 |
||||
|
||||
|
||||
|
||||
Previous Article | Next Article
Volume 16, Number 19, Issue of October 1, 1996 pp. 5923-5941
Copyright ©1996 Society for NeuroscienceAre the Interlaminar Zones of the Ferret Dorsal Lateral Geniculate Nucleus Actually Part of the Perigeniculate Nucleus?
Maria V. Sanchez-Vives1, a, Thierry Bal2, a, Uhnoh Kim1, Marcus von Krosigk3, and David A. McCormick1 1 Section of Neurobiology, Yale University School of Medicine, New Haven, Connecticut 06510, 2 Institut Alfred Fessard, Centre National de la Recherche Scientifique, Gif Sur Yvette Cedex 91198, France, and 3 Kinsmen Laboratory of Neurological Research, University of British Columbia, Vancouver, British Columbia, Canada V6T 1W5
ABSTRACT
The ferret dorsal lateral geniculate nucleus (LGNd) contains interneurons within the interlaminar zones situated between the laminae corresponding to the ipsi- and contralateral eyes. We found that a subset of these neurons exhibits electrophysiological properties similar to those previously reported for perigeniculate (PGN) neurons, including the generation of rhythmic sequences of rebound low-threshold Ca2+ spikes at a frequency of 1-4 Hz after the intracellular injection of a hyperpolarizing current pulse. These ``PGN-like'' interlaminar interneurons innervated restricted regions of the A-laminae, inhibited thalamocortical cells through GABAA, and perhaps GABAB, receptors, and were excited by axon collaterals from thalamocortical cells. This reciprocal relationship is identical to that formed by PGN cells and allowed the PGN-like interlaminar neurons to participate in the generation of spindle waves and other network oscillations. Pharmacologically, PGN-like interlaminar interneurons were also similar to PGN neurons: both generated a prolonged depolarization in response to the local application of serotonin, 1S,3R-ACPD, and CCK8S, and a rapid depolarization followed by a more prolonged hyperpolarization in response to acetylcholine.
Examination of parvalbumin and calbindin staining in the ferret LGNd revealed that both PGN and a subset of interlaminar neurons were parvalbumin-positive. In contrast, calbindin-positive cells were relatively absent in the PGN and sparsely present in the interlaminar zones, but were numerous in the A and C laminae. Our results indicate that the interlaminar zone in between laminae A and A1 and A1 and C in the ferret LGNd possesses a cell type that is electrophysiologically, pharmacologically, anatomically, immunocytochemically, and functionally similar to neurons in the PGN.
Key words: spindle waves; GABA; thalamus; interneurons; thalamic reticular nucleus; thalamocortical cell collaterals
INTRODUCTION
The dorsal lateral geniculate nucleus (LGNd) of many carnivores is a multilayer structure consisting of bands (laminae) of neurons that are characterized by their retinal innervation and receptive-field properties (Guillery, 1970; Sanderson, 1974
The role of the GABAergic neurons located in the interlaminar zone in the function of the LGNd is unknown. Electron microscopic examination of synaptic profiles on interlaminar zone GABAergic neurons reveals that these cells may receive strong innervation from thalamocortical cells, cortical cells, and other GABAergic neurons, similar to PGN cells (Montero, 1989a
MATERIALS AND METHODS
For the preparation of slices, male or female ferrets, ~2-4 months old, were deeply anesthetized with sodium pentobarbital (30 mg/kg) and killed by decapitation. The forebrain was rapidly removed, and the hemispheres were separated with a midline incision. Sagittal slices (400 µm thick) were formed on a vibratome (Ted Pella, Inc.). A modification of the technique developed by Aghajanian and Rasmussen (1989)Intracellular recording electrodes were formed on a Sutter Instruments P-80 micropipette puller from medium-walled glass (WPI, 1BF100) and beveled on a Sutter Instruments beveler. Micropipettes were filled with 1.2 M K-acetate and 2% biocytin for intracellular labeling of recorded neurons and had resistances of between 60 and 100 M
. Cells were included in the present study if they exhibited a stable resting membrane potential for at least 10 min (typically 45-90 min), were able to generate a repetitive barrage of action potentials after depolarization, exhibited input resistances of at least 30 M
Drugs were applied with the pressure-pulse technique in which the delivery of a brief pulse of nitrogen (10-100 msec, 200-350 kPa) was applied to the micropipette to extrude volumes of 2-20 pl. Application of glutamate was performed at varying locations and depths within the slice to determine the best response. Other agonists and antagonists were applied to the surface of the slice either within 50 µm of the entry point of the recording electrode or as indicated in the figures. The latency for activation of neurons with local application of glutamate was estimated by performing extracellular multiple-unit recordings adjacent to the glutamate-applying micropipette within the slice. These applications revealed that pressure-pulse application of glutamate resulted in the activation of action potentials at a minimum latency of 20-25 msec, followed by an increase in the intensity of neuronal discharge peaking at ~35-40 msec. Therefore, monosynaptic connections between neurons excited by local application of glutamate and recipient cells will have a minimum latency of 20-30 msec. This latency may be longer if the location of the excited neuron is not immediately adjacent to the local application of glutamate. The detection of monosynaptic connections was facilitated by the relative lack of polysynaptic excitatory connections between thalamocortical cells in the LGNd.
Parvalbumin and calbindin immunocytochemistry was performed to examine the distribution of neurons positive for these Ca2+-binding proteins. Ferrets (2-3 months old) were deeply anesthetized with intraperitoneal injections of sodium pentobarbital (40 mg/kg) and perfused through the heart briefly with 0.85% sodium chloride in 10 mM PB followed by 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.2. After the brains were removed from the skull, they were post-fixed overnight at 4°C in 4% paraformaldehyde and 20% sucrose. The brains were cut into 20-µm-thick sections with a freezing microtome. The sections were incubated with gentle agitation for 48-72 hr at 4°C with monoclonal anti-parvalbumin or anti-calbindin (Sigma, St Louis, MO) diluted 1:500 or 1:200, respectively, in PBS containing 1% normal goat serum and 0.3% Triton X-100. After the sections were rinsed three times with PBS, they were incubated for 1 hr at room temperature with biotinylated goat anti-mouse Ig diluted 1:100 in PBS containing 1% normal goat serum. The sections were rinsed and incubated in a 1:50 dilution of avidin-biotin complex (Vectastain Elite, Vector Laboratories, Burlingame, CA) for 1 hr. After being rinsed, the sections were processed with 3.3
-diaminobenzidine (DAB), coverslipped, and photographed. Adjacent sections were processed for parvalbumin, calbindin, and Nissl bodies. In addition, immunocytochemical labeling of GABA was also performed on some sections (Schwartz and Meineke, 1992
RESULTS
Intracellular recording from neurons in sagittal slices of the ferret LGNd maintained in vitro in which the microelectrode was within or adjacent to the interlaminar zones revealed four electrophysiologically distinct cell types. The most prominent cell type exhibited electrophysiological features previously associated with thalamocortical neurons (Fig. 1A) (Jahnsen and Llinás, 1984a
Fig. 1. Four electrophysiologically distinct types of neurons were recorded in or adjacent to the interlaminar zones. A, Thalamocortical neurons generated a train of action potentials, with a delayed onset, in response to depolarization and a rebound burst of action potentials after hyperpolarization. These cells also exhibited a strong ``sag'' in the electrotonic response to hyperpolarization. B, ``Classic'' interneurons exhibited a train of action potentials in response to a depolarizing current pulse and a rebound response of one or a few action potentials in response to a hyperpolarizing current pulse. These interneurons were recorded throughout the laminae and within the interlaminar zones. C, ``Chirping'' interneurons generated repetitive burst discharges at 10-15 Hz in response to depolarizing or as a rebound response to hyperpolarizing current pulses. So far these neurons have only been recorded in or adjacent to the interlaminar regions of the LGNd. D, PGN-like interneurons exhibited a burst of action potentials in response to depolarization from a membrane potential of61 to
[View Larger Version of this Image (23K GIF file)]
A third category of neuron exhibited electrophysiological features that have been associated previously with neurons in the perigeniculate nucleus or the thalamic reticular nucleus (n = 15) (Mulle et al., 1986
Finally, on rare occasions, an additional type of interneuron was recorded that was distinct from the other categories (n = 6). These neurons also exhibited relatively short-duration action potentials (0.28-0.32 msec at half-amplitude) as did the other two types of interneurons, but were distinct in that these cells generated rhythmic bursts of action potentials at frequencies of 12-15 Hz after the intracellular injection of a hyperpolarizing or depolarizing current pulses (Fig. 1C). In addition, the frequency of action potential generation within each of these bursts was unusually high, within the range of 600-750 Hz. Here we refer to these cells as ``chirping'' interneurons because of the unusual sound of their repetitive burst discharges over the audio monitor. Chirping interneurons were found only when the recording electrode appeared to be in the interlaminar zones and the intracellular injection of biocytin into these cells revealed on two occasions cell bodies that were within or adjacent to the interlaminar zones. In the present report, we focus on the electrophysiological, morphological, and interactions with thalamocortical neurons of the PGN-like interlaminar interneurons.
Electrophysiological properties of PGN-like interlaminar interneurons
Intracellular injection of depolarizing and hyperpolarizing current pulses as well as a slowly depolarizing and hyperpolarizing ramp of current into the PGN-like interlaminar interneurons revealed that these cells possess two distinct modes of action potential generation (Fig. 2). Tonic depolarization of these neurons positive to approximately
Fig. 2. Electrophysiological properties of PGN-like interlaminar cells. A, Intracellular injection of a hyperpolarizing current pulse after depolarization of the neuron into the single-spike firing mode with the injection of d.c. results in the generation of a rhythmic sequence of burst discharges that are interspaced by hyperpolarizing potentials. The first and second bursts are expanded for detail. In B, intracellular injection of a depolarizing current pulse results in the generation of a train of action potentials. A portion of the tonic firing is expanded for detail. In B, intracellular injection of a slowly depolarizing and hyperpolarizing current ramp illustrates the two firing modes of PGN-like interlaminar neurons. At a threshold of approximately
[View Larger Version of this Image (27K GIF file)]
Intracellular injection of a slowly depolarizing and hyperpolarizing current ramp illustrated well the two different firing modes (Fig. 2C). Slowly depolarizing PGN-like interlaminar neurons resulted in the activation of a rhythmic burst of action potentials at membrane potentials of approximately
Functional connectivity of interlaminar interneurons
We have reported previously that sagittal slices of the ferret LGNd spontaneously generate spindle waves at the rate of approximately one every 5-20 sec (von Krosigk et al., 1993
Fig. 3. PGN-like interlaminar interneurons participate in the generation of synchronized network oscillations. A, Simultaneous intracellular recording from a PGN-like interlaminar neuron and an extracellular multiple-unit recording from lamina A during the generation of a spindle wave. B, Expansion of the indicated part of the recording in A illustrates the arrival of barrages of EPSPs in the interlaminar neuron and the generation of low-threshold Ca2+ spike-mediated bursts of action potentials. C, Bath application of bicuculline-methiodide (20 µM) transforms the oscillation into a synchronized slow oscillation characterized by strong burst firing in both the extracellular recording and the interlaminar neuron. D, Expansion of the indicated portion of the recording in C.
[View Larger Version of this Image (29K GIF file)]
Close examination of the fast EPSPs arriving in PGN-like interlaminar interneurons during the generation of spindle waves (or when glutamate was applied to the A laminae) revealed that individual EPSPs were 0.7 to 4.2 mV (2.26 ± 1.06 mV; n = 50 in 1 cell) in amplitude at membrane potentials of
Fig. 4. PGN-like interlaminar interneurons receive rhythmic barrages of EPSPs at 0.5-4 Hz. A, Intracellular recording from a PGN-like interlaminar interneuron illustrating the regular occurrence of rhythmic barrages of EPSPs arriving at ~1.5 Hz (indicated by arrows). B, Expansion of the indicated (a, b) EPSP barrages illustrates that they are composed of 3-5 individual EPSPs that occurred at 125-280 Hz and overlapped temporally. These bursts of EPSPs presumably result from rhythmic bursts of action potentials in a single thalamocortical cell. C, Comparison of the action potential discharge of a bursting thalamocortical cell (chosen at random) with the EPSP barrage in the PGN-like interlaminar neuron.
[View Larger Version of this Image (14K GIF file)]
We have demonstrated previously that bath application of the GABAA receptor antagonist bicuculline transforms normal spindle waves into marked 2-3 Hz synchronized paroxysmal oscillations (von Krosigk et al., 1993
PGN-like interlaminar cells form disynaptic loops with thalamocortical neurons
One functionally important property of PGN neurons is that they form disynaptic reciprocal connections with thalamocortical cells in the A and C laminae of the ferret LGNd (von Krosigk et al., 1993In support of this hypothesis, we often observed the arrival of rhythmic barrages of EPSPs in interlaminar interneurons at a rate of ~1-4 Hz (1.54 ± 0.18 Hz) in between the generation of spindle waves (Fig. 4). Close examination of these PSP barrages revealed that they consisted of 3-5 EPSPs with an interburst frequency of 125-280 Hz, in similarity with the frequency range with which action potential are generated by thalamocortical cells during the generation of intrinsic bursts. This rhythmic pattern of EPSP barrages is similar to that which occurs spontaneously in a subpopulation of thalamocortical cells through intrinsic membrane mechanisms and which is known as the ``delta'' oscillation (McCormick and Pape, 1990a
Further support for the excitation of interlaminar interneurons by thalamocortical cells was obtained with the activation of the later through the local application of glutamate in the A laminae (Fig. 5). Local application of glutamate (~2-10 pl in a drop ~8-25 µm in diameter) within the A or A1 laminae resulted in the generation of barrages of EPSPs or IPSPs in interlaminar interneurons (Fig. 5A). The EPSPs arrived from lamina A1 at latencies of 30-40 msec, which suggests a direct monosynaptic connection (see Materials and Methods). Interestingly, the application of glutamate to lamina A1 excited these PGN-like interneurons in the interlaminar zone between A and A1 more often than did applications of glutamate to lamina A. IPSPs could also be activated from laminae A or A1, although these exhibited longer latencies (60-100 msec), suggesting that they are attributable to the activation of perigeniculate cells or PGN-like interlaminar interneurons by the excitation of thalamocortical cells with glutamate (Fig. 5A, lamina A). In addition to the initial barrage of synaptic potentials, local application of glutamate in the A laminae or PGN typically also activated spindle waves. These glutamate-initiated spindle waves resulted in the arrival of cyclical barrages of EPSPs in PGN-like interlaminar interneurons after the initial barrage of EPSPs or IPSPs (see Fig. 5B).
Fig. 5. Interlaminar interneurons are excited by thalamocortical cells and inhibited by PGN neurons. A, Activation of laminae A or A1, or the PGN with the local application of glutamate (glu; 0.5 mM in micropipette) during an intracellular recording from a PGN-like interlaminar neuron. Activation of the PGN resulted in barrages of IPSPs followed by the generation of a spindle wave, which appeared as barrages of EPSPs and the generation of burst discharges (full spindle not shown). Pressure application of glutamate to lamina A1 resulted in the rapid activation of EPSPs in the interlaminar neuron. In contrast, pressure application of glutamate to lamina A resulted in the activation of IPSPs at a latency of 80-100 msec, followed by the generation of a spindle wave. This long-latency inhibition probably results from the excitation of PGN or PGN-like interneurons by thalamocortical cells activated by glutamate. Each trace is the average of five consecutive glutamate applications with a 10-15 sec interval. B, Increasing the duration of the glutamate application in the PGN increases the amplitude and duration of the inhibition of the PGN-like interlaminar neuron. C, Activation of a burst of action potentials through the intracellular injection of a depolarizing or hyperpolarizing current pulse in this interlaminar neuron results in the generation of return or feedback EPSPs, indicating that the cell is disynaptically connected with thalamocortical cells. D, Illustration of the intracellular recording site and locations of glutamate applications corresponding to the recordings in A.
[View Larger Version of this Image (25K GIF file)]
Repeated application of glutamate in various regions of the A and A1 laminae during the intracellular recording from a PGN-like interlaminar interneuron revealed that these cells received presumed direct excitatory input from only a subportion of these laminae (Fig. 5). Indeed, it was often found that moving the glutamate-applying electrode by as little as 50 µm, either through the thickness of the slice or in the dorsal-ventral or anterior-posterior axis, could result in the abolition of the glutamate-activated EPSPs. This result suggests that the glutamate application remained localized to small regions of the laminae and that only localized regions project to the interlaminar interneurons.
In addition to receiving excitatory inputs from the A laminae, the PGN-like interlaminar interneurons were inhibited by application of glutamate in the PGN (Fig. 5B). Again, the region of the PGN that was effective in inhibiting interlaminar interneurons was relatively circumscribed, and repeated applications of glutamate at multiple locations were performed so as to identify these regions. In general, the regions of the A laminae and the PGN that provided synaptic inputs to the interlaminar interneurons were found to lie on the anterior-posterior axis, along the lines of retinotopic organization.
Intracellular injection of a depolarizing or hyperpolarizing current pulse into PGN-like interlaminar neurons, so as to generate a low-threshold Ca2+-spike-mediated burst of action potentials, often resulted in the appearance of a barrage of EPSPs (Figs. 5C, 6, 7). These return barrages of EPSPs were composed of a composite of small EPSPs similar to those that occur during the generation of a spindle wave (Fig. 6). The average latency for the generation of these return barrages of EPSPs was 140 ± 4 msec, which is similar to the interval between barrages of EPSPs during the generation of spindle waves (202 ± 38 msec; Fig. 6). These data indicate that interlaminar interneurons form disynaptic loops with thalamocortical neurons in the A and C laminae.
Fig. 6. Characteristics of return EPSPs in PGN-like interlaminar interneurons. A, Intracellular injection of a depolarizing current pulse in a PGN-like interlaminar interneuron located between laminae A and A1 results in the generation of return barrages of EPSPs as well as a short spindle wave. B, Expansion of the indicated portion in A. C, Several examples of return EPSPs generated by burst firing in this neuron are illustrated. The average latency for the onset of these return EPSPs is 140 msec from the first action potential in the burst. Also illustrated are barrages of EPSPs that occur during the generation of a spindle wave. Here the average latency between barrages is 202 msec.
[View Larger Version of this Image (21K GIF file)]
Fig. 7. The amplitude of feedback EPSPs in PGN-like interlaminar interneurons is decreased during the spindle wave refractory period. A, B, Intracellular injection of a depolarizing current pulse while the cell is hyperpolarized to
[View Larger Version of this Image (32K GIF file)]
We have reported recently that the ability of PGN neurons to induce rebound burst firing in thalamocortical cells, and a subsequent return barrage of EPSPs, is depressed after the generation of a spindle wave (Bal and McCormick, 1995
The ability of PGN-like interlaminar interneurons to activate return barrages of EPSPs after a burst of action potentials indicates that these cells inhibit thalamocortical neurons at least in part through the activation of GABAA receptors. We tested this hypothesis through the application of glutamate to the interlaminar zones while recording from thalamocortical cells in the neighboring A laminae (Fig. 8). Local application of glutamate to the interlaminar zone resulted in the generation of barrages of IPSPs in thalamocortical cells (Fig. 8A). Close examination of these barrages of IPSPs revealed on occasion that they consisted in part of discrete events occurring at frequencies of 50-70 Hz (Fig. 8A, enlargement).
Fig. 8. Activation of the interlaminar zone results in the inhibition of thalamocortical cells. A, Intracellular recording from a thalamocortical cell in lamina A1 during local application of glutamate in the interlaminar zone between A and A1 (see drawing in B). Glutamate application resulted in the generation of barrages of IPSPs that, on closer examination (expanded trace), appeared as a barrage of discrete IPSPs at 50-70 Hz. B, Local application of bicuculline (400 µM in micropipette) into the interlaminar zone (I.Z.) enhanced the amplitude of the IPSPs generated in this thalamocortical cell in response to identical local application of glutamate in the interlaminar zone. Local application of bicuculline to the region of the recorded thalamocortical cell substantially reduced the amplitude of the glutamate-evoked IPSP. Superimposed traces correspond to consecutive glutamate applications with a 15 sec interval. C, Application of increasing doses of glutamate in the interlaminar zone between A1 and C results in increasingly large IPSPs in a thalamocortical cell in lamina A1. These applications of glutamate could also activate spindle waves in thalamocortical and PGN networks. Bath application of bicuculline results in an abolition of these fast IPSPs and reveals the occurrence of slower, presumed GABAB-mediated, IPSPs. D, Intracellular recording from a PGN-like interlaminar neuron before and after local application of bicuculline (bic). Activation of the A1-lamina with the local application of glutamate (glu) results in a barrage of EPSPs and the activation of a burst discharge in the interlaminar neuron (1). Repetitive glutamate application (10-15 sec interval) results in EPSP barrages that are both sub and suprathreshold (2). Local application of bicuculline (0.4 mM in micropipette) results in a marked prolongation of the burst discharge in this interlaminar neuron (3, 4), even though the EPSP barrages induced by the glutamate application were of approximately the same amplitude (4).
[View Larger Version of this Image (30K GIF file)]
Local application of bicuculline methiodide (400 µm in micropipette) to the interlaminar zones resulted in an enhancement of the IPSPs evoked by glutamate application (Fig. 8B). These IPSPs were mediated in large part through the activation of GABAA receptors, because local application of bicuculline to the region of the recorded thalamocortical cell resulted in a substantial decrease in their amplitude (Fig. 8B). Increasing the dose of glutamate application in the interlaminar zones resulted in an increasing amplitude of the evoked IPSPs, including those evoked in normal bathing medium (Fig. 8C) and those observed after the bath application of bicuculline (Fig. 8C).
These results suggest that the duration of burst discharges in PGN-like interlaminar neurons is enhanced by the local application of bicuculline. To test this hypothesis, we activated localized regions of either lamina A or lamina A1 with local glutamate application and examined the effects of local application of bicuculline to the intralaminar zones on the response of the PGN-like interlaminar neuron (Fig. 8D). Local application of bicuculline resulted in a substantial enhancement of the burst discharges in the interlaminar neuron evoked by the glutamate evoked EPSP barrages (compare Fig. 8, D1 and D3).
Responses of interlaminar interneurons to neuromodulators
We have demonstrated previously that application of 5-HT or the glutamate metabotropic receptor agonist trans-(1S,3R)-1-amino-1,2-cyclopentanedicarboxylic acid (1S,3R-ACPD) to perigeniculate neurons results in pronounced excitation (McCormick and Wang, 1991Application of 5-HT (500 µM in micropipette) resulted in a prolonged depolarization of interlaminar interneurons (n = 3; Fig. 9A). This prolonged depolarization was associated with a switch from the burst firing mode to the single-spike mode of action potential generation (Fig. 9A), as well as a decreased responsiveness to the barrages of EPSPs occurring during the generation of a spindle wave (Fig. 9A). In contrast, application of 5-HT to intralaminar interneurons resulted either in no detectable response (n = 2) or in a prolonged hyperpolarization that was associated with an increase in membrane conductance (n = 2; Fig. 9C). As in the cat and guinea pig LGNd (McCormick and Pape, 1990b
Fig. 9. PGN-like interlaminar neurons are pharmacologically similar to PGN neurons, but not local interneurons. A, Intracellular recording from a PGN-like interlaminar interneuron during the generation of spindle waves. Local application of serotonin (5HT; 0.5 mM in micropipette) results in membrane depolarization, the occurrence of single-spike activity, and the inhibition of burst firing. B, Application of 5-HT to a PGN neuron exhibits the same response. C, Intracellular recording from a classical intralaminar interneuron during the generation of a spindle wave reveals that this cell exhibits only small (~1 mV) depolarizations during these events (arrows). Application of 5-HT to this cell results in membrane hyperpolarization. D, Local application of 1S,3R-ACPD (500 µM in micropipette) results in a prolonged depolarization and switch from the burst to single-spike mode of action potential generation. E, Local application of acetylcholine (ACh) to a PGN-like interlaminar interneuron results in a rapid excitation followed by a more prolonged hyperpolarization. F, Local application of the peptide CCK8S (CCK) to a PGN-like interlaminar interneuron results in a slow depolarization of the membrane potential.
[View Larger Version of this Image (37K GIF file)]
Similar to 5-HT, application of 1S,3R-ACPD (0.5 mM in micropipette) to PGN-like interlaminar interneurons also resulted in a pronounced depolarization (Fig. 9D) as well as the activation of tonic activity. The application of ACh to PGN-like interlaminar interneurons was associated with a rapid depolarization and the generation of action potentials followed by a more prolonged hyperpolarization (Fig. 9E). In contrast, application of ACh to local interneurons resulted in the hyperpolarizing response only (data not shown). Finally, local application of the neuropeptide cholecystokinin-8-sulfate (CCK-8-S) (Fig. 9F) to PGN-like interlaminar interneurons resulted in a prolonged depolarization of the membrane potential, as found for cells in the thalamic reticular or perigeniculate nuclei (Cox et al., 1995
PGN-like interlaminar interneurons densely innervate the relay laminae
Four PGN-like interlaminar neurons were intracellularly filled with biocytin. The cell bodies of these neurons were situated within the interlaminar zones either between laminae A and A1 (n = 3; Fig. 10) or between laminae A1 and C (n = 1). These cells exhibited two distinct types of process extending into the local neuropil. Presumed dendritic processes were substantially thicker than apparent axonal processes and exhibited a relatively smooth appearance. These dendritic processes extended away from the cell body in the dorsal and ventral directions with a preference for the plane of the interlaminar zone. In the cell reconstructed in Figure 10, the presumed dendritic processes remain largely within the interlaminar zone between laminae A and A1. In all four cells, the dendritic processes extended from 100 to 200 µm away from the cell body in both the dorsal and the ventral directions. A second type of process, presumed axon collaterals, was composed of highly branched, thin-beaded processes that extended into the local neuropil as well as into the A laminae and even into the PGN (Fig. 10). The cell reconstructed in Figure 10 exhibited both posteriorly and anteriorly oriented axonal branches. In the posterior direction, these axonal fibers extensively innervate laminae A1 and C (Fig. 10). In the anterior direction, there is a much sparser innervation of lamina A. These axonal branches were observed to give rise to occasional axonal swellings (``beads'') as it passed through lamina A, and then to bifurcate within the PGN (Fig. 10). In the other three cells, presumed axonal collaterals densely innervated the local neuropil within the field of the dendritic arbor within the interlaminar zone as well as extending into the adjacent lamina. In two of the three cells there appeared to be a preference for the innervation of the more posterior laminae, i.e., lamina A1 for one cell and lamina C for the other. The other cell, located between laminae A and A1, gave rise to a dense innervation of lamina A and only a sparse innervation of lamina A1. Interestingly, this cell also received strong barrages of EPSPs when lamina A was activated locally with glutamate application.
Fig. 10. Morphological properties of a PGN-like interlaminar interneuron. A, Dark-field illumination of a PGN-like interlaminar interneuron. B, Camera lucida reconstruction of the cell in A. The cell body and dendrites were located in the A-A1 interlaminar zone (IZ). The axon densely innervated lamina A1 and also innervated lamina C. One branch of the axon passed through lamina A and into the PGN. Recordings from this neuron are illustrated in Figures 2, 4, 8D, and 9D.
[View Larger Version of this Image (74K GIF file)]
Thalamocortical cells give rise to axon collaterals in the interlaminar zones
Close examination of axons emanating from thalamocortical cells revealed on occasion axon collaterals within the interlaminar zones between A and A1 (n = 6) or between A1 and C (n = 1), with the main branch of the axon extending toward the PGN and optic radiation (Fig. 11). These axonal branches appeared as fine processes extending into the interlaminar zones from the parent axon as it traveled through the laminae toward the optic radiation. Each process typically took a circuitous route in the local neuropil of the interlaminar zone and could give rise to additional axon collaterals (Fig. 11). Each axon collateral exhibited repeated swellings at intervals of ~2-8 µm, presumably representing presynaptic terminals. The full extent of innervation of the interlaminar zone in the dorsal-ventral plane varied from 50 to 100 µm, and the average number of swellings was 28 ± 14 (n = 7). No thalamocortical neurons were observed to give rise to axon collaterals in an interlaminar zone that was posterior to the location of their cell body (i.e., not on the trajectory between their cell body and the optic radiation). The same axons that provided collaterals within the interlaminar zones also gave rise to collaterals within the perigeniculate nucleus (Fig. 11). Collaterals within the perigeniculate nucleus typically were slightly more extensive than those in the interlaminar zones, extending from 100 to 300 µm in the dorsal-ventral plane (for example, see Fig. 11).
Fig. 11. Thalamocortical cells can give rise to axon collaterals in the interlaminar zones and in the perigeniculate nucleus. A, Camera lucida reconstruction of a thalamocortical cell in lamina A1. B, Dark-field illumination of this thalamocortical neuron. C, Photograph of axon collaterals in the PGN. D, Photograph of axon collaterals in the interlaminar zone between laminae A and A1. This thalamocortical cell gave rise to axon collaterals in both the interlaminar zone (A, B, D) and the PGN (A-C). The reconstruction in A is of the section photographed in B.
[View Larger Version of this Image (86K GIF file)]
Examination of the axonal arbors of perigeniculate neurons in the ferret LGNd sometimes revealed dense innervations of the interlaminar zones (n = 6; Fig. 12). The axonal arbors of some PGN neurons were found to selectively innervate restricted regions of both the A and C laminae as well as either the interlaminar zones between A and A1 or between A1 and C (Fig. 12).
Fig. 12. Perigeniculate neurons can innervate the interlaminar zones. A-C, Dark-field illumination of sequential 75 µm sections through the axonal innervation of the A and C laminae and interlaminar zones by a PGN neuron. D-F, Higher-magnification photographs of the axon collaterals in the laminae and interlaminar zones (IZ). This PGN neuron densely innervates lamina A (D) and the interlaminar zone between A and A1 (E) as well as between A1 and C (F). Double arrowheads in B and C are for orientation in E and F, respectively.
[View Larger Version of this Image (116K GIF file)]
Parvalbumin- and calbindin-containing cells in the interlaminar region
Previous examination of parvalbumin and calbindin cells in the cat LGNd have demonstrated that PGN cells are positive for parvalbumin, but not for calbindin (Demeulemeester et al., 1991
Fig. 13. Parvalbumin- and calbindin-immunoreactive cells in the ferret geniculate. A, Nissl stain of a sagittal slice of the ferret geniculate. The interlaminar zone between A and A1 is clearly seen as a relatively cell-sparse region. Note that a similar region occurs between lamina A and the PGN. B, Calbindin-immunoreactive cells in an adjacent section. Calbindin-positive cells are found throughout the A and C laminae but not in the PGN. Occasional calbindin-positive cells are found in the interlaminar zones. C, Parvalbumin-positive cells are found throughout the PGN and in the interlaminar zone between laminae A and A1. The indicated area is illustrated in Figure 14. Sections A-C are adjacent 20 µm sections. The asterisks indicate the same blood vessel. D, GABA-immunoreactive cells in the ferret LGNd. GABA-immunoreactive cells are found in the PGN and throughout the intralaminar and interlaminar regions of the LGNd.
[View Larger Version of this Image (139K GIF file)]
Fig. 14. Photograph of parvalbumin-positive cells in the interlaminar zone between A and A1 (indicated by arrowheads). B, Close-up of two of the parvalbumin-positive cells. Note that the initial segments of the dendrites are parallel to the interlaminar zone.
[View Larger Version of this Image (195K GIF file)]
Examination of Nissl-stained sagittal sections of the ferret LGNd revealed not only clearly defined interlaminar zones between laminae A and A1, but also a relatively cell-free zone between lamina A and the PGN (Fig. 13A). Examination of this zone between lamina A and the PGN revealed that it was similar to the interlaminar zone between A and A1 in that it contained parvalbumin-positive cells as well as, on rare occasions, calbindin-positive cells. Intracellular recordings from this zone reveal a mixture of cells with the electrophysiological properties of thalamocortical or perigeniculate cells.
DISCUSSION
Based on the types of synaptic contacts found with electron microscopic examination of GABAergic interlaminar interneurons in the cat LGNd, as well as the orientation of primary dendrites and soma size, Montero (1989a)Electrophysiologically, PGN-like interlaminar interneurons and PGN neurons both exhibit two modes of action potential generation. At membrane potentials negative to approximately
Anatomically, PGN-like interlaminar interneurons exhibit dendritic arbors and axonal interconnections that are similar to those of PGN cells. The dendritic arbor of our small sample of PGN-like interlaminar interneurons was often oriented parallel to the interlaminar zones, similar to the orientation of the dendritic arbors of perigeniculate neurons (Szentágothai, 1972
The axonal interconnections of both PGN-like interlaminar interneurons and PGN cells reveal that both form disynaptic loops with thalamocortical cells. PGN-like interlaminar interneurons densely innervated laminae adjacent to the interlaminar zone in which their cell body is located. Our examination of intracellularly filled thalamocortical cells reveals that some of these cells give rise to axon collaterals that form putative synaptic contacts in the interlaminar zone between A and A1 and between A1 and C, in agreement with previous examination of the intrageniculate axonal projections of thalamocortical cells (Friedlander et al., 1981
Interestingly, we found that interlaminar interneurons are inhibited by activation of perigeniculate neurons, in a manner similar to the inhibition of perigeniculate cells by other perigeniculate neurons (Bal et al., 1995b
Pharmacologically, PGN-like interlaminar interneurons and PGN neurons were quite similar, yet distinct from intralaminar interneurons. Both PGN and PGN-like interlaminar interneurons were strongly depolarized by 5-HT, by the glutamate metabotropic receptor agonist 1S,3R-ACPD, and by CCK8S, and they also exhibited a rapid excitatory response, followed by a more prolonged hyperpolarizing response, to ACh (McCormick and Wang, 1991
Functionally, we have demonstrated that both PGN and PGN-like intralaminar interneurons from disynaptic loops with thalamocortical cells, such that the activation of a burst of action potentials in a PGN-like interlaminar or PGN interneuron can generate a rebound burst of action potentials in several thalamocortical cells, which then results in the generation of a return barrage of EPSPs (Bal et al., 1995b
The timing of the disynaptic loop between PGN-like interlaminar interneurons and thalamocortical cells indicates that these cells inhibit thalamocortical neurons through GABAA receptors (see Bal et al., 1995a
As with perigeniculate cells, the presence of disynaptic loops between PGN-like interlaminar cells and thalamocortical neurons allows for the interlaminar cells to actively participate in the generation of spindle waves and bicuculline-induced paroxysmal oscillations (Bal et al., 1995a
The generation of spindle waves and the bicuculline-induced slow oscillation are associated with a relative refractory period of 5-20 sec during which the threshold for activation of additional network oscillations is dramatically increased (Kim et al., 1995
The examination of the distribution of neurons that contain parvalbumin, a low molecular weight calcium-binding protein, in the rat, guinea pig, cat, and primate LGNd have demonstrated marked species differences. However, the PGN in all species is positive for the presence of parvalbumin (Stichel et al., 1988
As in other species, we found that the cells of the ferret perigeniculate nucleus stain intensely for the presence of parvalbumin, and not for calbindin. However, we also found parvalbumin-positive neurons to be present in the interlaminar zones of the ferret LGNd and to be relatively sparse within the relay laminae. Calbindin-positive cells were relatively sparse in the ferret PGN but were abundantly present within the relay laminae. Calbindin-positive cells were also relatively sparse within the interlaminar zones, although some were clearly present. Although we have not yet directly examined this, we propose that the PGN-like interlaminar interneurons that we have characterized here may be represented by the parvalbumin-containing neurons, similar to those of the perigeniculate nucleus.
Developmentally, the ferret PGN and LGNd exhibit distinct origins with the perigeniculate forming from the ventral thalamus and the LGNd arising from the dorsal thalamus (see Mitrofanis, 1994
The anatomical or physiological properties of interlaminar zone GABAergic neurons have only been examined in the cat (Montero, 1989a
In conclusion, we suggest that there exist in the interlaminar zones in the carnivore LGNd neurons that are functionally equivalent to perigeniculate neurons in every regard. Indeed, we propose that these neurons are actually part of the perigeniculate nucleus. One possibility that requires further examination is that the PGN actually forms a continuous sheet of GABAergic neurons that gives rise to laminae or ``fingers'' that penetrate within the body of the LGNd such that the layers of PGN and LGNd are intercalated.
FOOTNOTES
Received May 22, 1996; revised July 1, 1996; accepted July 8, 1996.
a The first two authors contributed equally to this research.Correspondence should be addressed to Dr. David A. McCormick, Section of Neurobiology, Yale University School of Medicine, New Haven, CT 06510.
This research was supported by grants from National Institutes of Health, the Klingenstein Fund, the Sloan Foundation, and the Medical Research Council. M.V.S.V. is a fellow of NATO and the Epilepsy Foundation of America. We thank Françoise Bal for her assistance in the camera lucida reconstruction of neurons, Anita Luthi for her comments on this manuscript, and Kendall Lee for his contribution. The original data included in the manuscript and a program for viewing and analyzing this data are located at the following web site: http://info.med.yale.edu/neurobio/mccormick/mccormick.
REFERENCES
- Aghajanian GK, Rasmussen K (1989) Intracellular studies in the facial nucleus illustrating a simple new method for obtaining viable motoneurons in adult rat brain slices. Synapse 3:331-338 . [ISI][Medline]
- Avanzini G, de Curtis M, Panzica F, Spreafico R (1989) Intrinsic properties of nucleus reticularis thalami neurones of the rat studied in vitro. J Physiol (Lond) 416:111-122 .
[Abstract/Free Full Text] - Avoli M, Gloor P, Kostopoulos G, Gotman J (1983) An analysis of penicillin-induced generalized spike and wave discharge using simultaneous recordings of cortical and thalamic single neurons. J Neurophysiol 50:819-837 .
[Abstract/Free Full Text] - Bal T, McCormick DA (1993) Ionic mechanisms of rhythmic burst firing and tonic activity in the nucleus reticularis thalami: a mammalian pacemaker. J Physiol (Lond) 468:669-691 .
[Abstract/Free Full Text] - Bal T, McCormick DA (1995) A mechanism for the waning of thalamic spindle and ``absence-seizure like'' oscillations in vitro. Soc Neurosci Abstr 21:11.
- Bal T, McCormick DA (1996) What stops synchronized thalamocortical oscillations? Neuron, in press.
- Bal T, von Krosigk M, McCormick DA (1995a) Synaptic and membrane mechanisms underlying synchronized oscillations in the ferret LGNd in vitro. J Physiol (Lond) 483:641-663 . [ISI][Medline]
- Bal T, von Krosigk M, McCormick DA (1995b) Role of the ferret perigeniculate nucleus in the generation of synchronized oscillations in vitro. J Physiol (Lond) 483:665-685 . [ISI][Medline]
- Contreras D, Curró Dossi R, Steriade M (1993) Electrophysiological properties of cat reticular thalamic neurones in vivo. J Physiol (Lond) 470:273-294 .
[Abstract/Free Full Text] - Cox CL, Huguenard JR, Prince DA (1995) Cholecystokinin depolarizes rat thalamic reticular neurons by suppressing a K+ conductance. J Neurophysiol 74:990-1000 .
[Abstract/Free Full Text] - De Biasi S, Arcelli P, Spreafico R (1994) Parvalbumin immunoreactivity in the thalamus of guinea pig: light and electron microscopic correlation with gamma-aminobutyric acid immunoreactivity. J Comp Neurol 348:556-569 . [ISI][Medline]
- Demeulemeester H, Vandesande F, Orbain GA, Heizmann CW, Pochet R (1989) Calbindin D-28K and parvalbumin immunoreactivity is confined to two separate neuronal subpopulations in the cat visual cortex whereas partial coexistence is shown in the dorsal lateral geniculate nucleus. Neurosci Lett 99:6-11 . [ISI][Medline]
- Demeulemeester H, Arckens L, Vandesande F, Orban GA, Heizmann CW, Pochet R (1991) Calcium binding proteins as molecular markers for cat geniculate neurons. Exp Brain Res 83:513-520 . [ISI][Medline]
- Domich L, Oakson G, Steriade M (1986) Thalamic burst patterns in the naturally sleeping cat: a comparison between cortically projecting and reticularis neurones. J Physiol (Lond) 379:429-449 .
[Abstract/Free Full Text] - Dubin MW, Cleland BG (1977) The organization of visual inputs to interneurons of the lateral geniculate nucleus of the cat. J Neurophysiol 40:410-427 .
[Abstract/Free Full Text] - Ferster D, LeVay S (1978) The axonal arborization of lateral geniculate neurons in the striate cortex of the cat. J Comp Neurol 182:923-944 . [ISI][Medline]
- Friedlander MJ, Lin CS, Stanford LR, Sherman SM (1981) Morphology of functionally identified neurones in the lateral geniculate nucleus of the cat. J Neurophysiol 46:80-129 .
[Free Full Text] - Guillery RW (1970) The laminar distribution of retinal fibers in the dorsal lateral geniculate nucleus of the cat: a new interpretation. J Comp Neurol 138:339-368 . [ISI][Medline]
- Hamos JE, Van Horn SC, Rackowski D, Uhlrich DJ, Sherman SM (1985) Synaptic connectivity of a local circuit neurone in lateral geniculate nucleus of the cat. Nature 317:618-621 . [Medline]
- Hendry SHC (1991) Delayed reduction in GABA and GAD immunoreactivity of neurons in the adult monkey dorsal lateral geniculate nucleus following monocular deprivation or enucleation. Exp. Brain Res 86:47-59.
- Hendry SHC, Yoshioka T (1994) A neurochemically distinct third channel in Macaque dorsal lateral geniculate nucleus. Science 264:575-577.
[Abstract/Free Full Text] - Horikawa K, Armstrong WE (1988) A versatile means of intracellular labeling: injection of biocytin and its detection with avidin conjugates. J Neurosci Methods 25:1-11 . [ISI][Medline]
- Hu B, Steriade M, Deschênes M (1989) The effects of brainstem peribrachial stimulation on perigeniculate neurons: the block of spindle waves. Neuroscience 31:1-12 . [ISI][Medline]
- Huguenard JR, Prince DA (1994) Intrathalamic rhythmicity studied in vitro nominal T current modulation causes robust anti-oscillatory effects. J Neurosci 14:5845-5502.
- Humphrey AL, Weller R (1987) Structural correlates of functionally distinct X-cells in the lateral geniculate nucleus of the cat. J Comp Neurol 268:448-468.
- Ide LS (1982) The fine structure of the perigeniculate nucleus in the cat. J Comp Neurol 210:317-334 . [ISI][Medline]
- Jahnsen H, Llinás R (1984a) Electrophysiological properties of guinea-pig thalamic neurons: an in vitro study. J Physiol (Lond) 349:205-226 .
[Abstract/Free Full Text] - Jahnsen H, Llinás R (1984b) Ionic basis for the electroresponsiveness and oscillatory properties of guinea-pig thalamic neurons in vitro. J Physiol (Lond) 349:227-247 .
[Abstract/Free Full Text] - Jones EG (1985) The thalamus. .
- Jones EG, Hendry SHC (1989) Differential calcium binding protein immunoreactivity distinguishes classes of relay neurons in monkey thalamic nuclei. Eur J Neurosci 1:222-246. [ISI][Medline]
- Jones HE, Sillito AM (1994) The length-response properties of cells in the feline perigeniculate nucleus. Eur J Neurosci 6:1199-1204 . [ISI][Medline]
- Kim U, Bal T, McCormick DA (1995) Spindle waves are propagating synchronized oscillations in the ferret LGNd in vitro. J Neurophysiol 74:1301-1323 .
[Abstract/Free Full Text] - Kozak W, Rodieck RW, Bishop PO (1965) Responses of single units in lateral geniculate nucleus of cat to moving visual patterns. J Neurophysiol 28:19-47.
[Free Full Text] - Lee K, McCormick DA (1995) Acetylcholine excites GABAergic neurons of the ferret perigeniculate nucleus through nicotinic receptors. J Neurophysiol 73:2123-2128 .
[Abstract/Free Full Text] - Lee K, Bal T, McCormick DA (1994) Waking up the sleeping slice: neuromodulation of spindle waves in vitro. Soc Neurosci Abstr 20:133.
- Linden DC, Guillery RW, Cucchiaro J (1981) The dorsal lateral geniculate nucleus of the normal ferret and its postnatal development. J Comp Neurol 203:189-211 . [ISI][Medline]
- Luth HJ, Winkelmann E, Celio MR (1993) Light- and electron microscopic localization of parvalbumin, Calbindin D-28K and calretinin in the dorsal lateral geniculate nucleus of the rat. J Hirnforsch 34:47-56 . [Medline]
- McCormick DA, Pape HC (1988) Acetylcholine inhibits identified interneurons in the cat lateral geniculate nucleus. Nature 334:246-248 . [Medline]
- McCormick DA, Pape HC (1990a) Properties of a hyperpolarization-activated cation current and its role in rhythmic oscillation in thalamic relay neurons. J Physiol (Lond) 431:291-318 .
[Abstract/Free Full Text] - McCormick DA, Pape HC (1990b) Noradrenergic and serotonergic modulation of a hyperpolarization-activated cation current in thalamic relay neurons. J Physiol (Lond) 431:319-342 .
[Abstract/Free Full Text] - McCormick DA, Wang Z (1991) Serotonin and noradrenaline excite GABAergic neurones of the guinea pig and cat thalamic reticular nucleus. J Physiol (Lond) 442:235-255 .
ABSTRACT
