Developmental effects of oxytocin on GABAergic neurons in the olfactory brain regions.

Oxytocin affects social recognition, interactions, and behavior in adults. Despite growing data on the role of oxytocin in the sensory systems, its effects on early olfactory system development remain poorly understood. The present study aimed to investigate the developmental impact of oxytocin on selected parameters of the GABAergic system in olfactory brain regions. We found a significant increase in the expression of GABAergic markers and scaffolding proteins in the olfactory bulb during the early stages of development in both male and female rats, regardless of oxytocin treatment administered on postnatal days 2 and 3 (P2 and P3, 5 µg/pup). Oxytocin administration markedly reduced the expression of the scaffolding protein Gephyrin in male rats and it led to a significant increase in the number of GABAergic synaptic puncta in the piriform cortex of male rats at P5, P7, and P9. Our data suggest that the developmental action of oxytocin in relation to the GABAergic system may represent a mechanism by which the plasticity and maturation of olfactory brain regions are regulated.

Activity-dependent extracellular potassium changes in unmyelinated versus myelinated areas in olfactory regions of the isolated female guinea-pig brain.

The potassium released in the extracellular space during neuronal activity is rapidly removed by glia and neurons to maintain tissue homeostasis. Oligodendrocyte-derived myelin axonal coating contributes to potassium buffering and is therefore crucial to control brain excitability. We studied activity-dependent extracellular potassium ([K+]o) changes in the piriform cortex (PC), a region that features highly segregated bundles of myelinated and unmyelinated fibers. Four-aminopyridine (4AP; 50 µM) treatment or patterned high-frequency stimulations (hfST) were utilized to generate [K+]o changes measured with potassium-sensitive electrodes in the myelinated lateral olfactory tract (LOT), in the unmyelinated PC layer I and in the myelinated deep PC layers in the ex vivo isolated guinea-pig brain. Seizure-like events induced by 4AP are initiated by the abrupt [K+]o rise in the layer I formed by unmyelinated fibers (Uva et al., 2017). Larger [K+]o shifts occurred in unmyelinated layers compared to the myelinated LOT. LOT hfST that mimicks pre-seizure discharges also generated higher [K+]o changes in unmyelinated PC layer I than in LOT and deep PC layers. The treatment with the Kir4.1 potassium channel blocker BaCl2 (100 µM) enhanced the [K+]o changes generated by hfST in myelinated structures. Our data show that activity-dependent [K+]o changes are intrinsically different in myelinated vs unmyelinated cortical regions. The larger [K+]o shifts generated in unmyelinated structures may represent a vehicle for seizure generation.

Effects of Kv1.3 knockout on pyramidal neuron excitability and synaptic plasticity in piriform cortex of mice.

Kv1.3 belongs to the voltage-gated potassium (Kv) channel family, which is widely expressed in the central nervous system and associated with a variety of neuropsychiatric disorders. Kv1.3 is highly expressed in the olfactory bulb and piriform cortex and involved in the process of odor perception and nutrient metabolism in animals. Previous studies have explored the function of Kv1.3 in olfactory bulb, while the role of Kv1.3 in piriform cortex was less known. In this study, we investigated the neuronal changes of piriform cortex and feeding behavior after smell stimulation, thus revealing a link between the olfactory sensation and body weight in Kv1.3 KO mice. Coronal slices including the anterior piriform cortex were prepared, whole-cell recording and Ca2+ imaging of pyramidal neurons were conducted. We showed that the firing frequency evoked by depolarization pulses and Ca2+ influx evoked by high K+ solution were significantly increased in pyramidal neurons of Kv1.3 knockout (KO) mice compared to WT mice. Western blotting and immunofluorescence analyses revealed that the downstream signaling molecules CaMKII and PKCα were activated in piriform cortex of Kv1.3 KO mice. Pyramidal neurons in Kv1.3 KO mice exhibited significantly reduced paired-pulse ratio and increased presynaptic Cav2.1 expression, proving that the presynaptic vesicle release might be elevated by Ca2+ influx. Using Golgi staining, we found significantly increased dendritic spine density of pyramidal neurons in Kv1.3 KO mice, supporting the stronger postsynaptic responses in these neurons. In olfactory recognition and feeding behavior tests, we showed that Kv1.3 conditional knockout or cannula injection of 5-(4-phenoxybutoxy) psoralen, a Kv1.3 channel blocker, in piriform cortex both elevated the olfactory recognition index and altered the feeding behavior in mice. In summary, Kv1.3 is a key molecule in regulating neuronal activity of the piriform cortex, which may lay a foundation for the treatment of diseases related to piriform cortex and olfactory detection.

Sexually dimorphic control of affective state processing and empathic behaviors.

Recognizing the affective states of social counterparts and responding appropriately fosters successful social interactions. However, little is known about how the affective states are expressed and perceived and how they influence social decisions. Here, we show that male and female mice emit distinct olfactory cues after experiencing distress. These cues activate distinct neural circuits in the piriform cortex (PiC) and evoke sexually dimorphic empathic behaviors in observers. Specifically, the PiC → PrL pathway is activated in female observers, inducing a social preference for the distressed counterpart. Conversely, the PiC → MeA pathway is activated in male observers, evoking excessive self-grooming behaviors. These pathways originate from non-overlapping PiC neuron populations with distinct gene expression signatures regulated by transcription factors and sex hormones. Our study unveils how internal states of social counterparts are processed through sexually dimorphic mechanisms at the molecular, cellular, and circuit levels and offers insights into the neural mechanisms underpinning sex differences in higher brain functions.

Olfactory threat extinction in the piriform cortex: An age-dependent employment of NMDA receptor-dependent long-term depression.

Extinction of threat memory is a measure of behavioral flexibility. In the absence of additional reinforcement, the extinction of learned behaviors allows animals and humans to adapt to their changing environment. Extinction mechanisms and their therapeutic implications for maladaptive learning have been extensively studied. However, how aging affects extinction learning is much less understood. Using a rat model of olfactory threat extinction, we show that the extinction of olfactory threat memory is impaired in aged Sprague-Darley rats. Following extinction training, long-term depression (LTD) in the piriform cortex (PC) was inducible ex vivo in aged rats and was NMDA receptor (NMDAR)-independent. On the other hand, adult rats acquired successful olfactory threat extinction, and LTD was not inducible following extinction training. Neuronal cFos activation in the posterior PC correlated with learning and extinction performance in rats. NMDAR blockade either systemically or locally in the PC during extinction training prevented successful extinction in adult rats, following which NMDAR-dependent LTD became inducible ex vivo. This suggests that extinction learning employs NMDAR-dependent LTD mechanisms in the PC of adult rats, thus occluding further LTD induction ex vivo. The rescue of olfactory threat extinction in aged rats by D-cycloserine, a partial NMDAR agonist, suggests that the impairment in olfactory threat extinction of aged animals may relate to NMDAR hypofunctioning and a lack of NMDAR-dependent LTD. These findings are consistent with an age-related switch from NMDAR-dependent to NMDAR-independent LTD in the PC. Optimizing NMDAR function in sensory cortices may improve learning and flexible behavior in the aged population.

Aging differentially affects LTCC function in hippocampal CA1 and piriform cortex pyramidal neurons.

Aging is associated with cognitive decline and memory loss in humans. In rats, aging-associated neuronal excitability changes and impairments in learning have been extensively studied in the hippocampus. Here, we investigated the roles of L-type calcium channels (LTCCs) in the rat piriform cortex (PC), in comparison with those of the hippocampus. We employed spatial and olfactory tasks that involve the hippocampus and PC. LTCC blocker nimodipine administration impaired spontaneous location recognition in adult rats (6-9 months). However, the same blocker rescued the spatial learning deficiency in aged rats (19-23 months). In an odor-associative learning task, infusions of nimodipine into either the PC or dorsal CA1 impaired the ability of adult rats to learn a positive odor association. Again, in contrast, nimodipine rescued odor associative learning in aged rats. Aged CA1 neurons had higher somatic expression of LTCC Cav1.2 subunits, exhibited larger afterhyperpolarization (AHP) and lower excitability compared with adult neurons. In contrast, PC neurons from aged rats showed higher excitability and no difference in AHP. Cav1.2 expression was similar in adult and aged PC somata, but relatively higher in PSD95- puncta in aged dendrites. Our data suggest unique features of aging-associated changes in LTCCs in the PC and hippocampus.

Oxytocin via oxytocin receptor excites neurons in the endopiriform nucleus of juvenile mice.

The neuropeptide oxytocin (OXT) modulates social behaviors across species and may play a developmental role for these behaviors and their mediating neural pathways. Despite having high, stable levels of OXT receptor (OXTR) ligand binding from birth, endopiriform nucleus (EPN) remains understudied. EPN integrates olfactory and gustatory input and has reciprocal connections with several limbic areas. Because the role of OXTR signaling in EPN is unknown, we sought to provide anatomical and electrophysiological information about OXTR signaling in mouse EPN neurons. Using in situ hybridization, we found that most EPN neurons co-express Oxtr mRNA and the marker for VGLUT1, a marker for glutamatergic cells. Based on high levels of OXTR ligand binding in EPN, we hypothesized that oxytocin application would modulate activity in these cells as measured by whole-cell patch-clamp electrophysiology. Bath application of OXT and an OXTR specific ligand (TGOT) increased the excitability of EPN neurons in wild-type, but not in OXTR-knockout (KO) tissue. These results show an effect of OXT on a mainly VGLUT1+ cell population within EPN. Given the robust, relatively stable OXTR expression in EPN throughout life, OXTR in this multi-sensory and limbic integration area may be important for modulating activity in response to an array of social or other salient stimuli throughout the lifespan and warrants further study.

A hypothesis concerning distinct schemes of olfactory activation evoked by perceived versus nonperceived input.

SignificanceThe authors propose that odors are consciously perceived or not, depending on whether the olfactory cortex succeeds in activating the endopiriform nucleus-a structure that, in turn, is capable of activating multiple downstream brain areas. The authors further propose that the cellular mechanisms of endopiriform nucleus activation are an attenuated form of cellular events that occur during epileptic seizure initiation. If correct, the authors' hypothesis could help explain the mechanisms of action of certain general anesthetics.

Disinhibitory Circuitry Gates Associative Synaptic Plasticity in Olfactory Cortex.

Inhibitory microcircuits play an essential role in regulating cortical responses to sensory stimuli. Interneurons that inhibit dendritic or somatic integration act as gatekeepers for neural activity, synaptic plasticity, and the formation of sensory representations. Conversely, interneurons that selectively inhibit other interneurons can open gates through disinhibition. In the anterior piriform cortex, relief of inhibition permits associative LTP of excitatory synapses between pyramidal neurons. However, the interneurons and circuits mediating disinhibition have not been elucidated. In this study, we use an optogenetic approach in mice of both sexes to identify the inhibitory interneurons and disinhibitory circuits that regulate LTP. We focused on three prominent interneuron classes: somatostatin (SST), parvalbumin (PV), and vasoactive intestinal polypeptide (VIP) interneurons. We find that LTP is gated by the inactivation SST or PV interneurons and by the activation of VIP interneurons. Further, VIP interneurons strongly inhibit putative SST cells during LTP induction but only weakly inhibit PV interneurons. Together, these findings suggest that VIP interneurons mediate a disinhibitory circuit that gates synaptic plasticity during the formation of olfactory representations.SIGNIFICANCE STATEMENT Inhibitory interneurons stabilize neural activity during sensory processing. However, inhibition must also be modulated to allow sensory experience shape neural responses. In olfactory cortex, inhibition regulates activity-dependent increases in excitatory synaptic strength that accompany odor learning. We identify two inhibitory interneuron classes that act as gatekeepers preventing excitatory enhancement. We demonstrate that driving a third class of interneurons inhibits the gatekeepers and opens the gate for excitatory enhancement. All three inhibitory neuron classes comprise disinhibitory microcircuit motifs found throughout the cortex. Our findings suggest that a common disinhibitory microcircuit promotes changes in synaptic strength during sensory processing and learning.

Induction of Bdnf from promoter I following electroconvulsive seizures contributes to structural plasticity in neurons of the piriform cortex.

BACKGROUND: Electroconvulsive therapy (ECT) efficacy is hypothesized to depend on induction of molecular and cellular events that trigger neuronal plasticity. Investigating how electroconvulsive seizures (ECS) impact plasticity in animal models can help inform our understanding of basic mechanisms by which ECT relieves symptoms of depression. ECS-induced plasticity is associated with differential expression of unique isoforms encoding the neurotrophin, brain-derived neurotrophic factor (BDNF). HYPOTHESIS: We hypothesized that cells expressing the Bdnf exon 1-containing isoform are important for ECS-induced structural plasticity in the piriform cortex, a highly epileptogenic region that is responsive to ECS. METHODS: We selectively labeled Bdnf exon 1-expressing neurons in mouse piriform cortex using Cre recombinase dependent on GFP technology (CRE-DOG). We then quantified changes in dendrite morphology and density of Bdnf exon 1-expressing neurons. RESULTS: Loss of promoter I-derived BDNF caused changes in spine density and morphology in Bdnf exon 1-expressing neurons following ECS. CONCLUSIONS: Promoter I-derived Bdnf is required for ECS-induced dendritic structural plasticity in Bdnf exon 1-expressing neurons.

Age-Dependent Contributions of NMDA Receptors and L-Type Calcium Channels to Long-Term Depression in the Piriform Cortex.

In the hippocampus, the contributions of N-methyl-D-aspartate receptors (NMDARs) and L-type calcium channels (LTCCs) to neuronal transmission and synaptic plasticity change with aging, underlying calcium dysregulation and cognitive dysfunction. However, the relative contributions of NMDARs and LTCCs in other learning encoding structures during aging are not known. The piriform cortex (PC) plays a significant role in odor associative memories, and like the hippocampus, exhibits forms of long-term synaptic plasticity. Here, we investigated the expression and contribution of NMDARs and LTCCs in long-term depression (LTD) of the PC associational fiber pathway in three cohorts of Sprague Dawley rats: neonatal (1-2 weeks), young adult (2-3 months) and aged (20-25 months). Using a combination of slice electrophysiology, Western blotting, fluorescent immunohistochemistry and confocal imaging, we observed a shift from an NMDAR to LTCC mediation of LTD in aged rats, despite no difference in the amount of LTD expression. These changes in plasticity are related to age-dependent differential receptor expression in the PC. LTCC Cav1.2 expression relative to postsynaptic density protein 95 is increased in the associational pathway of the aged PC layer Ib. Enhanced LTCC contribution in synaptic depression in the PC may contribute to altered olfactory function and learning with aging.

CB1 receptors in corticotropin-releasing factor neurons selectively control the acoustic startle response in male mice.

The endocannabinoid system is an important regulator of the hormonal and behavioral stress responses, which critically involve corticotropin-releasing factor (CRF) and its receptors. While it has been shown that CRF and the cannabinoid type 1 (CB1) receptor are co-localized in several brain regions, the physiological relevance of this co-expression remains unclear. Using double in situ hybridization, we confirmed co-localization in the piriform cortex, the lateral hypothalamic area, the paraventricular nucleus, and the Barrington's nucleus, albeit at low levels. To study the behavioral and physiological implications of this co-expression, we generated a conditional knockout mouse line that selectively lacks the expression of CB1 receptors in CRF neurons. We found no effects on fear and anxiety-related behaviors under basal conditions nor after a traumatic experience. Additionally, plasma corticosterone levels were unaffected at baseline and after restraint stress. Only acoustic startle responses were significantly enhanced in male, but not female, knockout mice. Taken together, the consequences of depleting CB1 in CRF-positive neurons caused a confined hyperarousal phenotype in a sex-dependent manner. The current results suggest that the important interplay between the central endocannabinoid and CRF systems in regulating the organism's stress response is predominantly taking place at the level of CRF receptor-expressing neurons.

Target-specific control of piriform cortical output via distinct inhibitory circuits.

Information represented by principal neurons in anterior piriform cortex (APC) is regulated by local, recurrent excitation and inhibition, but the circuit mechanisms remain elusive. Two types of layer 2 (L2) principal neurons, semilunar (SL), and superficial pyramidal (SP) cells, are parallel output channels, and the control of their activity gates the output of APC. Here, we examined the hypothesis that recurrent inhibition differentially regulates SL and SP cells. Patterned optogenetic stimulation revealed that the strength of recurrent inhibition is target- and layer-specific: L1 > L3 for SL cells, but L3 > L1 for SP cells. This target- and layer-specific inhibition was largely attributable to the parvalbumin (PV), but not somatostatin, interneurons. Intriguingly, olfactory experience selectively modulated the PV to SP microcircuit while maintaining the overall target and laminar specificity of inhibition. Together, these results indicate the importance of target-specific inhibitory wiring for odor processing, implicating these mechanisms in gating the output of piriform cortex.

Adenosine A2A receptor neurons in the olfactory bulb mediate odor-guided behaviors in mice.

Depression, rapid eye movement (REM) sleep behavior disorder, and altered olfaction are often present in Parkinson's disease. Our previous studies demonstrated the role of the olfactory bulb (OB) in causing REM sleep disturbances in depression. Furthermore, adenosine A2A receptors (A2AR) which are richly expressed in the OB, play an important role in the regulation of REM sleep. Caffeine, an adenosine A1 receptors and A2AR antagonist, and other A2AR antagonists were reported to improve olfactory function and restore age-related olfactory deficits. Therefore, we hypothesized that the A2AR neurons in the OB may regulate olfaction or odor-guided behaviors in mice. In the present study, we employed chemogenetics to specifically activate or inhibit neuronal activity. Then, buried food test and olfactory habituation/dishabituation test were performed to measure the changes in the mice's olfactory ability. We demonstrated that activation of OB neurons or OB A2AR neurons shortened the latency of buried food test and enhanced olfactory habituation to the same odors and dishabituation to different odors; inhibition of these neurons showed the opposite effects. Photostimulation of ChR2-expressing OB A2AR neuron terminals evoked inward current in the olfactory tubercle (OT) and the piriform cortex (Pir), which was blocked by glutamate receptor antagonists 2-amino-5-phosphonopentanoic acid and 6-cyano-7nitroquinoxaline-2,3-dione. Collectively, these results suggest that the OB mediates olfaction via A2AR neurons in mice. Moreover, the excitatory glutamatergic release from OB neurons to the OT and the Pir were found responsible for the olfaction-mediated effects of OB A2AR neurons.

Chronic loss of inhibition in piriform cortex following brief, daily optogenetic stimulation.

It is well established that seizures beget seizures, yet the cellular processes that underlie progressive epileptogenesis remain unclear. Here, we use optogenetics to briefly activate targeted populations of mouse piriform cortex (PCx) principal neurons in vivo. After just 3 or 4 days of stimulation, previously subconvulsive stimuli trigger massive, generalized seizures. Highly recurrent allocortices are especially prone to "optokindling." Optokindling upsets the balance of recurrent excitation and feedback inhibition. To understand how this balance is disrupted, we then selectively reactivate the same neurons in vitro. Surprisingly, we find no evidence of heterosynaptic potentiation; instead, we observe a marked, pathway-specific decrease in feedback inhibition. We find no loss of inhibitory interneurons; rather, decreased GABA synthesis in feedback inhibitory neurons appears to underlie weakened inhibition. Optokindling will allow precise identification of the molecular processes by which brain activity patterns can progressively and pathologically disrupt the balance of cortical excitation and inhibition.

Perturbation of in vivo Neural Activity Following α-Synuclein Seeding in the Olfactory Bulb.

BACKGROUND: Parkinson's disease (PD) neuropathology is characterized by intraneuronal protein aggregates composed of misfolded α-Synuclein (α-Syn), as well as degeneration of substantia nigra dopamine neurons. Deficits in olfactory perception and aggregation of α-Syn in the olfactory bulb (OB) are observed during early stages of PD, and have been associated with the PD prodrome, before onset of the classic motor deficits. α-Syn fibrils injected into the OB of mice cause progressive propagation of α-Syn pathology throughout the olfactory system and are coupled to olfactory perceptual deficits. OBJECTIVE: We hypothesized that accumulation of pathogenic α-Syn in the OB impairs neural activity in the olfactory system. METHODS: To address this, we monitored spontaneous and odor-evoked local field potential dynamics in awake wild type mice simultaneously in the OB and piriform cortex (PCX) one, two, and three months following injection of pathogenic preformed α-Syn fibrils in the OB. RESULTS: We detected α-Syn pathology in both the OB and PCX. We also observed that α-Syn fibril injections influenced odor-evoked activity in the OB. In particular, α-Syn fibril-injected mice displayed aberrantly high odor-evoked power in the beta spectral range. A similar change in activity was not detected in the PCX, despite high levels of α-Syn pathology. CONCLUSION: Together, this work provides evidence that synucleinopathy impacts in vivo neural activity in the olfactory system at the network-level.

Piriform cortex alterations in the Ts65Dn model for down syndrome.

The piriform cortex is involved in olfactory information processing, that is altered in Down Syndrome. Moreover, piriform cortex has a crucial involvement in epilepsy generation and is one of the first regions affected in Alzheimer's Disease, both maladies being prevalent among Down Syndrome individuals. In this work, we studied the alterations in neuronal morphology, synaptology and structural plasticity in the piriform cortex of the Ts65Dn mouse model, which is the most used model for the study of this syndrome and mimics some of their alterations. We have observed that Ts65Dn piriform cortex displays: a reduction in dendritic arborisation, a higher density of inhibitory synapses (GAD67), a lower density of excitatory synapses (vGLUT1) and a higher density of inhibitory postsynaptic puncta (gephyrin). Under electron microscopy the excitatory presynaptic and postsynaptic elements were larger in trisomic mice than in controls. Similar results were obtained using confocal microscopy. There were less immature neurons in piriform cortex layer II in addition to a reduction in the expression of PSA-NCAM in the neuropil that subsequently can reflect impairment in structural plasticity. These data support the idea of an impaired environment with altered ratio of inhibition and excitation that involves a reduction in plasticity and dendritic atrophy, providing a possible substrate for the olfactory processing impairment observed in DS individuals.

Sequential pattern of sublayer formation in the paleocortex and neocortex.

The piriform cortex (paleocortex) is the olfactory cortex or the primary cortex for the sense of smell. It receives the olfactory input from the mitral and tufted cells of the olfactory bulb and is involved in the processing of information pertaining to odors. The piriform cortex and the adjoining neocortex have different cytoarchitectures; while the former has a three-layered structure, the latter has a six-layered structure. The regulatory mechanisms underlying the building of the six-layered neocortex are well established; in contrast, less is known about of the regulatory mechanisms responsible for structure formation of the piriform cortex. The differences as well as similarities in the regulatory mechanisms between the neocortex and the piriform cortex remain unclear. Here, the expression of neocortical layer-specific genes in the piriform cortex was examined. Two sublayers were found to be distinguished in layer II of the piriform cortex using Ctip2/Bcl11b and Brn1/Pou3f3. The sequential expression pattern of Ctip2 and Brn1 in the piriform cortex was similar to that detected in the neocortex, although the laminar arrangement in the piriform cortex exhibited an outside-in arrangement, unlike that observed in the neocortex.

Serotonergic afferents from the dorsal raphe decrease the excitability of pyramidal neurons in the anterior piriform cortex.

The olfactory system receives extensive serotonergic inputs from the dorsal raphe, a nucleus involved in control of behavior, regulation of mood, and modulation of sensory processing. Although many studies have investigated how serotonin modulates the olfactory bulb, few have focused on the anterior piriform cortex (aPC), a region important for olfactory learning and encoding of odor identity and intensity. Specifically, the mechanism and functional significance of serotonergic modulation of the aPC remain largely unknown. Here we used pharmacologic, optogenetic, and fiber photometry techniques to examine the serotonergic modulation of neural activity in the aPC in vitro and in vivo. We found that serotonin (5-HT) reduces the excitability of pyramidal neurons directly via 5-HT2C receptors, phospholipase C, and calcium-activated potassium (BK) channels. Furthermore, endogenous serotonin attenuates odor-evoked calcium responses in aPC pyramidal neurons. These findings identify the mechanism underlying serotonergic modulation of the aPC and shed light on its potential role.

Functional Integration of Neuronal Precursors in the Adult Murine Piriform Cortex.

The extent of functional maturation and integration of nonproliferative neuronal precursors, becoming neurons in the adult murine piriform cortex, is largely unexplored. We thus questioned whether precursors eventually become equivalent to neighboring principal neurons or whether they represent a novel functional network element. Adult brain neuronal precursors and immature neurons (complex cells) were labeled in transgenic mice (DCX-DsRed and DCX-CreERT2 /flox-EGFP), and their cell fate was characterized with patch clamp experiments and morphometric analysis of axon initial segments. Young (DCX+) complex cells in the piriform cortex of 2- to 4-month-old mice received sparse synaptic input and fired action potentials at low maximal frequency, resembling neonatal principal neurons. Following maturation, the synaptic input detected on older (DCX-) complex cells was larger, but predominantly GABAergic, despite evidence of glutamatergic synaptic contacts. Furthermore, the rheobase current of old complex cells was larger and the maximal firing frequency was lower than those measured in neighboring age-matched principal neurons. The striking differences between principal neurons and complex cells suggest that the latter are a novel type of neuron and new coding element in the adult brain rather than simple addition or replacement for preexisting network components.