Development and evaluation of a standardized method and atlas for contouring primary and permanent dentition.

OBJECTIVES: Radiation toxicity of the dentition may present significant treatment-related morbidity in the paediatric head and neck cancer population. However, clear dose-effect relationships remain undetermined and must be predicated upon accurate structure delineation and dosimetry at the individual tooth level. Radiation oncologists generally have limited familiarity or experience with relevant dental anatomy. METHODS: We therefore developed a detailed CT atlas of permanent and primary dentition. After studying this atlas, five radiation oncology clinicians delineated all teeth for each of eight different cases (selected for breadth of dental maturity and anatomical variability). They were asked to record confidence in their contours on a per-tooth basis as well as the duration of time required per case. Contour accuracy and interclinician variability were assessed by Hausdorff distance and Dice similarity coefficient. All analyses were performed using R v. 3.1.1 and the RadOnc v. 1.0.9 package. RESULTS: Participating clinicians delineated teeth with varying degrees of completeness and accuracy, stratified primarily by the age of the subject. On a per-tooth basis, delineation of permanent dentition was feasible for incisors, canines, premolars and first molars among all subjects, even at the youngest ages. However, delineation of second and third molars was less consistent, commensurate with approximate timing of tooth development. Within each tooth contour, uncertainty was the greatest at the level of the dental roots. CONCLUSIONS: Delineation of individual teeth is feasible and serves as a necessary precursor for dental dose assessment and avoidance. Among the paediatric radiation oncology community in particular, this atlas may serve as a useful tool and reference.

Eyeblink conditioning leads to fewer synapses in the rabbit cerebellar cortex.

Eyeblink conditioning involves the pairing of a conditioned stimulus (tone) to an aversive unconditioned stimulus (air puff). Although the circuitry that underlies this form of learning is well defined, synaptic changes in these structures have not been fully investigated. This experiment examined synaptic structural plasticity in the cerebellar cortex, a structure that has been found to modulate the acquisition and timing of the conditioned response. Long-term depression of Purkinje cells (PCs) in the cerebellar cortex has been proposed as a mechanism for releasing inhibition of the interpositus nuclei, a structure critical for the formation of the CR. Adult albino rabbits were randomly allocated to either a paired, unpaired, or exposure-only condition. The results showed a significant decrease in the number of excitatory synapses in the outer layer of the cerebellar cortex in the conditioned rabbits compared with controls. This finding suggests that a reduction in the number of excitatory synapses may contribute to the lasting depression of PC activity that is associated with eyeblink conditioning.

The role of the cerebellum in classical conditioning of discrete behavioral responses.

The cerebellum and its associated circuitry constitutes the entire essential neuronal system for classical conditioning of eye-blink and other discrete responses (e.g. limb flexion) learned with an aversive unconditioned stimulus (US) using the standard delay paradigm where the conditioned stimulus (CS) and the US coterminate. Evidence reviewed here strongly supports the following conclusions. The CS pathway involves sensory relay nuclei projections to the pontine nuclei and its mossy fiber projections to the cerebellar cortex and nuclei. The US pathway involves activation of the inferior olive (dorsal accessory olive for eye blink) and its climbing fiber projections to the cerebellar cortex and nuclei. The conditioned response (CR) pathway involves the cerebellar interpositus nucleus, the superior cerebellar peduncle pathway to the magnocellular red nucleus and rubral projections to premotor and motor nuclei generating the behavioral response. Anatomical data, neuronal unit recordings, electrical stimulation, lesions and methods of reversible inactivation all strongly support the hypothesis that the essential memory trace for the learning of these discrete conditioned responses is formed and stored in the cerebellar interpositus nucleus. Neuronal/synaptic plasticity is also established in the cerebellar cortex in this form of learning but the role of the cortex is less clear. We argue that the cortex plays a key role in normal acquisition and adaptive timing of the conditioned response, under certain circumstances, but it remains unclear exactly what features of conditioning are being encoded in the cerebellar cortex in this basic form of associative learning and memory.

Dissociaton of conditioned eye and limb responses in the cerebellar interpositus.

Thompson and colleagues have demonstrated that the lateral interpositus nucleus of the cerebellum is the essential locus for the classical conditioning of the somatic eyeblink response. Preliminary studies reported that lesioning the cerebellar interpositus nucleus ipsilateral to the side of training also appears to abolish conditioned limb flexion responses. Previous studies have suggested that the interpositus nucleus is somatotopically organized with the eye being represented laterally and the hindlimb medially. Presently, we employed a double dissociation paradigm to examine the effects of muscimol (a GABA(A) agonist) injections on eyeblink versus limb flexion conditioned responses in the ipsilateral cerebellar interpositus nucleus of New Zealand white rabbits. For eyeblink conditioning, the conditioned stimulus (CS) was a 14-V lamp bulb and the unconditioned stimulus (US) was a 3-psi corneal airpuff to the left eye. For limb flexion conditioning, the CS was a 1-kHz, 85-95 dB SPL tone and the US was a 3- to 5-mA shock to the upper left hindlimb. Upon training on both responses to a 60-100% criterion, the rabbits were then tested on eyeblink and limb flexion responses after injections of muscimol (0.1-0.3 mul of a 0.01- to 1.0-M solution) into either the lateral (eyeblink) or medial (limb flexion) interpositus nucleus. We have been able to successfully decrease or abolish the percent conditioned responses (CRs) of both the eyeblink and limb flexion conditioning selectively without affecting the other. These results thus lend further support for the notion of the existence of a somatotopic map in the interpositus nucleus for learning.

Cerebellar substrates for error correction in motor conditioning.

The authors evaluate a mapping of Rescorla and Wagner's (1972) behavioral model of classical conditioning onto the cerebellar substrates for motor reflex learning and illustrate how the limitations of the Rescorla-Wagner model are just as useful as its successes for guiding the development of new psychobiological theories of learning. They postulate that the inhibitory pathway that returns conditioned response information from the cerebellar interpositus nucleus back to the inferior olive is the neural basis for the error correction learning proposed by Rescorla and Wagner (Gluck, Myers, & Thompson, 1994; Thompson, 1986). The authors' cerebellar model expects that behavioral processes described by the Rescorla-Wagner model will be localized within the cerebellum and related brain stem structures, whereas behavioral processes beyond the scope of the Rescorla-Wagner model will depend on extracerebellar structures such as the hippocampus and related cortical regions. Simulations presented here support both implications. Several novel implications of the authors' cerebellar error-correcting model are described including a recent empirical study by Kim, Krupa, and Thompson (1998), who verified that suppressing the putative error correction pathway should interfere with the Kamin (1969) blocking effect, a behavioral manifestation of error correction learning. The authors also discuss the model's implications for understanding the limits of cerebellar contributions to associative learning and how this informs our understanding of hippocampal function in conditioning. This leads to a more integrative view of the neural substrates of conditioning in which the authors' real-time circuit-level model of the cerebellum can be viewed as a generalization of the long-term memory module of Gluck and Myers' (1993) trial-level theory of cerebellar-hippocampal interaction in motor conditioning.

Cyclic changes in estradiol regulate synaptic plasticity through the MAP kinase pathway.

Hippocampal synaptic structure and function exhibit marked variations during the estrus cycle of female rats. Estradiol activates the mitogen-activated protein (MAP) kinase pathway in numerous cell types, and MAP kinase has been shown to play a critical role in the mechanisms underlying synaptic plasticity. Here, we report that endogenous estrogen produces a tonic phosphorylation/activation of extracellular signal-regulated kinase 2 (ERK2)/MAP kinase throughout the female rat brain and an increase in tyrosine phosphorylation of NR2 subunits of N-methyl-D-aspartate (NMDA) receptors. Moreover, cyclic changes in estrogen levels during the estrus cycle of female rats are associated with corresponding changes in the levels of activation of ERK2, the state of tyrosine phosphorylation of NR2 subunits of NMDA receptors, and the magnitude of long-term potentiation in hippocampus. Thus, cyclic changes in female sexual hormones result in marked variations in the state of activation of a major cellular signaling pathway critical for learning and memory and in a cellular model of learning and memory.

Associative fear conditioning of enkephalin mRNA levels in central amygdalar neurons.

The central nucleus of the amygdala (CEA) is required for the expression of learned fear responses. This study used in situ hybridization to show that mRNA levels of the neuropeptide enkephalin are increased in CEA neurons after rats are placed in an environment that they associate with an unpleasant experience. In contrast, mRNA levels of another neuropeptide, corticotropin releasing hormone, do not change under the same conditions in the CEA of the same rats. Conditioned neuropeptide levels in amygdalar circuits may act as a reversible "gain control" for long-term modulation of subsequent fear responses.

Potentiation of conditioned freezing following dorsomedial prefrontal cortex lesions does not interfere with fear reduction in mice.

In Experiment 1, an auditory conditioned stimulus (CS) was paired with footshock, except when it was preceded by another stimulus (a visual conditioned inhibitor [CI]). After conditioning, all mice displayed less CS-evoked freezing when the CI-CS compound was presented than when the CS was presented alone. However, lesions of the dorsomedial prefrontal cortex (dmPFC) potentiated CS-evoked freezing on each of the 2 sessions (i.e., CI CS and CS alone). In Experiment 2, mice were submitted to fear extinction (CS-alone presentation for 3 days). Lesioned mice exhibited a higher level of freezing behavior than controls on each of the 3 sessions. However, lesioned mice and controls displayed the same rate of reduction of freezing over the 3 days of extinction. These data in mice support previous studies in rats, which suggests that the dmPFC is not critical for either conditioned inhibition or extinction of acquired freezing behavior.

Learning- and cerebellum-dependent neuronal activity in the lateral pontine nucleus.

The effects of inactivation of cerebellar deep nuclei and the lateral pontine nucleus on classical eyeblink conditioning with tone or lateral reticular nucleus (LRN) stimulation as conditioned stimuli (CSs) were examined. Inactivation of cerebellar deep nuclei abolished eyeblink conditioned responses (CRs) when the CS was either a tone or LRN stimulation. Inactivation of the lateral pontine nucleus prevented only the acquisition and retention of tone-evoked eyeblink CRs. Multiple-unit recording demonstrated that when LRN stimulation was used as the CS, inactivation of the interpositus nucleus abolished learning-related neuronal activity in the lateral pontine nucleus, whereas inactivation of pontine nucleus had little effect on similar activity in the interpositus nucleus. Thus, the learning-induced neuronal activity in the lateral pontine nucleus was most likely driven by the cerebellar interpositus nucleus.

Identification of variants and dual promoters of murine serine/threonine kinase KKIAMRE.

KKIAMRE is a serine/threonine protein kinase whose transcripts increase in the deep cerebellar nuclei of the rabbit after eyeblink conditioning, a model of associative learning and memory. We here characterized the expression, isoforms, and promoters of murine KKIAMRE gene. The expression of KKIAMRE was detected, by in situ hybridization and immunohistochemistry, in neurons in various brain regions including deep cerebellar nuclei. The gene spans approximately 40 kb and consists of 15 exons. Analysis of cDNA clones revealed multiple variants, having diversity in the putative carboxy-terminal regulatory domain, generated by alternative splicing and intraexonal termination. Furthermore, they had alternative 5' noncoding sequences. Primer extension, RNase protection, and transient expression assays revealed that two alternative promoters linked to distinct noncoding exons direct the expression of KKIAMRE. The gene was mapped on chromosomes 5 and 4 in mouse and human, respectively.

Intracerebellar conditioning--Brogden and Gantt revisited.

In 1942, Brogden and Gantt reported that electrical stimulation of cerebellar white matter elicited specific behavioral responses (limb flexion, eyeblink, etc.) and that these movements so elicited could easily be conditioned to a neural tone CS, using standard Pavlovian procedures. This early evidence for the key role of the cerebellum in learning of discrete movements has in recent years been replicated and much extended. It is now clear that the cerebellum is the essential structure for associative learning of discrete movements elicited by peripheral aversive or intracerebellar stimuli and that the memory traces so formed are stored in the cerebellum.

The tyrosine kinase and mitogen-activated protein kinase pathways mediate multiple effects of estrogen in hippocampus.

Estrogen replacement therapy in women is associated with improvement of cognitive deficits and reduced incidence of Alzheimer's disease. The present study indicates that estrogen is neuroprotective against N-methyl-d-aspartate (NMDA)- and kainate-mediated neurotoxicity, an effect mediated by tyrosine kinase/mitogen-activated protein kinase (MAPK) pathways. Estrogen also stimulates tyrosine phosphorylation of NMDA receptors via an src tyrosine kinase/MAPK pathway. Finally, estrogen-mediated enhancement of long-term potentiation in hippocampal slices is mediated by activation of an src tyrosine kinase pathway. Thus, estrogen, by activating an src tyrosine kinase and the extracellular signal-related protein kinase/MAPK signaling pathway, both enhances NMDA receptor function and long-term potentiation and retains neuroprotective properties against excitotoxicity. These findings warrant further evaluation of the usefulness of estrogenic compounds for the treatment of Alzheimer's disease and other neurodegenerative diseases.

17beta-estradiol suppresses expression of long-term depression in aged rats.

It has been recently reported that the female steroid hormone 17beta-estradiol enhances synaptic transmission and the magnitude of long-term potentiation (LTP) in adult rodent hippocampus. Moreover, 17beta-estradiol ameliorates cognitive and memory function in postmenopausal women. Since aging is associated with an alteration of synaptic plasticity (e.g., higher susceptibility to long-term depression [LTD]), we examined whether 17beta-estradiol alters the expression of LTD in aged rats. We now report that the induction of LTD recorded from CA1 hippocampal neurons of aged rats is suppressed by 17beta-estradiol treatment, which produced only a minimal effect in suppressing LTD in adult rats. These results suggest that estrogen may act to improve memory by suppressing forgetfulness via a synaptic mechanism, such as LTD.

The amygdala modulates prefrontal cortex activity relative to conditioned fear.

Animals learn that a tone can predict the occurrence of an electric shock through classical conditioning. Mice or rats trained in this manner display fear responses, such as freezing behaviour, when they hear the conditioned tone. Studies using amygdalectomized rats have shown that the amygdala is required for both the acquisition and expression of learned fear responses. Freezing to a conditioned tone is enhanced following damage to the dorsal part of the medial prefrontal cortex, indicating that this area may be involved in fear reduction. Here we show that prefrontal neurons reduce their spontaneous activity in the presence of a conditioned aversive tone as a function of the degree of fear. The depression in prefrontal spontaneous activity is related to amygdala activity but not to the freezing response itself. These data indicate that, in the presence of threatening stimuli, the amygdala controls both fear expression and prefrontal neuronal activity. They suggest that abnormal amygdala-induced modulation of prefrontal neuronal activity may be involved in the pathophysiology of certain forms of anxiety disorder.

Impaired cerebellar synapse maturation in waggler, a mutant mouse with a disrupted neuronal calcium channel gamma subunit.

The waggler, a neurological mutant mouse with a disrupted putative neuronal Ca(2+) channel gamma subunit, exhibits a cerebellar granule cell-specific brain-derived neurotrophic factor deficit, severe ataxia, and impaired eyeblink conditioning. Here, we show that multiple synapses of waggler cerebellar granule cells are arrested at an immature stage during development. Synaptic transmission is reduced at parallel fiber-Purkinje cell synapses. The Golgi cell-granule cell synaptic currents show immature kinetics associated with reduced gamma-aminobutyric acid type A receptor alpha6 subunit expression in granule cells. In addition, the mossy fiber-granule cell synapses exhibit N-methyl-D-aspartate (NMDA) receptor-mediated excitatory postsynaptic currents (EPSCs), but not alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated EPSCs. Our results suggest that voltage-dependent Ca(2+) channels are involved in synapse maturation. This deficient synaptic transmission in the waggler cerebellum may account for their behavioral deficits.

Learning induces a CDC2-related protein kinase, KKIAMRE.

To elucidate molecular mechanisms in learning and memory, we analyzed expression of mRNAs in brains of rabbits undergoing eyeblink conditioning. Infusion of the transcription inhibitor actinomycin D into the cerebellar interpositus nucleus reversibly blocked learning but not performance of the conditioned response. Differential display PCR analysis of cerebellar interpositus RNAs from trained and pseudotrained rabbits identified a 207 bp band that was induced with learning. The fragment was used to isolate a cDNA from a lambdagt11 rabbit brain library containing a 1698 bp open reading frame. The deduced amino acid sequence contains the KKIAMRE motif, which is conserved among cell division cycle 2 (cdc2)-related kinases. These results suggest that there is a new category of cdc2-related kinases in the brain whose function may be important in learning and memory.

Transgenic brain-derived neurotrophic factor modulates a developing cerebellar inhibitory synapse.

Brain-derived neurotrophic factor (BDNF) has been shown to promote synapse formation and maturation in neurons of many brain regions, including inhibitory synapses. In the cerebellum, the Golgi cell-granule cell GABAergic synaptic responses undergo developmental transition from slow-decaying to fast-decaying kinetics, which parallels a developmental increase of GABA(A) receptor alpha6 subunit expression in the cerebellar granule cells. In culture, BDNF accelerates the expression of GABA(A) receptor alpha6 subunit expression in granule cells. Here we examined synaptic GABA(A) response kinetics in BDNF transgenic mice. The mutant mouse, which carries a BDNF transgene driven by a beta-actin promoter, overexpresses BDNF (two- to fivefold increase compared with wild types) in all brain regions. Recordings of the spontaneous GABA(A) responses indicate that the decay time constant of the GABAergic responses decreases during early postnatal development; this transition is accelerated in the BDNF transgenic mouse. The amplitude of the spontaneous GABA(A) responses was also larger in the transgenic mouse than in the wild-type mouse. However, the frequency of the spontaneous GABA(A) responses were not different between the two groups. Our results suggest that BDNF may modulate GABAergic synapse maturation in the cerebellum.