Age-related dendritic hypertrophy and sexual dimorphism in rat basolateral amygdala

Little research has examined the influence of aging or sex on anatomical measures in the basolateral amygdala. We quantified spine density and dendritic material in Golgi-Cox stained tissue of the basolateral nucleus in young adult (3-5 months) and aged (20-24 months) male and female Long-Evans rats. Dendritic branching and spine density were measured in principal neurons. Age, but not sex, influenced the dendritic tree, with aged animals displaying significantly more dendritic material. Previous findings from our laboratory in the same set of subjects indicate an opposite effect of aging on dendritic material in the medial prefrontal cortex and hippocampus. We also report here a sex difference across ages in dendritic spine density, favoring males.     

Relationship of calcium-binding protein containing neurons and projection neurons in the rat basolateral amygdala

Two-laser and two-color approaches were used to observe the colocalization of the calcium-binding proteins, calbindin D28k and parvalbumin, and the retrograde tracer, Fluoro-Gold (FG) in the basolateral amygdala of the rat. The study was performed on five adult rats into which FG was injected to the frontal association cortex. Then, the localization of the retrogradely labeled neurons in the basolateral amygdala was compared with the localization of the neurons labeled by calcium-binding proteins. The present study showed that most of the retrogradely labeled neurons in the posterior part of the basolateral amygdala are also calbindin-positive. Even though a lot of parvalbumin-positive endings were present at the surface of the retrogradely labeled cells, we did not observe the colocalization of the parvalbumin and projective neurons.     

Immunohistochemical identification of gamma-aminobutyric acid-containing neurons in the rat basolateral amygdala

The peroxidase-antiperoxidase immunohistochemical technique was used together with an antiserum to gamma-aminobutyric acid (GABA) to identify GABA-containing structures in the rat basolateral amygdala (ABL). Morphological characteristics of GABA-positive neurons in ABL indicate that they correspond to class II, and perhaps class III, local circuit neurons observed in previous Golgi studies. GABA-positive punctate structures resembling axon terminals were observed both in the neuropil and forming peri-cellular baskets around large unlabeled perikarya in ABL. These results suggest that the strong intrinsic inhibition noted in electrophysiological studies of ABL is due primarily to synapses of GABAergic class II neurons with class I projection neurons.     

Colocalization of calcium-binding proteins and GABA in neurons of the rat basolateral amygdala

The basolateral amygdala contains subpopulations of non-pyramidal neurons that express the calcium-binding proteins parvalbumin, calbindin-D28k (calbindin) or calretinin. Although little is known about the exact functions of these proteins, they have provided useful markers of specific neuronal subpopulations in studies of the neuronal circuitry of the cerebral cortex and other brain regions. The purpose of the present study was to investigate whether basolateral amygdalar non-pyramidal neurons containing parvalbumin, calbindin, or calretinin exhibit immunoreactivity for GABA, and to determine if calretinin is colocalized with parvalbumin or calbindin in the rat basolateral amygdala. Pyramidal neurons were distinguished from non-pyramidal neurons on the basis of staining intensity. Using immunofluorescence confocal laser scanning microscopy, as well as the 'mirror technique' on immunoperoxidase-stained sections, it was found that there was virtually no colocalization of calretinin with parvalbumin or calbindin, but that the great majority of basolateral amygdalar non-pyramidal neurons containing parvalbumin, calbindin, or calretinin exhibited GABA immunoreactivity. Calbindin-positive neurons constituted almost 60% of the GABA-containing population in both subdivisions of the basolateral nucleus and more than 40% of the GABA-containing population in the lateral nucleus. Parvalbumin-positive neurons constituted 19-43% of GABA-immunoreactive neurons in the basolateral amygdala, depending on the nucleus. Calretinin-positive non-pyramidal neurons constituted about 20% of the GABA-positive neuronal population in each nucleus of the basolateral amygdala.These findings indicate that non-pyramidal neurons containing parvalbumin, calbindin, or calretinin comprise the majority of GABA-containing neurons in the basolateral amygdala, and that the calretinin subpopulation is distinct from non-pyramidal subpopulations containing parvalbumin and calbindin. These separate neuronal populations may play unique roles in the inhibitory circuitry of the amygdala.     

Parvalbumin-immunoreactive neurons and GABAergic neurons of the basal forebrain project to the rat basolateral amygdala

The basal forebrain (BF) contains a diffuse array of cholinergic and non-cholinergic neurons that project to the cerebral cortex and basolateral nuclear complex of the amygdala (BLC). Previous studies have shown that the GABAergic subpopulation of non-cholinergic corticopetal BF neurons selectively innervates cortical interneurons. Although several investigations in both rodents and primates have indicated that some BF neurons projecting to the BLC are non-cholinergic, there have been no studies that have attempted to identify the neurochemical phenotype(s) of these neurons. The present study combined Fluorogold retrograde tract tracing with immunohistochemistry for two markers of BF GABAergic neurons, parvalbumin (PV) or glutamic acid decarboxylase (GAD), to determine if a subpopulation of BF GABAergic cells projects to the BLC. Injections of Fluorogold confined to the rat BLC, and centered in the basolateral nucleus, produced extensive retrograde labeling in the ventral pallidum and substantia innominata regions of the BF. Although the great majority of retrogradely labeled neurons were not double-labeled, about 10% of these neurons, located mainly along the ventral aspects of the fundus striati and globus pallidus, exhibited immunoreactivity for PV or GAD. The results of this investigation contradict the long-held belief that there is no extra-amygdalar source of GABAergic inputs to the BLC, and indicate that the cortex-like BLC, in addition to the cortex proper, receives inhibitory inputs from the basal forebrain.     

Types of neurons and some dendritic patterns of basolateral amygdala in humans--a golgi study.

Classification of the neurons in the human basolateral amygdala is performed on preparations impregnated by the Golgi technique. Three different neuronal types are found in the nuclei of the basolateral amygdala: Type I--Pyramidal cells, with numerous dendritic spines and two subtypes (slender and squat); Type II--Modified pyramidal cells, sparsely spinous with rare dendritic spines and two subtypes (single apical and double apical) and; Type III--Non-pyramidal cells, with few dendritic spines and three subtypes (bipolar, multipolar and gliaform). The analysis of the primary dendritic branches pointed out the occasional presence of dendritic bundles (fascicular dendritic arrangement) with their predomination in the parvicellular division of the basal nucleus and paralaminar nucleus. Additionally, the presence of dendrodendritic contacts, indicated by light microscopy, was also found in the parvicellular division of the basal nucleus and especially in the paralaminar nucleus.     

Postnatal maturation of GABAergic modulation of sensory inputs onto lateral amygdala principal neurons

Key points

• Throughout life, fear learning is indispensable for survival and neural plasticity in the lateral amygdala underlies this learning and storage of fear memories.
• During development, properties of fear learning continue to change into adulthood, but currently little is known about changes in amygdala circuits that enable these behavioural transitions.
• In recordings from neurons in lateral amygdala brain slices from infant up to adult mice, we show that spontaneous and evoked excitatory and inhibitory synaptic transmissions mature into adolescence.
• At this time, increased inhibitory activity and signalling has the ability to restrict the function of excitation by presynaptic modulation, and may thus enable precise stimulus associations to limit fear generalization from adolescence onward.
• Our results provide a basis for addressing plasticity mechanisms that underlie altered fear behaviour in young animals.

Abstract

Convergent evidence suggests that plasticity in the lateral amygdala (LA) participates in acquisition and storage of fear memory. Sensory inputs from thalamic and cortical areas activate principal neurons and local GABAergic interneurons, which provide feed‐forward inhibition that tightly controls LA activity and plasticity via pre‐ and postsynaptic GABA_A and GABA_B receptors. GABAergic control is also critical during fear expression, generalization and extinction in adult animals. During rodent development, properties of fear and extinction learning continue to change into early adulthood. Currently, few studies have assessed physiological changes in amygdala circuits that may enable these behavioural transitions. To obtain first insights, we investigated changes in spontaneous and sensory input‐evoked inhibition onto LA principal neurons and then focused on GABA_B receptor‐mediated modulation of excitatory sensory inputs in infant, juvenile, adolescent and young adult mice. We found that spontaneous and sensory‐evoked inhibition increased during development. Physiological changes were accompanied by changes in dendritic morphology. While GABA_B heteroreceptors were functionally expressed on sensory afferents already early in development, they could only be physiologically recruited by sensory‐evoked GABA release to mediate heterosynaptic inhibition from adolescence onward. Furthermore, we found GABA_B‐mediated tonic inhibition of sensory inputs by ambient GABA that also emerged in adolescence. The observed increase in GABAergic drive may be a substrate for providing modulatory GABA. Our data suggest that GABA_B‐mediated tonic and evoked presynaptic inhibition can suppress sensory input‐driven excitation in the LA to enable precise stimulus associations and limit generalization of conditioned fear from adolescence onward.

Abbreviations

BA

basal nucleus of the amygdala

BLA

basolateral complex of the amygdala

CS

conditioned stimulus

LA

lateral nucleus of the amygdala

LTP

long‐term potentiation

PND

postnatal day

PPD

paired‐pulse depression

PPR

paired‐pulse ratio

sEPSC

spontaneous excitatory postsynaptic current

sIPSC

spontaneous inhibitory postsynaptic current

US

unconditioned stimulus

     

Morphological and electrophysiological properties of principal neurons in the rat lateral amygdala in vitro

In this study, we characterize the electrophysiological and morphological properties of spiny principal neurons in the rat lateral amygdala using whole cell recordings in acute brain slices. These neurons exhibited a range of firing properties in response to prolonged current injection. Responses varied from cells that showed full spike frequency adaptation, spiking three to five times, to those that showed no adaptation. The differences in firing patterns were largely explained by the amplitude of the afterhyperpolarization (AHP) that followed spike trains. Cells that showed full spike frequency adaptation had large amplitude slow AHPs, whereas cells that discharged tonically had slow AHPs of much smaller amplitude. During spike trains, all cells showed a similar broadening of their action potentials. Biocytin-filled neurons showed a range of pyramidal-like morphologies, differed in dendritic complexity, had spiny dendrites, and differed in the degree to which they clearly exhibited apical versus basal dendrites. Quantitative analysis revealed no association between cell morphology and firing properties. We conclude that the discharge properties of neurons in the lateral nucleus, in response to somatic current injections, are determined by the differential distribution of ionic conductances rather than through mechanisms that rely on cell morphology.     

Muscarinic activation of a non-selective cationic conductance in pyramidal neurons in rat basolateral amygdala

In the present study, a cationic membrane conductance activated by the acetylcholine agonist carbachol was characterized in vitro in neurons of the basolateral amygdala. Extracellular perfusion of the K+ channel blockers Ba2+ and Cs+ or loading of cells with cesium acetate did not affect the carbachol-induced depolarization. Similarly, superfusion with low-Ca2+ solution plus Ba2+ and intracellular EGTA did not affect the carbachol-induced depolarization, suggesting a Ca2+-independent mechanism. On the other hand, the carbachol-induced depolarization was highly sensitive to changes in extracellular K+ or Na+. When the K+ concentration in the perfusion medium was increased from 4.7 to 10 mM, the response to carbachol increased in amplitude. In contrast, lowering the extracellular Na+ concentration from 143.2 to 29 mM abolished the response in a reversible manner. Results of coapplication of carbachol and atropine, pirenzepine or gallamine indicate that the carbachol-induced depolarization was mediated by muscarinic cholinergic receptors, but not the muscarinic receptor subtypes M1, M2 or M4, specifically. These data indicate that, in addition to the previously described reduction of a time- and voltage-independent K+ current (IKleak), a voltage- and time-dependent K+ current (IM), a slow Ca2+-activated K+ current (sIahp) and the activation of a hyperpolarization-activated inward rectifier K+ current (IQ), carbachol activated a Ca2+-independent non-selective cationic conductance that was highly sensitive to extracellular K+ and Na+ concentrations.     

Regulation of conditioned responses of basolateral amygdala neurons

The basolateral amygdala (BLA) is a component of a system that drives and modulates affective behavior. Some forms of affective behavior are regulated by the prefrontal cortex (PFC) and enhanced by dopamine (DA). By using intracellular and extracellular electrophysiological techniques in anesthetized rats, our studies attempt to uncover cellular mechanisms that allow for regulation of affect by PFC-induced inhibition of BLA output and plasticity, as well as mechanisms by which DA enhances affective behavior via modulation of BLA neuronal excitability, afferent input and plasticity. We have found that stimulation of medial PFC (mPFC) results in a profound inhibition of BLA output, manifest as a suppression of spontaneous, intracellular current-driven or sensory cortical afferent-driven spike firing of BLA projection neurons. This inhibition is mediated by excitation of GABAergic interneurons of the BLA. Activation of DA receptors attenuates this inhibitory action of the mPFC, while enhancing other (i.e., sensory-related) inputs by increases in postsynaptic excitability of BLA projection neurons. Furthermore, Pavlovian conditioning procedures that pair an odor with a footshock result in enhanced odor-evoked postsynaptic potentials. This plasticity of odor-evoked responses is blocked by antagonism of DA receptors and by stimulation of mPFC. Our data indicate that the mPFC exerts regulatory control over BLA via suppression of spontaneous and sensory-driven activity, as well as BLA plasticity. Activation of DA receptors suppresses the inhibitory influence of the mPFC, allowing sensory-driven BLA activity and plasticity. Functionally, in the presence of high DA levels, which suppresses mPFC-evoked inhibition, one source of affective control will be dampened. Furthermore, sensory-related inputs will be further enhanced by the increased excitability of BLA neurons. This situation is expected to maximize affective responses to sensory stimuli, as well as plasticity.     

Dopamine modulates excitability of basolateral amygdala neurons in vitro

The amygdala plays a role in affective behaviors, which are modulated by the dopamine (DA) innervation of the basolateral amygdala complex (BLA). Although in vivo studies indicate that activation of DA receptors alters BLA neuronal activity, it is unclear whether DA exerts direct effects on BLA neurons or whether it acts via indirect effects on BLA afferents. Using whole cell patch-clamp recordings in rat brain slices, we investigated the site and mechanisms through which DA regulates the excitability of BLA neurons. Dopamine enhanced the excitability of BLA projection neurons in response to somatic current injections via a postsynaptic effect. Dopamine D1 receptor activation increased excitability and evoked firing, whereas D2 receptor activation increased input resistance. Current- and voltage-clamp experiments in projection neurons showed that D1 receptor activation enhanced excitability by modulating a 4-aminopyridine- and alpha-dendrotoxin-sensitive, slowly inactivating K+ current. Furthermore, DA and D1 receptor activation increased evoked firing in fast-spiking BLA interneurons. Consistent with a postsynaptic modulation of interneuron excitability, DA also increased the frequency of spontaneous inhibitory postsynaptic currents recorded in projection neurons without changing release of GABA. These data demonstrate that DA exerts direct effects on BLA projection neurons and indirect actions via modulation of interneurons that may work in concert to enhance the neuronal response to large, suprathreshold inputs, while suppressing weaker inputs.     

Dopaminergic innervation of interneurons in the rat basolateral amygdala

The basolateral nuclear complex of the amygdala (BLC) receives a dense dopaminergic innervation that plays a critical role in the formation of emotional memory. Dopamine has been shown to influence the activity of BLC GABAergic interneurons, which differentially control the activity of pyramidal cells. However, little is known about how dopaminergic inputs interface with different interneuronal subpopulations in this region. To address this question, dual-labeling immunohistochemical techniques were used at the light and electron microscopic levels to examine inputs from tyrosine hydroxylase-immunoreactive (TH+) dopaminergic terminals to two different interneuronal populations in the rat basolateral nucleus labeled using antibodies to parvalbumin (PV) or calretinin (CR). The basolateral nucleus exhibited a dense innervation by TH+ axons. Partial serial section reconstruction of TH+ terminals found that at least 43-50% of these terminals formed synaptic junctions in the basolateral nucleus. All of the synapses examined were symmetrical. In both TH/PV and TH/CR preparations the main targets of TH+ terminals were spines and distal dendrites of unlabeled cells. In sections dual-labeled for TH/PV 59% of the contacts of TH+ terminals with PV+ neurons were synapses, whereas in sections dual-labeled for TH/CR only 13% of the contacts of TH+ terminals with CR+ cells were synapses. In separate preparations examined in complete serial sections for TH+ basket-like innervation of PV+ perikarya, most (76.2%) of TH+ terminal contacts with PV+ perikarya were synapses. These findings suggest that PV+ interneurons, but not CR+ interneurons, are prominent synaptic targets of dopaminergic terminals in the BLC.     

Parvalbumin-containing neurons in the rat basolateral amygdala: morphology and co-localization of Calbindin-D(28k)

Parvalbumin is a calcium-binding protein that is contained in certain neuronal populations in the brain. Although the exact function of parvalbumin is not clear, it has been found to be a useful marker for studying the connections of specific cell types in immunohistochemical studies. In the present investigation immunohistochemical techniques were used to study the morphology of parvalbumin-containing neurons in the rat basolateral amygdala. These neurons were found to be a morphologically heterogeneous subpopulation of non-pyramidal interneurons. Parvalbumin-positive axons in the basolateral amygdala were observed to form "pericellular baskets" that enveloped the perikarya of pyramidal neurons. In addition, some parvalbumin-immunoreactive axons formed "cartridges" that appeared to surround non-immunoreactive processes. The morphology of parvalbumin-positive neurons closely resembled that of neurons containing calbindin, a related calcium-binding protein. Analysis of adjacent sections stained for each protein using the mirror technique revealed that approximately 80% of parvalbumin neurons also contained calbindin, and that approximately 60% of calbindin neurons also contained parvalbumin.This study demonstrates that parvalbumin-containing neurons constitute an important subpopulation of non-pyramidal interneurons in the rat basolateral amygdala. The axonal configurations of these cells indicate that they may exert a potent inhibitory influence over pyramidal projection neurons. We suggest that parvalbumin-containing neurons can control emotional responses mediated by the basolateral amygdala by controlling the output from this important brain region.     

Activation of nicotinic acetylcholine receptors increases the frequency of spontaneous GABAergic IPSCs in rat basolateral amygdala neurons

The basolateral amygdala (BLA) is a critical component of the amygdaloid circuit, which is thought to be involved in fear conditioned responses. Using whole cell patch-clamp recording, we found that activation of nicotinic acetylcholine receptors (nAChRs) leads to an action potential-dependent increase in the frequency of spontaneous GABAergic currents in principal neurons in the BLA. These spontaneous GABAergic currents were abolished by a low-Ca2+/high-Mg2+ bathing solution, suggesting that they are spontaneous inhibitory postsynaptic currents (sIPSCs). Blockade of ionotropic glutamate receptors did not prevent this increased frequency of sIPSCs nor did blockade of alpha7 nAChRs. Among the nAChR agonists tested, cystisine was more effective at increasing the frequency of the sIPSCs than nicotine or 1,1-dimethyl-4-phenyl piperazinium iodide, consistent with a major contribution of beta4 nAChR subunits. The nicotinic antagonist, dihydro-beta-erythroidine, was less effective than d-tubocurarine in blocking the increased sIPSC frequency induced by ACh, suggesting that alpha4-containing nAChR subunits do not play a major role in the ACh-induced increased sIPSC frequency. Although alpha2/3/4/7 and beta2/4 nAChR subunits were found in the BLA by RT-PCR, the agonist and antagonist profiles suggest that the ACh-induced increase in sIPSC frequency involves predominantly alpha3beta4-containing nAChR subunits. Consistent with this, alpha-conotoxin-AuIB, a nAChR antagonist selective for the alpha3beta4 subunit combination, inhibited the ACh-induced increase in the frequency of sIPSCs. The observations suggest that nicotinic activation increases the frequency of sIPSCs in the BLA by acting mainly on alpha3beta4-containing nicotinic receptors on GABAergic neurons and may play an important role in the modulation of synaptic transmission in the amygdala.     

Dopaminergic innervation of pyramidal cells in the rat basolateral amygdala

Dopaminergic (DA) inputs to the basolateral nuclear complex of the amygdala (BLC) are critical for several important functions, including reward-related learning, drug-stimulus learning, and fear conditioning. Despite the importance of the DA projection to the BLC, very little is known about which neuronal subpopulations are innervated. The present study utilized dual-labeling immunohistochemistry at the electron microscopic level to examine DA inputs to pyramidal cells in the anterior basolateral amygdalar nucleus (BLa) in the rat. DA axon terminals and BLa pyramidal cells were labeled using antibodies to tyrosine hydroxylase (TH) and calcium/calmodulin-dependent protein kinase II (CaMK), respectively. Serial section reconstructions of TH-positive (TH+) terminals were performed to determine the extent to which these axon terminals formed synapses versus non-synaptic appositions in the BLa. Our results demonstrate that at least 77% of TH+ terminals form synapses in the BLa, and that 90% of these synapses are with pyramidal cells. The distal dendritic compartment received the great majority of these synaptic contacts, with CaMK+ distal dendrites and spines receiving one-third and one-half, respectively, of all synaptic inputs to pyramidal cells. Many spines receiving innervation from TH+ terminals also received asymmetrical synaptic inputs from putative excitatory terminals. In addition, TH+ terminals often formed non-synaptic appositions with axon terminals, most of which were putatively excitatory in that they were CaMK+ and/or made asymmetrical synapses. Thus, using CaMK as a marker, the present study demonstrates that pyramidal cells, especially their distal dendritic compartments, are the primary targets of dopaminergic inputs to the basolateral amygdala.     

Serotonergic modulation of neurotransmission in the rat basolateral amygdala.

Whole cell patch-clamp recordings were obtained from projection neurons and interneurons of the rat basolateral amygdala (BLA) to understand local network interactions in morphologically identified neurons and their modulation by serotonin. Projection neurons and interneurons were characterized morphologically and electrophysiologically according to their intrinsic membrane properties and synaptic characteristics. Synaptic activity in projection neurons was dominated by spontaneous inhibitory postsynaptic currents (IPSCs) that were multiphasic, reached 181 +/- 38 pA in amplitude, lasted 296 +/- 27 mS, and were blocked by the GABAA receptor antagonist, bicuculline methiodide (30 microM). In interneurons, spontaneous synaptic activity was characterized by a burst-firing discharge patterns (200 +/- 40 Hz) that correlated with the occurrence of 6-cyano-7-nitroquinoxaline-2,3-dione-sensitive, high-amplitude (260 +/- 42 pA), long-duration (139 +/- 19 mS) inward excitatory postsynaptic currents (EPSCs). The interevent interval of 831 +/- 344 mS for compound inhibitory postsynaptic potentials (IPSPs), and 916 +/- 270 mS for EPSC bursts, suggested that spontaneous IPSP/Cs in projection neurons are driven by burst of action potentials in interneurons. Hence, BLA interneurons may regulate the excitability of projection neurons and thus determine the degree of synchrony within ensembles of BLA neurons. In interneurons 5-hydroxytryptamine oxalate (5-HT) evoked a direct, dose-dependent, membrane depolarization mediated by a 45 +/- 6.9 pA inward current, which had a reversal potential of -90 mV. The effect of 5-HT was mimicked by the 5-HT2 receptor agonist, alpha-methyl-5-hydroxytryptamine (alpha-methyl-5-HT), but not by the 5-HT1A receptor agonist, (+/-) 8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT), or the 5-HT1B agonist, CGS 12066A. In projection neurons, 5-HT evoked an indirect membrane hyperpolarization ( approximately 2 mV) that was associated with a 75 +/- 42 pA outward current and had a reversal potential of -70 mV. The response was independent of 5-HT concentration, blocked by TTX, mimicked by alpha-methyl-5-HT but not by 8-OH-DPAT. In interneurons, 5-HT reduced the amplitude of the evoked EPSC and in the presence of TTX (0.6 microM) reduced the frequency of miniature EPSCs but not their quantal content. In projection neurons, 5-HT also caused a dose-dependent reduction in the amplitude of stimulus evoked EPSCs and IPSCs. These results suggest that acute serotonin release would directly activate GABAergic interneurons of the BLA, via an activation of 5-HT2 receptors, and increase the frequency of inhibitory synaptic events in projection neurons. Chronic serotonin release, or high levels of serotonin, would reduce the excitatory drive onto interneurons and may act as a feedback mechanism to prevent excess inhibition within the nucleus.     

Coexistence of GABA and peptide immunoreactivity in non-pyramidal neurons of the basolateral amygdala

Colocalization of γ-aminobutyric acid (GABA) immunoreactivity with somatostatin (SOM), neuropeptide Y (NPY), cholecystokinin (CCK), and vasoactive intestinal polypeptide (VIP) immunoreactivity was demonstrated in non-pyramidal neurons of the basolateral amygdala using a two-color immunoperoxidase procedure. Approximately 80–90% of SOM- and NPY-positive neurons in the basolateral amygdala were also immunoreactive for GABA. Virtually all large CCK-positive neurons also exhibited GABA-like immunoreactivity. About one-half of VIP-positive neurons and small CCK-positive cells were also immunoreactive for GABA.