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GABAA Receptor-Mediated Tonic Inhibition in Thalamic Neurons

Authors:
David W Cope
David W Cope
David W Cope
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Stuart Hughes at Vertex Pharmaceuticals
Stuart Hughes
Vincenzo Crunelli at Cardiff University
Vincenzo Crunelli
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Abstract

Tonic GABAA receptor-mediated inhibition is typically generated by delta subunit-containing extrasynaptic receptors. Because the delta subunit is highly expressed in the thalamus, we tested whether thalamocortical (TC) neurons of the dorsal lateral geniculate nucleus (dLGN) and ventrobasal complex exhibit tonic inhibition. Focal application of gabazine (GBZ) (50 microM) revealed the presence of a 20 pA tonic current in 75 and 63% of TC neurons from both nuclei, respectively. No tonic current was observed in GABAergic neurons of the nucleus reticularis thalami (NRT). Bath application of 1 microM GABA increased tonic current amplitude to approximately 70 pA in 100% of TC neurons, but it was still not observed in NRT neurons. In dLGN TC neurons, the tonic current was sensitive to low concentrations of the delta subunit-specific receptor agonists allotetrahydrodeoxycorticosterone (100 nM) and 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol (THIP) (100 nM) but insensitive to the benzodiazepine flurazepam (5 microM). Bath application of low concentrations of GBZ (25-200 nM) preferentially blocked the tonic current, whereas phasic synaptic inhibition was primarily maintained. Under intracellular current-clamp conditions, the preferential block of the tonic current with GBZ led to a small depolarization and increase in input resistance. Using extracellular single-unit recordings, block of the tonic current caused the cessation of low-threshold burst firing and promoted tonic firing. Enhancement of the tonic current by THIP hyperpolarized TC neurons and promoted burst firing. Thus, tonic current in TC neurons generates an inhibitory tone. Its modulation contributes to the shift between different firing modes, promotes the transition between different behavioral states, and predisposes to absence seizures.
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164 Current Literature in Basic Science
ATHALAMIC SLEEP TONIC
GABA
A
Receptor-Mediated Tonic Inhibition in Thalamic Neurons
Cope DW, Hughes SW, Crunelli V
J Neurosci 2005;25:11553–11563
Tonic GABA
A
receptor-mediated inhibition is typically gen-
erated by δ subunit-containing extrasynaptic receptors.
Because the δ subunit is highly expressed in the tha-
lamus, we tested whether thalamocortical (TC) neurons
of the dorsal lateral geniculate nucleus (dLGN) and ven-
trobasal complex exhibit tonic inhibition. Focal applica-
tion of gabazine (GBZ) (50 µM) revealed the presence
of a 20 pA tonic current in 75 and 63% of TC neu-
rons from both nuclei, respectively. No tonic current was
observed in GABAergic neurons of the nucleus reticu-
laris thalami (NRT). Bath application of 1 µM GABA in-
creased tonic current amplitude to 70 pA in 100% of
TC neurons, but it was still not observed in NRT neu-
rons. In dLGN TC neurons, the tonic current was sensitive
to low concentrations of the δ subunit-specific receptor
agonists allotetrahydrodeoxycorticosterone (100 nM) and
4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol (THIP) (100
nM) but insensitive to the benzodiazepine flurazepam (5
µM). Bath application of low concentrations of GBZ (25–
200 nM) preferentially blocked the tonic current, whereas
phasic synaptic inhibition was primarily maintained. Un-
der intracellular current-clamp conditions, the preferential
block of the tonic current with GBZ led to a small depolar-
ization and increase in input resistance. Using extracellular
single-unit recordings, block of the tonic current caused
the cessation of low-threshold burst firing and promoted
tonic firing. Enhancement of the tonic current by THIP hy-
perpolarized TC neurons and promoted burst firing. Thus,
tonic current in TC neurons generates an inhibitory tone. Its
modulation contributes to the shift between different firing
modes, promotes the transition between different behav-
ioral states, and predisposes to absence seizures.
COMMENTARY
T
onic inhibition is a remarkable form of signaling in the
CNS, which contrasts in both form and function from
classic phasic inhibitory signaling. The latter, as described in any
neurobiology text, is the result of neural activity of inhibitory
interneurons, fusion of GABA filled vesicles at synapses, tran-
sient (millisecond) increases in synaptic GABA concentration
to millimolar levels, gating (opening) of GABA
A
receptors, and
chloride ion flux into postsynaptic neurons. These phasic re-
sponses, which have effects whose durations are on the order
of milliseconds to tens of milliseconds, influence the timing,
synchrony, and number of spikes produced in the neuron(s)
receiving the inhibition. Phasic inhibition has surgical-like pre-
cision in terms of timing and focality of inhibitory signaling—it
is localized to the synapse at which GABA is released. If phasic
inhibition is a scalpel, designed to sculpt inputs by selectively
eliminating brief periods of input from specific dendrites, then
tonic inhibition is, pardon the expression, somewhat of a sledge-
hammer.
Tonic inhibition is the steady activation of extrasynap-
tic GABA
A
receptors, which produces a corresponding steady
GABA
A
conductance. Tonic inhibition, thus, increases the elec-
trical conductivity of the neuronal membrane and serves as
a short circuit, such that electrical signals traveling down the
membrane are shunted to the extracellular fluid “ground” and
lose their efficacy. Tonic inhibition can be considered a time-
invariant “veto” of synaptic and intrinsic signals. The veto ex-
erted by tonic inhibition is not absolute; that is, the degree
of tonic inhibition and the efficacy of the shunt will vary de-
pending on the resting level of GABA in the extracellular fluid.
Changes in the efficacy of tonic inhibition have been reported
as a consequence of modulation of the extrasynaptic GABA
A
re-
ceptors by neuromodulators, such as alcohol and neurosteroids
(1), which alter the sensitivity of extrasynaptic GABA receptors
and thus, increase their openings in response to ambient GABA
concentrations.
As far as is known, tonic inhibition does not distinguish
between one dendrite and another and certainly does not change
much over time. However, to date, tonic inhibition has only
been studied in recordings from the soma; it might be that
in different dendrites, tonic inhibition increases or decreases as
GABA released from localized synapses spreads to extrasynaptic
receptors on that particular dendrite. GABA might even be
Epilepsy Currents, Vol. 6, No. 5 (September/October) 2006 pp. 164–166
Blackwell Publishing, Inc.
C
American Epilepsy Society
Current Literature in Basic Science 165
released to specific locations in the extracellular space by the
reversed action of GABA transporters on neuronal and axonal
membranes (2). Recordings from the soma would reveal only
the average GABA conductance from all the dendrites, and so
at this time, there is no specific evidence that tonic inhibition
has any time or location specificity.
What is the point of this sledgehammer approach to inhi-
bition? Certainly, anesthesiologists and epileptologists can pro-
vide a ready answer: tonic inhibition, by reducing or blocking
excitatory synaptic input, is a great way to put neurons to sleep.
Recently, Belelli and colleagues (3) as well as Cope et al., re-
viewed here, have examined thalamic neurons in vitro to deter-
mine whether tonic GABA inhibition might play a role in the
sleep/wake cycle and in the generation of absence seizures (3).
The investigators used a standard method to evaluate tonic
inhibition, which is to evaluate the overall conductance of a
neuron before and after GABA
A
receptors are blocked. Unfor-
tunately, there are no drugs that perfectly select between phasic
and tonic inhibition, so blocking tonic inhibition usually re-
duces phasic inhibition as well. However, because tonic inhibi-
tion is generated by a low concentration of GABA reaching the
extrasynaptic receptors, whereas phasic inhibition is generated
by a very high concentration of GABA in the synaptic cleft, low
concentrations of competitive GABA antagonists are relatively
selective for tonic versus phasic inhibition.
When tonic inhibition was compared with phasic inhibi-
tion, Cope et al. confirmed a finding that is perhaps surprising
but previously had been observed in the cerebellar slices: more
than 90% of the total GABA conductance recorded in vitro
arises from tonic inhibition, leaving less than 10% that arises
from phasic inhibition. While the conductance change underly-
ing tonic inhibition is not large compared with the peak conduc-
tance changes that occur during synaptic GABA release, tonic
inhibition is always present. Thus, the 90% fraction reflects the
steady presence of a small conductance change compared with
the intermittent presence of a much larger conductance change.
The relative contributions of these two types of inhibition to
physiological activity in vivo, where both the intensity of synap-
tic activity as well as the resting levels of extracellular GABA are
likely to be different than in isolated brain slices, remains to be
seen.
Tonic inhibition is not observed in all types of neurons
(4), and both Cope et al. and Belelli et al. established that
this finding also is true in the thalamus. Thus, GABA-releasing
neurons in the nucleus reticularis thalami (nRT) do not ex-
hibit tonic inhibition, whereas thalamocortical relay neurons
in both the dorsal lateral geniculate nucleus and ventrobasal
complex, exhibit tonic inhibition. nRT neurons inhibit the tha-
lamocortical neurons, so agents that increase tonic inhibition
will selectively affect the thalamocortical principal neurons. For
example, benzodiazepines modulate synaptic receptors contain-
ing a γ 2 subunit but do not affect nonsynaptic receptors that
contain a δ subunit. These δ-subunit–containing GABA
A
re-
ceptors are ideal for subserving tonic inhibition, because they
are sensitive to low concentrations of GABA and are resistant
to desensitization (5). Another study showed that membrane
noise, presumably an index of tonic-receptor activation, was
decreased in mice relay neurons of deficient in the GABA
A
re-
ceptor δ subunit (6). Both Cope et al. and Belelli et al. studies
demonstrated that while benzodiazepines do not alter tonic in-
hibition (consistent with the presence of a δ subunit in the
GABA
A
receptors that subserve tonic inhibition), they do en-
hance phasic inhibition. Furthermore, the direct GABA ag-
onist 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol (THIP),
to which such δ-subunit–containing receptors appear to be se-
lectively sensitive, preferentially increased tonic inhibition in
the thalamocortical neurons.
Because thalamocortical neurons express high levels of T-
type calcium channels, membrane hyperpolarization enables
subsequent calcium-dependent burst firing. Thus, enhance-
ment of tonic inhibition by THIP causes the thalamocortical
neurons to begin bursting. As burst firing in thalamocortical
cells underlies sleep (7), the finding that GABA agonists induce
sleep-related behavior in the thalamic slices is interesting and
plausible. This finding by Cope and colleagues was augmented
by the finding of Belelli et al. that THIP administration in
vivo enhances slow-wave sleep activity. In absence epilepsy, tha-
lamocortical neuron burst firing also is prominent; therefore,
the efficacy of benzodiazepines in treatment of absence seizures
makes sense, as the tonic inhibition that promotes bursting
is not sensitive to benzodiazepines. Benzodiazepine efficacy in
absence seizures likely is related to selective enhancement of in-
hibitory signaling in the nRT, which decreases inhibitory output
of this nucleus and suppresses bursting of thalamocortical relay
neurons (8,9).
These new results from Cope et al. suggest one possible
function of tonic inhibition: rapid switching of neurons from
one state (tonic firing that subserves wakefulness) to another
state (burst firing that subserves sleep...and absence seizures).
Changes in ambient GABA concentration would then trigger
the switch. Why this effect might occur via GABA, rather than
a neuromodulator (e.g., acting on potassium conductances), re-
mains unknown. Perhaps, the ability to quickly exit sleep states
by rapidly altering GABA release or uptake provides a more
robust mechanism to become “instantly awake” than would be
possible using neuromodulators acting through second messen-
ger systems.
What do these findings mean for epilepsy treatment? If
benzodiazepines are effective therapy for absence seizures by
virtue of their lack of effect on tonic inhibition in thalamo-
cortical neurons, in addition to the specificity of their effect
on intrathalamic phasic signaling, one would predict that less
166 Current Literature in Basic Science
selective GABA agents should be similarly less effective ther-
apies for absence. This hypothesis seems to hold up well; for
example, barbiturates are effective but nonselective modula-
tors of GABA
A
receptors, and are not effective in the treat-
ment of absence. Tiagabine is a GABA reuptake inhibitor that
increases the concentration of extracellular GABA. Tiagabine
would be expected to enhance tonic inhibition in the thalam-
ocortical neurons and, thus, would not be expected to be an
effective treatment for absence. Accordingly, tiagabine has been
reported to induce absence status in children (10) and enhance
spike wave activity experimentally (11). Thus, the differential
action of selected allosteric GABA modulators on phasic versus
tonic inhibition may have important consequences not only
for the physiology and pharmacology of sleep, but also for the
treatment of epilepsy.
by Kevin J. Staley, MD
and John R. Huguenard, PhD
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... GABAARs are heteropentameric chloride channels, and constituent subunits influence 68 physiological and pharmacological receptor properties. GABAARs containing the δ 69 subunit, encoded by the Gabrd gene, are typically localized to 70 extrasynaptic/perisynaptic sites of certain neuron types, where they participate in tonic 71 and slow synaptic inhibition (Nusser et al., 1998;Cope et al., 2005). By contrast, other 72 subunit combinations mediate fast, phasic inhibition. ...
... δ containing GABAARs have been 73 extensively studied in principal cell types that show high δ subunit expression, including 74 hippocampal dentate gyrus granule cells (DGC) (Wei et al., 2003;Sun et al., 2004Sun et al., , 75 2018Sun et al., , 2020, cerebellar granule cells (CGC) (Nusser et al., 1998;Rossi et al., 2003;76 Rudolph et al., 2020), and thalamocortical cells (TC) Cope et al., 77 2005). In these cell types, receptors containing δ subunits also contain α4 (DGC & TC) 78 or α6 (CGC) subunits, and their activities can modulate cell excitability and transitions 79 between firing modes (Nusser et al., 1998;Sur et al., 1999;Cope et al., 2005;Glykys et 80 al., 2008). 81 ...
... GABAARs containing δ subunits have been primarily studied in select principal cell 531 types (Nusser et al., 1998;Stell and Mody, 2002;Cope et al., 2005;Brickley and Mody, 532 2012). PV+ interneurons also express functional δ subunit containing GABAARs, yet 533 their role in controlling PV+ related brain activity remains unclear (Glykys et al., 2007). ...
A role for δ subunit-containing GABA-A receptors on parvalbumin positive neurons in maintaining electrocortical signatures of sleep states
Preprint
  • Mar 2024
GABA A receptors containing δ subunits have been shown to mediate tonic/slow inhibition in the CNS. These receptors are typically found extrasynaptically and are activated by relatively low levels of ambient GABA in the extracellular space. In the mouse neocortex, δ subunits are expressed on the surface of some pyramidal cells as well as on parvalbumin positive (PV+) interneurons. An important function of PV+ interneurons is the organization of coordinated network activity that can be measured by EEG; however, it remains unclear what role tonic/slow inhibitory control of PV+ neurons may play in shaping oscillatory activity. After confirming a loss of functional δ mediated tonic currents in PV cells in cortical slices from mice lacking Gabrd in PV+ neurons (PV δcKO), we performed EEG recordings to survey network activity across wake and sleep states. PV δcKO mice showed altered spectral content of EEG during NREM and REM sleep that was a result of increased oscillatory activity in NREM and the emergence of transient high amplitude bursts of theta frequency activity during REM. Viral reintroduction of Gabrd to PV+ interneurons in PV δcKO mice rescued REM EEG phenotypes, supporting an important role for δ subunit mediated inhibition of PV+ interneurons for maintaining normal REM cortical oscillations. Significance statement The impact on cortical EEG of inhibition on PV+ neurons was studied by deleting a GABA A receptor subunit selectively from these neurons. We discovered unexpected changes at low frequencies during sleep that were rescued by viral reintroduction.
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... In contrast, application of the agonist THIP (1 µM, a concentration previously shown to be selective for δ subunit-containing receptors [45]) evoked currents of similar magnitude in RR and RQ cortical neurons (mean ± SEM in pA, N) (RR: 21.4 ± 5.7, 4; RQ: 23.8 ± 2.2, 5; p = 0.67; Fig. 3b). A similar profile of effect was observed with allopregnanolone (ALLO: 30 nM; Fig. 3c), a neurosteroid that also selectively activates δ subunit-containing GABA A receptors [46,47]. ...
... Distinct from cortical layer 2/3 neurons, thalamic relay neurons rely solely on δ subunit-containing GABA A receptors to produce inhibitory tonic currents [7,45,48]. We found that RQ relay neurons in the ventrobasal thalamus responded to THIP (1 µM) with 47% of the current produced in RR thalamic neurons (mean ± SEM in pA, N) (RR: 131.7 ± 31.2, 5; RQ: 69.3 ± 22.4, 4, p < 0.05; Fig. 3d). ...
... The link between absence seizures and increased δ subunit-associated GABA A receptor activation in thalamic relay neurons is well established [8][9][10]. The current leading hypothesis from this evidence is that persistent hyperpolarization of thalamic relay neurons favors T-type Ca 2+ channel availability [7,45], making these neurons more susceptible to rhythmic bursting and insensitive to sensory in-put, considered to be a necessary condition for SWD generation [9,10]. Consistent with this hypothesis, ethosuximide and valproic acid, two different T-type Ca 2+ channel blockers, decrease thalamic relay bursting and are currently the main treatment options to treat absence epilepsy. ...
Cortical Tonic Inhibition Gates the Expression of Spike-and-Wave Discharges Associated with Absence Epilepsy
Article
  • Jan 2024
Objective: Absence seizures result from aberrant thalamocortical processing that confers synchronous, bilateral spike-and-wave discharges (SWDs) and behavioral arrest. Previous work has demonstrated that SWDs can result from enhanced thalamic tonic inhibition, consistent with the mechanism of first-line antiabsence drugs that target thalamic low-voltage-activated calcium channels. However, nearly half of patients with absence epilepsy are unresponsive to first-line medications. In this study we evaluated the role of cortical tonic inhibition and its manipulation on absence seizure expression. Methods: We used video-electroencephalogram (EEG) monitoring to show that mice with a γ-aminobutyric acid type A (GABAA) receptor mutation (γ2R43Q) display absence seizures. Voltage-clamp recordings in brain slices from wild type and γ2R43Q mice were used to evaluate the amount of tonic inhibition and its selective pharmacological modulation. Finally, we determined whether modulating tonic inhibition controls seizure expression. Results: γ2R43Q mice completely lack tonic inhibition in principal neurons of both layer 2/3 cortex and ventrobasal thalamus. Blocking cortical tonic inhibition in wild type mice is sufficient to elicit SWDs. Tonic inhibition in slices from γ2R43Q mice could be rescued in a dose-dependent fashion by the synthetic neurosteroid ganaxolone. Low-dose ganaxolone suppressed seizures in γ2R43Q mice. Conclusions: Our data suggest that reduced cortical tonic inhibition promotes absence seizures and that normal function can be restored via selective pharmacological rescue. These results, together with previous findings, suggest that deviations of tonic inhibition either above or below an optimal set point can contribute to absence epilepsy. Returning the thalamocortical system to this set point may provide a novel treatment for refractory absence epilepsy.
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... Extrasynaptic GABAA receptors have a particular configuration, harboring δ instead of γ subunit. The presence of δ subunit (and lack of γ subunit) in the GABAA receptor (δGABAA receptors) results in high affinity towards even low concentrations of ambient 25:75 GABA and slow dissociation kinetics, allowing significant neuromodulation and provides a balance between excitation and inhibition [14][15][16][17]. Tonic inhibition decreases the size and duration of an excitatory postsynaptic potential, which narrows the temporal and spatial window for signal integration and reduces the likelihood of action potential generation [11]. ...
... Tonic inhibition decreases the size and duration of an excitatory postsynaptic potential, which narrows the temporal and spatial window for signal integration and reduces the likelihood of action potential generation [11]. δGABAA receptors are found ubiquitously in nearly all parts of the central nervous and trigeminal systems [11,15,[18][19][20][21][22]. Previous studies have demonstrated the expression of δGABAA receptors in all neuroglial cells [23,24]. ...
Extrasynaptic δGABAA receptors mediate resistance to migraine-like phenotype in rats
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Full-text available
  • May 2024
  • J HEADACHE PAIN
Background GABA, a key inhibitory neurotransmitter, has synaptic and extrasynaptic receptors on the postsynaptic neuron. Background GABA, which spills over from the synaptic cleft, acts on extrasynaptic delta subunit containing GABAA receptors. The role of extrasynaptic GABAergic input in migraine is unknown. We investigated the susceptibility to valid migraine-provoking substances with clinically relevant behavioral readouts in Genetic Absence Epilepsy of Rats Strasbourg (GAERS), in which the GABAergic tonus was altered. Subsequently, we screened relevant GABAergic mechanisms in Wistar rats by pharmacological means to identify the mechanisms. Methods Wistar and GAERS rats were administered nitroglycerin (10 mg/kg) or levcromakalim (1 mg/kg). Mechanical allodynia and photophobia were assessed using von Frey monofilaments and a dark-light box. Effects of GAT-1 blocker tiagabine (5 mg/kg), GABAB receptor agonist baclofen (2 mg/kg), synaptic GABAA receptor agonist diazepam (1 mg/kg), extrasynaptic GABAA receptor agonists gaboxadol (4 mg/kg), and muscimol (0.75 mg/kg), T-type calcium channel blocker ethosuximide (100 mg/kg) or synaptic GABAA receptor antagonist flumazenil (15 mg/kg) on levcromakalim-induced migraine phenotype were screened. Results Unlike Wistar rats, GAERS exhibited no reduction in mechanical pain thresholds or light aversion following nitroglycerin or levcromakalim injection. Ethosuximide did not reverse the resistant phenotype in GAERS, excluding the role of T-type calcium channel dysfunction in this phenomenon. Tiagabine prevented levcromakalim-induced mechanical allodynia in Wistar rats, suggesting a key role in enhanced GABA spillover. Baclofen did not alleviate mechanical allodynia. Diazepam failed to mitigate levcromakalim-induced migraine phenotype. Additionally, the resistant phenotype in GAERS was not affected by flumazenil. Extrasynaptic GABAA receptor agonists gaboxadol and muscimol inhibited periorbital allodynia in Wistar rats. Conclusion Our study introduced a rat strain resistant to migraine-provoking agents and signified a critical involvement of extrasynaptic δGABAergic receptors. Extrasynaptic δ GABAA receptors, by mediating constant background inhibition on the excitability of neurons, stand as a novel drug target with a therapeutic potential in migraine. Graphical abstract
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... Combined with the morphological classification of TC neurons, our observations are consistent with the view that LGd interneuron dendrites are less likely to form F2 profiles with Y-type TC neurons (Famiglietti, 1970;Rafols and Valverde, 1973;Wilson et al., 1984;Hamos et al., 1985;Sherman, 2004) and confirm our previous reports that slow IPSCs were rarely observed onto Y-type TC neurons (Bright et al., 2011). Tonic inhibition has been described in many studies of TC neuron excitability (Belelli et al., 2005;Cope et al., 2005;Chandra et al., 2006;Bright et al., 2007Bright et al., , 2011Herd et al., 2013) and we have shown that tonic inhibition is a feature of both X-and Y-type TC neurons (Bright et al., 2007(Bright et al., , 2011. The dynamic-clamp experiments employed in this study demonstrate how the different types of inhibition present in the thalamus have distinct influences on various aspects of information transfer by thalamocortical neurons. ...
The type of inhibition provided by thalamic interneurons alters the input selectivity of thalamocortical neurons
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A fundamental problem in neuroscience is how neurons select for their many inputs. A common assumption is that a neuron's selectivity is largely explained by differences in excitatory synaptic input weightings. Here we describe another solution to this important problem. We show that within the first order visual thalamus, the type of inhibition provided by thalamic interneurons has the potential to alter the input selectivity of thalamocortical neurons. To do this, we developed conductance injection protocols to compare how different types of synchronous and asynchronous GABA release influence thalamocortical excitability in response to realistic patterns of retinal ganglion cell input. We show that the asynchronous GABA release associated with tonic inhibition is particularly efficient at maintaining information content, ensuring that thalamocortical neurons can distinguish between their inputs. We propose a model where alterations in GABA release properties results in rapid changes in input selectivity without requiring structural changes in the network.
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... It is worth noting that the expression of α4β3δ GABAAR is prominent in brain regions associated with the regulation of sleep, particularly the neocortex and the thalamic ventrobasal complex (VB) (Pirker et al., 2000;Peng et al., 2002). This specific receptor subtype exhibits high sensitivity to gaboxadol, leading to enhanced tonic currents in thalamic relay neurons (Faulhaber et al., 1997;Cope et al., 2005;Winsky-Sommerer et al., 2007). These currents contribute to the production of cortical delta activity that is characteristic of NREM sleep (Brickley, 2018). ...
Nature’s brewery to bedtime: The role of hops in GABAA receptor modulation and sleep promotion
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  • Jan 2024
Insomnia, a prevalent health challenge, often requires pharmacological interventions to improve sleep onset, maintenance, and quality. Benzodiazepines and Z-drugs, like other positive modulators, enhance the inhibitory effects of gamma-aminobutyric acid (GABA) by stabilizing the open conformation of the GABAA receptor (GABAAR) chloride ion channels, facilitating the transition to sleep. However, prolonged use raises concerns, including dependence and cognitive issues. Among herbal alternatives, Humulus lupulus (hops) is gaining attention due to its role as a natural relaxant, sleep aid, and brewing component. Neuroactive phytochemicals in hops may modulate GABAARs differently from benzodiazepines. This research uncovers these hop constituents and potential therapeutic mechanisms. The α-acid humulone and hop prenylflavonoids (PFs), including xanthohumol/isoxanthohumol, 6/8-prenylnaringenin, enhanced GABA-induced displacement of [3H]EBOB, a GABAAR function marker, in the low micromolar range. These potent effects were flumazenil-insensitive and α6β3δ subtype-selective. Molecular docking at the α1β2γ2 isoform identified the extracellular α+/β− interface as the PF binding site. An additional 6-prenylnaringenin site was recognized at the extracellular α+/γ2− interface, aligning with its inhibition of [3H]flunitrazepam and [3H]Ro 15-4513 binding. Given humulone’s prominence and relatively high potency, its activity was confirmed electrophysiologically, where it enhanced GABA-evoked currents in the sedation-mediating α1β3γ2 subtype. In mice, humulone reduced locomotor activity, shortened sleep onset induced by pentobarbital, and prolonged sleep duration induced by either pentobarbital or ethanol. Moreover, [3H]EBOB binding assays showed synergies between humulone and ethanol, and additive interactions with PFs, suggesting enhanced alcohol intoxication in hop-rich beers. In summary, we revealed positive modulators of GABAARs that act independently of the classical benzodiazepine site. 6-prenylnaringenin also acts as a silent modulator with the potential to block benzodiazepine responses. Humulone plays a pivotal role in the sedative and sleep-promoting properties of hops. These findings offer novel mechanistic insights into hop neuroactive constituents and potential avenues for sleep aid optimization.
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... GABAARs in αβδ con guration are ideally suited to convey tonic inhibitory input. [45][46][47] In addition, the presence of δ subunit in the GABAA receptor (δGABAAR) results in the extrasynaptic localization of the receptor. 48 Therefore, we investigated the role of extrasynaptic GABAARs. ...
GAERS are resistant to migraine-provoking agents: The role of extrasynaptic GABAA receptors
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Full-text available
  • Dec 2023
Background Migraine headache is a global health concern, and the role of GABA-related mechanisms is ill-defined in migraine pathogenesis. The role of GABAergic system has never been examined in a migraine model with clinically relevant behavioral readouts. We aimed to investigate the susceptibility to valid and depicted migraine-provoking substances in Genetic Absence Epilepsy of Rats Strasbourg (GAERS), in which GABAergic tonus was altered. Methods Male Wistar and GAERS rats were administered nitroglycerin (10 mg/kg) or levcromakalim (1 mg/kg). Rats were tested for mechanical allodynia with von Frey monofilaments and light aversion using a dark-light box. Effects of tiagabine (5 mg/kg), baclofen (2 mg/kg), diazepam (1 mg/kg), gaboxadol (4 mg/kg), and muscimol (0.75 mg/kg), ethosuximide (100 mg/kg) or flumazenil (15 mg/kg) on levcromakalim induced migraine phenotype were screened. Results In contrast to Wistar rats, mechanical periorbital and hind paw pain thresholds were not reduced in GAERS rats after nitroglycerin or levcromakalim injection. Additionally, GAERS were free of the light aversive effects of these substances as opposed to Wistar rats. Ethosuximide did not reverse the resistant phenotype in GAERS, indicating T-type calcium channel dysfunction did not contribute to this phenomenon. Tiagabine, GABA reuptake inhibitor, prevented levcromakalim-induced mechanical allodynia in Wistar rats, suggesting a key role of enhanced GABA spillover. GABAB receptor agonist baclofen did not alleviate mechanical allodynia. Synaptic GABAA receptor agonist diazepam failed to mitigate levcromakalim-induced migraine phenotype. Additionally, the resistant phenotype by levcromakalim in GAERS was not affected by flumazenil, a synaptic GABAA receptor antagonist. Extrasynaptic GABAA receptor agonists gaboxadol and muscimol successfully inhibited periorbital mechanical allodynia in Wistar rats. Conclusion Our study introduces a migraine-resistant rat strain and signifies a critical role of GABAergic system in migraine phenotype. Also, detailed pharmacological evaluation demonstrates extrasynaptic GABAA receptors as a novel drug target with a therapeutic potential in migraine susceptibility.
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Molecular Biology of the Central Auditory System and Tinnitus
Chapter
  • Mar 2024
Tinnitus is an auditory precept without any external input. In most cases, initial damage to the cochlea results in a change in peripheral auditory input that, in turn, triggers a change in central auditory mechanisms to compensate for this loss. This compensation, a combination of homeostatic and synaptic plasticity, results in an increased ‘gain’ in central auditory structures that may underlie the initiation of tinnitus. Changes at the molecular, cellular, and synaptic level in both auditory and nonauditory areas have been noted in animal models of tinnitus. Current research efforts to cure or clinically target to alleviate the tinnitus symptoms are focused on these areas and target these changes, including ion channel activators. Future studies should focus on unraveling the mechanisms of tinnitus precept induction and circuitry distribution for better treatment options.
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Cortical Auditory Evoked Potential Indices of Impaired Sensory Gating in People With Chronic Tinnitus
Article
  • Jan 2024
  • Ear Hear
Objectives: The primary aim of this study was to evaluate whether there is cortical auditory evoked potential (CAEP) evidence of impaired sensory gating in individuals with tinnitus. On the basis of the proposed mechanism of tinnitus generation, including a thalamocortical inhibitory deficit, it was hypothesized that individuals with tinnitus would lack the normal inhibitory effect on the second CAEP response in a paired-click sensory gating paradigm, resulting in larger sensory gating ratios in individuals with tinnitus relative to age-, sex-, and hearing-matched controls. Further, this study assessed the relative predictive influence of tinnitus presence versus other related individual characteristics (hearing loss, age, noise exposure history, and speech perception in noise) on sensory gating. Design: A paired-click CAEP paradigm was used to measure sensory gating outcomes in an independent group's experimental design. Adults who perceived chronic unilateral or bilateral tinnitus were matched with control group counterparts without tinnitus by age, hearing, and sex (n = 18; 10 females, eight males in each group). Amplitude, area, and latency sensory gating ratios were determined for measured P1, N1, and P2 responses evoked by the first and second click in the paradigm and compared between groups by independent t tests. The relative influence of tinnitus (presence/absence), age (in years), noise exposure history (subjective self-report), hearing loss (pure-tone audiometric thresholds), and speech perception in noise (signal to noise ratio-50) on sensory gating was determined based on the proportional reduction in error associated with each variable using multiple regression. Results: A significantly larger was identified in the tinnitus group relative to the control group, consistent with the hypothesis of poorer sensory gating and poorer thalamocortical inhibition in individuals with chronic tinnitus. On the basis of the proportional reduction in error, the influence of tinnitus presence better predicted compared with other related individual characteristics (age, noise exposure history, hearing loss, and speech perception in noise). Conclusions: Results consistent with poorer sensory gating, including a larger , were found for the tinnitus group compared with the controls. This finding supported a thalamocortical inhibitory deficit in the tinnitus group and suggests that individuals with tinnitus may have poorer sensory gating. However, the tinnitus group did differ from controls in meaningful ways including having worse pure-tone thresholds in the extended high-frequency region, lower high-frequency distortion product otoacoustic emissions, and poorer speech perception in noise. Although tinnitus best predicted sensory gating outcomes, the specific effects of tinnitus presence versus absence and other individual characteristics on sensory gating cannot be completely separated.
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Enhancing GABAergic Tonic Inhibition Reduces Seizure-Like Activity in the Neonatal Mouse Hippocampus and Neocortex
Article
  • Jan 2024
  • J NEUROSCI
Approximately one-third of neonatal seizures do not respond to first-line anticonvulsants, including phenobarbital, which enhances phasic inhibition. Whether enhancing tonic inhibition decreases seizure-like activity in the neonate when GABA is mainly depolarizing at this age is unknown. We evaluated if increasing tonic inhibition using THIP [4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol, gaboxadol], a δ-subunit–selective GABA A receptor agonist, decreases seizure-like activity in neonatal C57BL/6J mice (postnatal day P5–8, both sexes) using acute brain slices. Whole-cell patch-clamp recordings showed that THIP enhanced GABAergic tonic inhibitory conductances in layer V neocortical and CA1 pyramidal neurons and increased their rheobase without altering sEPSC characteristics. Two-photon calcium imaging demonstrated that enhancing the activity of extrasynaptic GABA A Rs decreased neuronal firing in both brain regions. In the 4-aminopyridine and the low-Mg ²⁺ model of pharmacoresistant seizures, THIP reduced epileptiform activity in the neocortex and CA1 hippocampal region of neonatal and adult brain slices in a dose-dependent manner. We conclude that neocortical layer V and CA1 pyramidal neurons have tonic inhibitory conductances, and when enhanced, they reduce neuronal firing and decrease seizure-like activity. Therefore, augmenting tonic inhibition could be a viable approach for treating neonatal seizures.
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Intracellular recordings in thalamic neurones during spontaneous spike and wave discharges in rats with absence epilepsy
Article
Full-text available
  • Jun 1998
In vivo extracellular and intracellular recordings were performed from thalamocortical (TC) neurones in a genetic model of absence epilepsy (genetic absence epilepsy rats from Strasbourg) during spontaneous spike and wave discharges (SWDs). Extracellularly recorded single units ( n = 14 ) fired either a single action potential or a high frequency burst of up to three action potentials, concomitantly with the spike component of the spike‐wave complex. Three main events characterized the intracellular activity of twenty‐six out of twenty‐eight TC neurones during SWDs: a small amplitude tonic hyperpolarization that was present throughout the SWD, rhythmic sequences of EPSP/IPSPs occurring concomitantly with the spike‐wave complexes, and a small tonic depolarization at the end of the SWD. The rhythmic IPSPs, but not the tonic hyperpolarization, were mediated by activation of GABA A receptors since they reversed in polarity at ‐68 mV and appeared as depolarizing events when recording with KCl‐filled electrodes. The intracellular activity of the remaining two TC neurones consisted of rhythmic low threshold Ca ²⁺ potentials, with a few EPSP/IPSP sequences present at the start of the SWD. These results obtained in a well‐established genetic model of absence epilepsy do not support the hypothesis that the intracellular activity of TC neurones during SWDs involves rhythmic sequences of GABA B IPSPs and low threshold Ca ²⁺ potentials.
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The distribution of 13 GABAA receptor subunit mRNAs in the rat brain. I. Telencephalon, diencephalon, mesencephalon
Article
Full-text available
  • Mar 1992
The expression patterns of 13 GABAA receptor subunit encoding genes (alpha 1-alpha 6, beta 1-beta 3, gamma 1-gamma 3, delta) were determined in adult rat brain by in situ hybridization. Each mRNA displayed a unique distribution, ranging from ubiquitous (alpha 1 mRNA) to narrowly confined (alpha 6 mRNA was present only in cerebellar granule cells). Some neuronal populations coexpressed large numbers of subunit mRNAs, whereas in others only a few GABAA receptor-specific mRNAs were found. Neocortex, hippocampus, and caudate-putamen displayed complex expression patterns, and these areas probably contain a large diversity of GABAA receptors. In many areas, a consistent coexpression was observed for alpha 1 and beta 2 mRNAs, which often colocalized with gamma 2 mRNA. The alpha 1 beta 2 combination was abundant in olfactory bulb, globus pallidus, inferior colliculus, substantia nigra pars reticulata, globus pallidus, zona incerta, subthalamic nucleus, medial septum, and cerebellum. Colocalization was also apparent for the alpha 2 and beta 3 mRNAs, and these predominated in areas such as amygdala and hypothalamus. The alpha 3 mRNA occurred in layers V and VI of neocortex and in the reticular thalamic nucleus. In much of the forebrain, with the exception of hippocampal pyramidal cells, the alpha 4 and delta transcripts appeared to codistribute. In thalamic nuclei, the only abundant GABAA receptor mRNAs were those of alpha 1, alpha 4, beta 2, and delta. In the medial geniculate thalamic nucleus, alpha 1, alpha 4, beta 2, delta, and gamma 3 mRNAs were the principal GABAA receptor transcripts. The alpha 5 and beta 1 mRNAs generally colocalized and may encode predominantly hippocampal forms of the GABAA receptor. These anatomical observations support the hypothesis that alpha 1 beta 2 gamma 2 receptors are responsible for benzodiazepine I (BZ I) binding, whereas receptors containing alpha 2, alpha 3, and alpha 5 contribute to subtypes of the BZ II site. Based on significant mismatches between alpha 4/delta and gamma mRNAs, we suggest that in vivo, the alpha 4 subunit contributes to GABAA receptors that lack BZ modulation.
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Selective Modulation of Tonic and Phasic Inhibitions in Dentate Gyrus Granule Cells
Article
  • May 2002
In some nerve cells, activation of GABA A receptors by GABA results in phasic and tonic conductances. Transient activation of synaptic receptors generates phasic inhibition, whereas tonic inhibition originates from GABA acting on extrasynaptic receptors, like in cerebellar granule cells, where it is thought to result from the activation of extrasynaptic GABA A receptors with a specific subunit composition (α 6 β x δ). Here we show that in adult rat hippocampal slices, extracellular GABA levels are sufficiently high to generate a powerful tonic inhibition in δ subunit–expressing dentate gyrus granule cells. In these cells, the mean tonic current is approximately four times larger than that produced by spontaneous synaptic currents occurring at a frequency of ∼10 Hz. Antagonizing the GABA transporter GAT-1 with NO-711 (2.5 μM) selectively enhanced tonic inhibition by 330% without affecting the phasic component. In contrast, by prolonging the decay of inhibitory postsynaptic currents (IPSCs), the benzodiazepine agonist zolpidem (0.5 μM) augmented phasic inhibition by 66%, while leaving the mean tonic conductance unchanged. These results demonstrate that a tonic GABA A receptor–mediated conductance can be recorded from dentate gyrus granule cells of adult rats in in vitro slice preparations. Furthermore, we have identified distinct pharmacological tools to selectively modify tonic and phasic inhibitions, allowing future studies to investigate their specific roles in neuronal function.
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Preferential Coassembly of a4 and d Subunits of the g-Aminobutyric AcidA Receptor in Rat Thalamus
Article
  • Jul 1999
ABSTRACT Pharmacological,study,of rat thalamic,g-aminobutyric,acidA (GABAA) receptors,revealed,the presence,of two distinct pop- ulations, namely, diazepam-sensitive and diazepam-insensitive [, ion channels (for reviews, see Sieghart, 1995; McKernan and Whiting, 1996), which are pentameric,assemblies,of the 14 different subunits,cloned,to date (a1‐ 6, b1‐3, g1‐3, d, and e). The combination of a and g subunits,has,been,shown,to confer,specific functional,and pharmacological properties, in particular the affinity and
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Synchronized activities of coupled oscillators in the cerebral cortex and thalamus at different levels of vigilance [published erratum appears in Cereb Cortex 1997 Dec;7(8):779]
Article
  • Sep 1997
  • CEREB CORTEX
The cerebral cortex and thalamus constitute a unified oscillatory machine displaying different spontaneous rhythms that are dependent on the behavioral state of vigilance. In vivo multi-site recordings from a variety of neocortical areas and related thalamic nuclei in cat, including dual simultaneous intracellular recordings, demonstrate that corticofugal volleys are effective in synchronizing fast (20‐50 Hz) and low-frequency (<15 Hz) oscillations in thalamocortical networks, characterizing activated and deafferented states. (i) Fast spontaneous oscillations depend on the depolarization of thalamic and cortical cells and appear in a sustained manner during waking and REM sleep. Corticothalamic neurons, discharging high-frequency (400 Hz) spike-bursts at 30‐40 Hz, are good candidates to synchronize fast oscillations in reentrant thalamocortical loops. Weakly synchronized, fast spontaneous oscillations may be reset and become robustly coherent after relevant sensory stimuli in waking or internal signals during the dreaming state. (ii) During quiescent sleep, the long-range synchronization of brain electrical activity results from synchronous hyperpolarizations in forebrain neurons. The corticothalamic inputs during the depolarizing component of the slow oscillation (<1 Hz) are effective in grouping the thalamic-generated sleep rhythms (spindles at 7‐14 Hz and delta at 1‐4 Hz) into complex wave-sequences. These inputs also control the shape of spindles, and favor the long-range synchronization and nearly simultaneous appearance of spindles. (iii) The cortical control of thalamic activity is also demonstrated in spike-wave seizures developing from sleep patterns. More than half of thalamocortical neurons are silent during spike-wave seizures, being tonically hyperpolarized, and display IPSPs (closely related to the paroxysmal depolarizing shifts of cortical cells) that are determined by the pattern of activities in thalamic reticular cells. All these data congruently show the power of cortical control upon thalamic oscillators.
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Comparative molecular neuroanatomy of cloned GABAA receptor subunits in rat CNS
Article
  • Dec 1992
Aminobutyric acid A (GABA A ) receptors in the mammalian central nervous system (CNS) are members of a family of ligand‐gated ion channels consisting of heterooligomeric glycoprotein complexes in synaptic and extrasynaptic membranes. Although molecular cloning studies have identified 5 subunits (with ∼40% amino acid homology) and isoforms thereof (∼70% homology), namely α1–6, β1–4, γ1–3, δ, and ρ, the subunit composition and stoichiometry of native receptors are not known. The regional distribution and cellular expression of GABA A receptor messenger RNAs (mRNAs) in the rat CNS have now been investigated by in situ hybridization histochemistry with subunit‐specific 35S ‐labelled oligonucleotide probes on adjacent cryostat sections. Whereas α1, β2, and γ2 transcripts were the most abundant and ubiquitous in the rat brain—correlating with the radioautographic distribution of GABA A receptors revealed by an ionophore ligand—others had a more restricted expression while often being abundant. For example, α2 transcripts were found only in the olfactory bulb, cerebral cortex, caudate putamen, hippocampal formation, and certain lower brain stem nuclei; α3 only in the olfactory bulb and cerebral cortex; α5 in the hippocampal formation; and α6 only in cerebellar granule cells. In addition, β1, β3, γ1, and δ mRNAs were also uniquely expressed in restricted brain regions. Moreover, in the spinal cord, α1–3, β2,3, and γ2 mRNAs were differently expressed in Rexed layers 2–9, with α2, β3, and γ2 transcripts most prominent in motoneurons of layer 9. Although differential protein trafficking could lead to the incorporation of some subunits into somatic membranes and others into dendritic membranes, some tentative conclusions as to the probable composition of native proteins in various regions of the CNS may be drawn. For example, according to the strength of the hybridization signal the following subunits might be expected to play a prominent role in GABAergic neurotransmission in the corresponding regions: α1, β2 (1,3), γ2 in olfactory bulb mitral cells; α2, β3, γ2, δ in caudate putamen; α1,3 (2,5), β2,3, γ2 in cerebral cortex; α1, β2, γ1,2 in pallidum; α1,2,5, β1–3, γ2 in hippocampus; α1,5, β3, γ2, δ in dentate gyrus; α1, β2, γ1,2 in substantia nigra zona reticularis; α1, β2, γ2 in cerebellar Purkinje cells; α1,6, β2,3, γ2, δ in granule cells; α1,2, β2,3, γ2 in vestibular and facial nuclei; and finally α2, β3, γ2 in motoneurons of Rexed layer 9 in the spinal cord. © 1992 Wiley‐Liss, Inc.
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A T-type Ca2+ current underlies low-threshold Ca2+ potentials in cells of the cat and rat lateral geniculate nucleus
Article
  • Jul 1989
1. The characteristics of a transient inward Ca2+ current (IT) underlying low-threshold Ca2+ potentials were studied in projection cells of the cat and rat dorsal lateral geniculate nucleus (LGN) in vitro using the single-electrode voltage-clamp technique. 2. In cat LGN slices perfused at 25 degrees C with a solution which included 1 mM-Ca2+ and 3 mM-Mg2+, IT could be evoked by depolarizing voltage steps to -55 mV from a holding potential (Vh) of -95 mV and was abolished by reducing [Ca2+]o from 1 to 0.1 mM. IT was also blocked by 8 mM-Mg2+ and 500 microM-Ni2+, but 500 microM-Cd2+ was a significantly less effective antagonist. 3. The inactivation of IT, which occurred at Vh positive to -65 mV, was removed as Vh approached -100 mV. The process of inactivation removal was also time dependent, with 800-1000 ms needed for total removal. Activation curves for IT showed a threshold of -70 mV and illustrated that IT was extremely voltage sensitive over the voltage range from -65 to -55 mV. 4. The decay phase of IT followed a single-exponential time course with a time constant of decay which was voltage sensitive and ranged from 20 to 100 ms. The mean peak conductance increase associated with IT was 8.4 nS (+/-0.9, S.E.M.). 5. In more 'physiological' conditions (35 degrees C and 1.5 mM-Ca2+, 1 mM-Mg2+) the voltage dependence of activation and inactivation were unaffected. However, the development and decay of IT proceeded more rapidly and only 500-600 ms were needed for total removal of inactivation. Under these conditions, the use of voltage ramps showed that depolarization rates of greater than 30 mV/s were necessary for IT activation. 6. The use of multiple voltage-step protocols illustrated that the process of inactivation removal was rapidly reversed by brief returns to a Vh of -50 mV. Furthermore, any delay in IT activation, once the LGN cell membrane potential was in the IT activation range, resulted in a current of reduced amplitude. 7. Although IT in rat LGN cells was briefer and had a shorter latency to peak, it was otherwise similar to that seen in cat LGN cells. 8. The characteristics of IT are very similar to those of the T-type Ca2+ currents of other excitable membranes. The properties of IT are discussed with respect to its role in generating the low-threshold Ca2+ potentials which are central to the oscillatory behaviour of thalamic projection cells.
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The THIP-induced model of bilateral synchronous spike and wave in rodents
Article
  • Feb 1987
  • NEUROPHARMACOLOGY
Intraperitoneal administration of 5-10 mg/kg of THIP (4,5,6,7 tetrahydroxyisoxazolo (4,5,c) pyridine 3-ol) induced a transient, reliable model of bilaterally synchronous spikes and waves in rats. The paroxysmal bursts elicited by THIP had similar topographical distribution to the spontaneously-occurring spike and wave discharges often observed in naive rats. The responsiveness to electrical stimulation of subcortical nuclei was different in the two models. Systemic administration of THIP provided a simple, reliable model of bilaterally synchronous spike and wave discharges that may involve the same cerebral structures as those involved in the generation of the spontaneous paroxysms. The administration of the drug increased the yield of paroxysmal responses and provided a stable and predictable 4 hr test situation that may easily be quantified.
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In vivo determination of extracellular concentration of amino acids in the rat hippocampus. A method based on brain dialysis and computerized analysis
Article
  • Nov 1986
  • BRAIN RES
Extracellular (EC) concentrations of amino acids were determined in the rat dentate gyrus by means of non-linear regression analysis of 'in vivo' brain dialysis data, considering a simple model of diffusion through a dialysis membrane. The apparent diffusion constants (K) of several amino acids were also calculated in the 'in vivo' situation. While putative amino acid neurotransmitters (glutamate, aspartate and gamma-aminobutyric acid (GABA) were present in the EC fluid at the low micromolar range (0.8-2.9 microM), glutamine was by far the most prominent (193.4 microM). The values of intra/extracellular concentration ratios formed 3 groups: high (greater than 2000) for putative neurotransmitters; low (less than 100) for serine, glutamine, arginine and alpha-alanine; and intermediate (about 400) for taurine. The 'in vivo' calculated K values proved useful for estimation of both basal and changing EC concentrations of amino acids in relatively brief perfusions. These data were evaluated in terms of the functional significance of absolute EC concentrations and tissue-EC fluid ratios. Present findings indicate the simultaneous existence of both an inhibitory and an excitatory tonus as well as the utility of high intra/extracellular concentration ratios in determination of the possible neurotransmitter role of specific amino acids.
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Neural elements containing glutamic acid decarboxylase (GAD) in the dorsal lateral geniculate nucleus of the rat; Immunohistochemical studies by light and electron microscopy
Article
  • Feb 1983
  • NEUROSCIENCE
Immunoreactive constituents of the dorsal lateral geniculate nucleus of adult albino rats were examined by light- and electron-microscopy, using the unlabelled antibody enzyme method, following treatment of brain slices with a purified antibody to glutamic acid decarboxylase. The neuropil of the dorsal lateral geniculate nucleus displayed a conspicuous granular immunoreactivity. In addition, the antibody was bound to a class of small neurons of characteristic morphology. These cells possessed few (commonly 2-4) sparsely branched, long dendrites from some of which immunoreactive appendages were traced. Many cells were bipolar in form, and the dendrites of some appeared to be preferentially orientated. The immunoreactive cells closely resembled intrinsic interneurons characterized in previous Golgi studies of this nucleus. By electron-microscopy, immunoreactive presynaptic elements were present both in the extraglomerular neuropil and in the synaptic glomeruli. The former were axon terminals containing flattened synaptic vesicles and making Gray type II axo-dendritic synaptic contact; they appeared to correspond to axon terminals whose origin in the thalamic reticular nucleus has been established in previous studies, but it is possible that some were axon terminals of intrinsic interneurons. The immunoreactive glomerular components also contained flattened vesicles, were presynaptic to presumptive projection cell dendrites, postsynaptic to retinal axon terminals, and participated in triplet (triadic) and other complex synaptic arrangements. They corresponded in all respects to the synaptic portions of the complex dendritic appendages of intrinsic interneurons, identified and characterized in previous studies. The finding that there are high levels of glutamic acid decarboxylase in the cell bodies, dendritic shafts and dendritic appendages of intrinsic interneurons in the dorsal lateral geniculate nucleus of the rat, and in the axon terminals of fibres projecting to this site from the thalamic reticular nucleus, allows us to conclude that the inhibitory inputs to the geniculo-cortical projection cells from both of these sources are probably mediated by gamma-aminobutyric acid.
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