoked by minimal stimulation in the presence of ionotropic glutamate receptor antagonists were blocked by bicuculline, indicating that they were GABAA IPSCs. Evoked GABAA IPSCs were potentiated by D1 receptor agonists. After application of D1 receptor agonists, miniature IPSCs [recorded in the presence of tetrodotoxin (TTX) and the Ca2+ channel blocker Cd2+] increased in frequency but not in amplitude. Effects of D1 receptor stimulation were mimicked by forskolin, as expected, if a cAMP-dependent mechanism was involved. The D1 antagonist SCH23390 blocked the effects of the agonists, and perfusion with SCH23390 resulted in a reduction of evoked IPSCs. In TTX and Cd2+, which prevented dopamine release, the D1 antagonist had no effect on miniature IPSCs. Blocking of monoamine uptake by imipramine increased the amplitude of evoked IPSCs. We conclude that dopamine released from dendrites of dopaminergic neurons enhances GABA release in the pars reticulata of the substantia nigra through D1 receptors presumably located on striatonigral afferents. These D1 receptors, thereby, can reinforce D1 receptor-mediated activation of striatal projection neurons that inhibit the inhibitory output neurons of the basal ganglia in substantia nigra.

Cajal-Retzius (CR) cells are among the earliest generated neurons and are thought to play a role in corticogenesis and early neuronal migration. However, the role of CR cells in an early cortical microcircuit is still rather unclear. We therefore have investigated the morphology and physiology of CR cells by using whole-cell patch-clamp recordings combined with intracellular biocytin filling in acute brain slices of postnatal day 5-11 rats. CR cells are characterized by a long horizontally oriented dendrite; the axonal collaterals form a dense horizontally oriented plexus in layer 1 and to a certain extent in layer 2/3, projecting over >2 mm of cortical surface. The bouton density is relatively high, and synaptic contacts are established preferentially with dendritic spines or shafts of excitatory neurons, presumably terminal tuft dendrites of pyramidal neurons. In turn, CR cells receive dense GABAergic and non-GABAergic input on somata, dendritic shafts, and spine-like appendages. Extracellular stimulation in layer 1 could activate both GABAergic and glutamatergic synaptic inputs. The GABAergic response was blocked by the GABA(A) receptor antagonist bicuculline. The glutamatergic response was mediated solely by NMDA receptors and was highly sensitive to ifenprodil, indicating that it was mediated mainly via NR1/NR2B subunit-containing receptors. NMDA EPSPs were apparent in 1 mm extracellular Mg2+, suggesting that this pure NMDA synapse is not silent functionally. Together, the long-range horizontal projection of the axon, the high density of synaptic boutons, and the functional synaptic input of CR cells suggest that they are an integral part of an early cortical network.

From an anatomical point of view the neocortex is subdivided into up to six layers depending on the cortical area. This subdivision has been described already by Meynert and Brodmann in the late 19/early 20. century and is mainly based on cytoarchitectonic features such as the size and location of the pyramidal cell bodies. Hence, cortical lamination is originally an anatomical concept based on the distribution of excitatory neuron. However, it has become apparent in recent years that apart from the layer-specific differences in morphological features, many functional properties of neurons are also dependent on cortical layer or cell type. Such functional differences include changes in neuronal excitability and synaptic activity by neuromodulatory transmitters. Many of these neuromodulators are released from axonal afferents from subcortical brain regions while others are released intrinsically. In this review we aim to describe layer- and cell-type specific differences in the effects of neuromodulator receptors in excitatory neurons in layers 2–6 of different cortical areas. We will focus on the neuromodulator systems using adenosine, acetylcholine, dopamine, and orexin/hypocretin as examples because these neuromodulator systems show important differences in receptor type and distribution, mode of release and functional mechanisms and effects. We try to summarize how layer- and cell type-specific neuromodulation may affect synaptic signaling in cortical microcircuits.

In this report, we describe a reliable protocol for biocytin labeling of neuronal tissue and diaminobenzidine (DAB)-based processing of brain slices. We describe how to embed tissues in different media and how to subsequently histochemically label the tissues for light or electron microscopic examination. We provide a detailed dehydration and embedding protocol using Eukitt that avoids the common problem of tissue distortion and therefore prevents fading of cytoarchitectural features (in particular, lamination) of brain tissue; as a result, additional labeling methods (such as cytochrome oxidase staining) become unnecessary. In addition, we provide correction factors for tissue shrinkage in all spatial dimensions so that a realistic neuronal morphology can be obtained from slice preparations. Such corrections were hitherto difficult to calculate because embedding in viscous media resulted in highly nonlinear tissue deformation. Fixation, immunocytochemistry and embedding procedures for light microscopy (LM) can be completed within 42-48 h. Subsequent reconstructions and morphological analyses take an additional 24 h or more.

In the neocortex, most excitatory, glutamatergic synapses are established during the first 4-5 weeks after birth. During this period profound changes in the properties of synaptic transmission occur. Excitatory postsynaptic potentials (EPSPs) at immature synaptic connections are profoundly and progressively reduced in response to moderate to high frequency (5-100 Hz) stimulation. With maturation, this frequency-dependent depression becomes progressively weaker and may eventually transform into a weak to moderate EPSP facilitation. In parallel to changes in the short-term plasticity, a reduction in the synaptic reliability occurs at most glutamatergic neocortical synapses: immature synapses show a high probability of neurotransmitter release as indicated by their low failure rate and small EPSP amplitude variation. This high reliability is reduced in mature synapses, which show considerably higher failure rates and more variable EPSP amplitudes. During early neocortical development synaptic vesicle pools are not yet fully differentiated and their replenishment may be slow, thus resulting in EPSP amplitude depression. The decrease in the probability of neurotransmitter release may be the result of an altered Ca 2+ control in the presynaptic terminal with a reduced Ca 2+ influx and/or a higher Ca 2+ buffering capacity. This may lead to a lower synaptic reliability and a weaker short-term synaptic depression with maturation.

Acetylcholine (ACh) is known to regulate cortical activity during different behavioral states, for example, wakefulness and attention. Here we show a differential expression of muscarinic ACh receptors (mAChRs) and nicotinic ACh receptors (nAChRs) in different layer 6A (L6A) pyramidal cell (PC) types of somatosensory cortex. At low concentrations, ACh induced a persistent hyperpolarization in corticocortical (CC) but a depolarization in corticothalamic (CT) L6A PCs via M 4 and M1 mAChRs, respectively. At ~ 1 mM, ACh depolarized exclusively CT PCs via α4β2 subunit-containing nAChRs without affecting CC PCs. Miniature EPSC frequency in CC PCs was decreased by ACh but increased in CT PCs. In synaptic connections with a presynaptic CC PC, glutamate release was suppressed via M4 mAChR activation but enhanced by nAChRs via α4β2 nAChRs when the presynaptic neuron was a CT PC. Thus, in L6A, the interaction of mAChRs and nAChRs results in an altered excitability and synaptic release, effectively strengthening CT output while weakening CC synaptic signaling.

An elementary feature of sensory cortices is thought to be their organisation into functional signal-processing units called 'cortical columns'. These elementary units process sensory information arriving from peripheral receptors; they are vertically oriented throughout all cortical layers and contain several thousands of excitatory and inhibitory synaptic connections. To understand how sensory signals are transformed into electrical activity in the neocortex it is necessary to elucidate the spatial-temporal dynamics of cortical signal processing and the underlying neurons and synaptic 'microcircuits'.In the somatosensory barrel cortex there appears to be a structural correlate for the 'functional' cortical column. Therefore, it has become an attractive model system to study the synaptic microcircuitry in the neocortex. Although many synaptic connections in whisker-related cortical 'columns' have been characterised over the past years our knowledge is far from complete, in particular with respect to inhibitory connections. In this chapter we will summarise recent data on different excitatory and inhibitory synaptic connections in a whisker-related 'column' of the somatosensory cortex and try to outline their function in the neuronal network. This requires an appreciation of the diverse types of excitatory and inhibitory neurons and their function within cortical columns and beyond. When necessary, we will also discuss the synaptic input from and to subcortical structures, in particular the thalamus. However, we will not provide a detailed description of the functional mechanisms of these connections; this is beyond the scope of this chapter.

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.