The Basal Ganglia are a highly interconnected system of subcortical nuclei. Many years ago they were often referred to as "The extrapyramidal motor system." However, converging evidence from several different types of research make it clear that the basal ganglia are, in the strictest sense, neither extrapyramidal nor simply a motor system. Normal functions of the basal ganglia subsume cognition, emotion, learning and memory and sensorimotor integration, in addition to control of voluntary movement. Disorders of the basal ganglia include Parkinson's disease, Huntington's disease, schizophrenia and other disorders of thought, emotion and voluntary movement.
The research in my laboratory is aimed at understanding the functional circuitry of the basal ganglia at a systems level. Most of the work uses a combination of approaches including in vivo extracellular single unit recording, in vivo and in vitro intracellular recording and staining, in vitro visualized whole cell recording, immunocytochemistry, tract tracing and light and electron microscopy.
We're particularly interested in the GABAergic inputs and how they control (perhaps independently) firing rate and firing pattern through activation of GABAA and GABAB receptors. Corollary projects include electrophysiological and morphological characterization of GABAergic neuron subtypes in the substantia nigra, and the cellular and subcellular localization of different GABAA and GABAB receptor subtypes on nigral neurons.
Electrophysiolgical and anatomical studies of the GABAergic neurons of the pars reticulata in vivo and in vitro
The pars reticulata, along with the internal segment of the globus pallidus, represent the output nuclei of the basal ganglia. We are trying to determine if there are different types of neurons in the pars reticulata, and if so, if they have distinct physiological properties and efferents and afferents.
Electrophysiological and anatomical studies of the physiology and function of the neostriatum.
Much recent evidence suggests that one of the most important sources of the intrinsic GABAergic signalling is from the fast-spiking and other interneurons, and that these interneurons may also be important sites of action of striatal neuromodulators including acetylcholine and dopamine. Recent work in our lab has revealed the existence of no fewer than 4 electrophysiologically distinct types of striatal interneurons that express tyrosine hydroxylase. These striatal interneurons are both GABAergic and dopamiminergic, and form afferent and efferent synaptic connections with spiny projection neurons.
Animal Models of Human Disease
Huntington's disease is an incurable, progressive neurodegenerative disease caused by a single gene dominant mutation. It leads to severe motor and cognitive disabilities and is invariably fatal. In collboration with the Abercrombie lab, we use in vivo single unit recordings to explore the electrophysiological changes that occur in the substantia nigra, subthalamic nucleus and globus palllidus of mutant mouse models of Huntington's disease, as well as the corresponding neuroanatomical changes.
Optogenetic Investigation of Basal Ganglia Circuitry
Using in vivo lentiviral transduction in vivo in transgenic mice expressing Cre under the control of different specific promotors, we can get expression of channelrhodopsin2 (ChR2) or halorhodopdin in specific neuronal subtypes, for example, dopaminergic neurons or cholinergic neurons. By illuminating transduced neurons with blue light, we can activate those neurons expressing ChR2 selectively, something that is impossible with conventional techniques. Transduction with halorhodopsin and illlumination with yellow light similarly activates a Cl- pump and hyperpolarizes and silences a neuron. We use these techniques in combination with in vitro recording to analyze basal ganglia circuitry and network properties
Alexandre and Tepper, unpublished
Reconstruction of an electrophysiologically identified nigrostriatal dopaminergic neuron intracellularly labeled with HRP following in vivo intracellular recording. Note the slow, regular spontaneous activity and the long, deep spke afterhyperpolarization in the upper right inset. The soma and laterally projecting dendrites are colored orange, the ventral dendrites projecting into pars reticulata are colored magenta, and the initial portion of the axon is colored yellow. The inset at lower right shows the position of the neuron with respect to the boundaries of substantia nigra. Note that the long ventral dendrite extends all the way throough pars reticulata and actually penetrates the crus cerebri. Modified from Tepper, Sawyer and Groves (1987) Electrophysiologically identified nigral dopaminergic neurons intracellularly labeled with HRP: Light microscopic analysis. J. Neuroscience 7:2794-1806.
Synapses onto dopaminergic neurons in substantia nigra
Symmetric small round vesicle-containing synapse made onto a dendrite approximately 125 microns from the soma of a substantia nigra dopaminergic neuron intracellularly stained in vivo with HRP.
Effects of blockade of GABA inputs on firing pattern of dopaminergic neurons
Autocorrelograms from one typical electrophysiologically identified dopaminergic neuron in substantia nigra illustrating the effects of local application of the selective GABAA receptor antagonist, bicuculline, and the selective GABAB receptor antagonist, CGP-55348A. Insets in all the autocorrelograms show the initial portion of the same autocorrelation at higher temporal resolution. A) Before application of any drug the neuron fired in a pacemaker pattern indicated by the regularity of interspike intervals in the oscilloscope trace to the right and several peaks in the autocorrelogram. B) After application of the GABAA receptor antagonist, bicuculline, the autocorrelogram shifted to an initial peak with a decay to a steady state level indicating a bursty firing pattern. To the right of the autocorrelogram is an oscilloscope trace from the same neuron showing two of the bursts observed after bicuculline application. Note the progressively decreasing spike amplitude typical of spontaneously occurring bursts. This effect lasted for approximately seven minutes. C) Application of the GABAB antagonist CGP-55845A increased the regularity of firing indicated by a more regular interspike interval seen in the oscilloscope trace and the increase in the number of peaks in the autocorrelogram. From Paladini and Tepper (1999) GABAA and GABAB antagonists differentially affect the firing pattern of substantia nigra dopaminergic neurons in vivo. Synapse 32:165-176.
Control of firing pattern of dopaminergic neurons in vivo
Effects of decreasing and increasing pallidal activity on the firing pattern of a dopaminergic neuron. A. Spike train (upper panel), autocorrelogram and first order interval histogram (inset) of a typical dopaminergic neuron showing random firing under control conditions. B. After muscimol-induced inhibition of globus pallidus the firing rate decreased and the firing pattern became pacemaker-like as can be seen in the spike train (upper panels) and the autocorrelograms. C. A subsequent infusion of bicuculline into the globus pallidus increased the firing rate of the neuron and not only reversed the pacemaker effect of the prior muscimol infusion but shifted the dopaminergic neuron into the burst firing mode, characterized by the single initial peak in the autocorrelogram, and increased proportion of spikes fired within bursts. From Celada, Paladini and Tepper (1999) GABAergic control of rat substantia nigra dopaminergic neurons: Role of globus pallidus and substantia nigra pars reticulata. Neuroscience 89:813-825.
Effects of manipulating endogenous burst firing of dopaminergic neurons on striatal dopamine levels
In a subsequent experiment the burst firing in DA neurons caused by pallidal excitation was replicated, this time with a microdialysis probe in the striatum. The burst firing, which was accompanied by only a relatively small 14% average increase in firing rate, produced a very large increase in basal DA levels in striatum, thus showing that changes in firing pattern elicited by alterations in pallidal output similar to those to occurring spontaneously in freely moving animals cause powerful modulation of dopamine release by disinhibiting the pars reticulata input to the dopamine neurons. Lee, Abercrombie, and Tepper (2004) Pallidal control of nigral dopaminergic neuron firing pattern and its relation to extracellular neostriatal dopamine. Neuroscience 129:481-489.
Local axonal arborization of a pars reticulata GABAergic projection neuron
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The axons of pars reticulata projection neurons issue local axon collaterals that arborize in a few discrete areas in the pars compacta and pars reticulata a shown in this example of a neuron juxtacellularly labeled in vivo after being antidromically identified as a nigrothalamic GABAergic neuron. F. Shah and J.M. Tepper, unpublished.
In vivo intracellular labeling of striatal spiny neurons
Neostriatal spiny projection neuron from an adult rat intracellularly stained in vivo with biocytin. The dendrites are densely spiny. Some of the local axon collaterals are also visible. From Tepper, Sharpe, Koós, and Trent (1998) Postnatal development of the rat neostriatum: Electrophysiological, light and electron microscopic studies. Dev. Neurosci. 20:125-145.
This is a reconstruction of a striatal fast-spiking interneuron stained with neurobiotin in vitro showing the incredibly dense local axon collateral field (yellow). The insets show intermediate and high magnification electron micrographs of a synapse made by the interneuron onto a nearby medium spiny neuron. Modified from Tepper and Bolam (2004) Functional diversity and specificity of neootriatal interneurons. Curr Opin. Neurobiol., 14:684-692.
Dual whole cell recording in vitro showing interneuronal modulation of action potential generation in striatal spiny neurons. (a) A single action potential elicited in a spiny neuron by current injection (upper black trace "c") is delayed by IPSPs evoked by single spikes (lower blue trace) or a spike doublet (red trace) of a fast spiking interneuron. The delay is variable (compare blue traces spikes 1, 2), and the spike doublet (red traces) is more effective than single spikes. The inset shows the IPSPs at higher gain. (b) The same experiment as in (a) conducted in a pair of an LTS interneuron (lower trace, same cell as in Fig. 1d) and a spiny neuron cell (upper traces). The LTS of the interneuron elicits three fast spikes (lower trace) evoking compound IPSPs (upper black traces) which prevent the firing of the GSP cell (red traces 2, 4) for approximately 20 ms. The momentary firing rate is decreased by 35%. The trials were performed in the order of numbering indicating the stability of the postsynaptic cell and the reliability of the inhibition. See Koós and Tepper (1999) Inhibitory control of neostriatal projection neurons by GABAergic interneurons. Nature Neuroscience 2:467-472.
Inhibitory effects of fast spiking interneurons on spiny neurons is modulated by presynaptic muscarinic receptors
Muscarinic receptor mediated inhibition of synaptic transmission between FS interneurons and MS neurons. Representative example of recording from a synaptically connected pair of an FS interneuron and an MS neuron. Single action potentials of the FS interneuron (arrowheads) elicited IPSCs (green sweeps) with variable amplitudes (left). Bath application of 10 µM muscarine strongly reduced the average amplitude of the postsynaptic response without affecting passive or active properties of the FS interneuron. Co-application of 10 µM atropine reversed the effect of muscarine (right). Red traces are population means. IPSCs are inward due to chloride loading. From Koós and Tepper (2002) Dual cholinergic control of fast spiking interneurons in the neostriatum. J. Neurosci.22:529-535.
Striatal fast spiking interneurons are powerfully excited by acetylcholine
Nicotinic responses of striatal fast-spiking interneurons. A. Bath application of 100 µM carbachol depolarized silent FS in-terneuron and in-duced persistent irregular bursty firing. B. Local pressure application of ACh (5 mM, 40 ms duration, thick bar) in the presence of CNQX and APV induced de-polarization which was blocked by bath application of 5 µM MEC. C. Local application of 5 mM ACh (thick bar) evoked depolarization which was unaffected by bath application of 100 nM MLA but which was almost completely blocked by subsequent application of 1µM MEC. D. Positive control, showing effectiveness of MLA on carbachol-induced de-polarization (at thick bar) of a hippocampal stratum radiatum interneuron known to express type 1 (MLA-sensitive) nicotinic receptors. See Koós and Tepper, (2002) Dual control of fast-spiking interneurons in the neostriatum. J. Neurosci. 22:529-535.
Difference in the chloride reversal potential in nigral GABAergic reticulata neurons vs dopaminergic neurons GABAA mediated responses
are different in nigral DAergic and pars reticulata neurons. The
DAergic neurons (TH+) lack the potassium-chloride cotransporter
(KCC2) which is responsible for maintaining the hyperplarizing
chloride gradient in most mature neurons. (LEFT) Light
micrographs demonstrate the localization of KCC2 in non-DAergic
cells of the rat SN. A-B) Segregation of TH-immunolabeled
neurons (brown) and KCC2-immunopositive dendrites (blue-black)
in both the SNc and SNr. Both somata and dendrites of the DAergic
neurons are labeled for TH (arrows). KCC-2 was found only in the
dendrites of non-DAergic cells (arrowheads). C-D) Double-immunofluorescent
staining shows a mutually exclusive distribution pattern for TH
and KCC2. None of the TH-positive (green) dendrites or somata
are outlined by KCC2 immunoreactivity (red). Scales: 50 µm
(A,C,D), 25 µm (B). (RIGHT) Pharmacologically
isolated GABAA receptor-mediated IPSPs in response
to stimulation of the SNr in vitro. A) Representative current
clamp recordings of a DAergic neuron at various membrane potentials
show an IPSP in response to stimulation of the SNr. Inset is
a plot of the IPSP amplitude versus the membrane potential. The
plot of the regression line revealed that the reversal potential
of the IPSP was 62.9 mV. B) Representative current
clamp recordings of IPSP in GABAergic SNr neuron shows significantly
more hyperpolarized reversal potential of75.8 mV. C, D)
In bicarbonate ion free slice buffer, the GABAA receptor mediated IPSP reversal potential in a representative DAergic neuron (C) becomes significantly less hyperpolarized at -50.2 mV, whereas there is no significant change in a non-DAergic neuron in which the IPSP reverses at -70.6 mV (D) indicating that the slightly hyperpolarizing Cl- gradient in DAergic neurns comes from the sodium-dependent anion exchanger (NDAE). Modified from Gulácsi, Lee, Sík, Viitanen, Kaila, Tepper and Freund (2003) Cell type-specific differences in chloride-regulatory mechanisms and GABAA receptor mediated inhibition in rat substantia nigra. J. Neurosci. 23:8237-8246.
A. Comparison of IPSPs recorded in spiny neurons in neostriatal slices under similar conditions of membrane potential, driving force and input resistance elicited from a FS interneuron (A1) and a spiny cell axon collateral (A2). Note difference in amplitude scales. B. Comparison of IPSCs recorded in spiny neurons in neostriatal slices under identical conditions (140 mM CsCl internal). The IPSCs from elicited from the FS interneuron (B1) are much larger than those from the spiny cell (B2). Note the frequent failures in the collateral IPSP in contrast to the high reliability of the FS synapse. From Tepper, Koós and Wilson (2004) GABAergic microcircuits in the neostriatum. TINS. 27:662-229.
Quantal analysis of IPSCs in neostriatal spiny neurons arising from another spiny neuron (left panels) or from a FS interneuron (right panels) under conditions that negate the effects of synaptic location (CsCl internal). Note the frequent failures in the spiny-spiny synapse and the absence failures in the FS-spiny synapse. Note also the very large, rapid synaptic depression for the spiny-spiny synapse compared to a slower, more modest depression in the FS-spiny synapse. Mean variance analysis shows that although the size of the unitary IPSC is about the same for each synapse, the FS-spiny IPSC is still several times larger due to the difference in N, the number of release sites. From Koós, Tepper and Wilson 2004. J. Neurosci. 24:7916-7922.
Schematic of the difference between spiny-spiny neuron collateral synapses and interneuron-spiny neuron synapses in striatum
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Schematic of the GABergic circuitry of the neostriatum. Although the basic properties of the axon collateral synapse and the feed-forward FS-spiny cell synapse are quite similar, the amplitude of the axon collateral synapse appears much smaller at the soma because each spiny cell makes very few synapses (~2-3) with each other spiny cell and these synapses are located distally, on the spiny regions of the dendrites. In contrast, each FS interneuron makes a larger number of synapses on each spiny cell (~ 7 or more) and these synapses are located pericellularly. From Tepper, Koós and Wilson (2004) GABAergic microcircuits in the neostriatum. TINS. 27:662-229.
Changes in substantia nigra pars reticulata in R6/2 mutant model of Huntington's disease
Nisssl stain reveals shrinkage of the substantia nigra in R6/2 mice at 8 weeks. The nucleus is smaller, there are fewer GABAergic neurons and their somata size is reduced in the transgenic mice (Tgn) compared to wild typle controls. Cell counts are unbiased stereological resuults using an optical disector method. Puddu, Abercrombie and Tepper, in preparation.
Ibanez-Sandoval et al. (2007) Soc. Neurosci. Abstr.
